EEEV replicase trans-amplifying rnas

Abstract

This disclosure includes trans-amplifying RNAs (taRNAs) encoding Eastern Equine Encephalitis Virus (EEEV) replicase and methods of use thereof for amplifying a ribonucleic acid (RNA) polynucleotide comprising or encoding a payload in a subject in trans without inducing substantial cytotoxicity in the liver of the subject.

Description

[0001] EEEV REPLICASE TRANS- AMPLIFYING RNAS

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit under 35 U. S. C. § 119(e) of US Provisional Application No. 63 / 728,441, filed December 5, 2024, entitled “EEEV REPLICASE TRANSAMPLIFYING RNAS”, the content of which is hereby incorporated by reference herein in its entirety for all purposes.

[0004] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0005] The contents of the electronic sequence listing (A141470009WO00-SEQ-KGC.xml; Size: 245,699 bytes; and Date of Creation: December 4, 2025) are herein incorporated by reference in their entirety.

[0006] BACKGROUND

[0007] Presence and expression of non-self-nucleic acids (e.g., viral or synthetic polynucleotides) in mammalian cells often leads to host immune responses and can cause undesired secondary effects, posing several major obstacles for ribonucleic acid (RNA)-based therapies.

[0008] SUMMARY

[0009] Trans amplifying ribonucleic acids (taRNAs) are useful for expressing payloads (e.g., RNA payloads, proteins). taRNAs comprise two different RNA polynucleotides: a first RNA encoding a replicase (i.e., a replicase construct); and a second RNA comprising or encoding a payload (i.e., a trans-replicating RNA (trRNA). The replicase, when expressed, replicates the trRNA in trans which in turn produces the payload. Administration of taRNAs has been associated with induction of certain host responses. While the cause of these host responses has largely been unclear, it has long been assumed to be related to amplification of RNA by replicases. The inventors have discovered that taRNAs encoding certain replicases (e.g., EEEV) are useful for evading expression of certain cytokines and attenuating liver cytotoxicity.

[0010] Additionally, taRNAs may comprise a nucleic acid encoding an immunomodulatory protein (IMP) on the first and / or second RNA). Provided herein are methods and compositions useful

[0011] 12673398.1 for amplifying and expressing RNA-encoded payloads in trans in subjects while avoiding typical side effects associated with taRNA administration.

[0012] Provided herein, in some aspects, are / ra / r.s-amplifying ribonucleic acids (RNA) (taRNAs) comprising a first RNA polynucleotide (i.e., a replicase construct) comprising a nucleic acid encoding an Eastern Equine Encephalitis Virus (EEEV) replicase; and a second RNA polynucleotide (i.e., a trRNA) comprising: a nucleic acid payload or a nucleic acid encoding a payload; wherein the first RNA polynucleotide and / or the second RNA polynucleotide further comprise a nucleic acid encoding an immune modulating protein (IMP). In some embodiments, the first RNA polynucleotide comprises the nucleic acid encoding the IMP. In some embodiments, the second RNA polynucleotide comprises the nucleic acid encoding the IMP.

[0013] In some embodiments, the IMP is a viral immune evasion protein (VIEP). In some embodiments, the VIEP is from vaccinia virus (VACV), herpes simplex virus (HSV), influenza virus, Toscana Virus (TOSV), or encephalomyocarditis virus (EMCV). In some embodiments, the VIEP is a VACV soluble IFN alpha / beta receptor B 18 (B18R), VACV RNA-binding protein E3 (E3L), VACVF1L, VACV N1L, HSV infected cell protein 34.5 (ISC34.5), HSV unique short 1 (US1), HSV unique short 11 (US11), influenza virus non-structural protein 1 (NS1), TOSV non- structural (NSs) protein, or EMCV Lpro. In some embodiments, the NS1 is from Influenza A / Puerto Rico / 8 / 34 influenza virus (PR8) (PR8 NS1). In some embodiments, the VIEP is PR8 NS1. In some embodiments, the VIEP is TOSV NSs. In some embodiments, the VIEP is EMCV Lpro.

[0014] In some embodiments, the IMP antagonizes a mammalian antiviral factor. In some embodiments, the IMP that antagonizes a mammalian antiviral factor is dominant negative mitochondrial antiviral-signaling protein (dnMAVS), microprotein in antiviral immunity 1 (MAVH), dominant negative protein kinase R (dnPKR), suppressor of cytokine signaling (SOCS), or dominant negative Zinc-finger Antiviral Protein (dnZAP). In some embodiments, the dnZAP is rat ZAP with cysteine-to-arginine mutation at position 88 (rZAPC88R). In some embodiments, the SOCS is SOCS1 or SOCS3.

[0015] In some embodiments, the first RNA polynucleotide comprises, from 8' to 6': (i) the nucleic acid encoding the EEEV replicase; and (ii) the nucleic acid encoding the IMP. In some embodiments, the nucleic acid encoding the EEEV replicase and the nucleic acid encoding the IMP are joined by a linker. In some embodiments, the linker is a nucleic acid encoding a 2A peptide, or an internal ribosome entry site (IRES) element. In some embodiments, 12673398.1 the 2A peptide is thosea asigna virus 2A (T2A), porcine teschovirus-1 2A (P2A), equine rhinitis A virus 2A (E2A), or Furin-2A. In some embodiments, the IRES element is an IRES from Coxsackievirus B3 (CVB3), encephalomyocarditis virus (EMCV), porcine kobuvirus (PKV), Manhattan Parechovirus (MPV), or tortoise rafivirus (TraV).

[0016] In some embodiments, the first RNA polynucleotide comprises, from 8' to 6': (i) the nucleic acid encoding the EEEV replicase; (ii) the linker; and (iii) the nucleic acid encoding the IMP.

[0017] In some embodiments, the first RNA polynucleotide comprises, from 8' to 6': (i) the nucleic acid encoding the IMP; (ii) the linker; and (iii) the nucleic acid encoding the EEEV replicase.

[0018] In some embodiments, the taRNA comprises a nucleic acid encoding a first IMP and a nucleic acid encoding a second IMP. In some embodiments, the first IMP and the second IMP are different IMPs. In some embodiments, the first RNA polynucleotide comprises, from 8' to 6': (i) the nucleic acid encoding the first IMP; (ii) a first linker; (iii) the nucleic acid encoding an EEEV replicase; (iv) a second linker; and (vi) the nucleic acid encoding the second IMP.

[0019] In some embodiments, the first RNA polynucleotide comprises, from 8' to 6': (i) the nucleic acid encoding the IMP, wherein the IMP comprises PR8 NS1 or TOSV NS; (ii) a nucleic acid encoding a 2A peptide; and (iii) the nucleic acid encoding the EEEV replicase.

[0020] In some embodiments, the first RNA polynucleotide comprises, from 8' to 6': (i) the nucleic acid the IMP, wherein the IMP comprises PR8 NS1 or TOSV NS; (ii) a nucleic acid encoding a 2A peptide; (iii) the nucleic acid encoding the EEEV replicase; (iv) a nucleic acid encoding a 2A peptide; and (vi) a nucleic acid encoding an additional IMP, wherein the IMP comprises EMCV Lpro.

[0021] In some embodiments, the second RNA polynucleotide further comprises a 8' untranslated region (UTR). In some embodiments, the 8' UTR comprises a conserved sequence element (CSE) which is cognate to the EEEV replicase.

[0022] In some embodiments, the payload comprises an antigen.

[0023] In some embodiments, the EEEV replicase is a wildtype EEEV replicase. In some embodiments, the EEEV replicase is an Opal-R EEEV replicase.

[0024] In some embodiments, the EEEV replicase comprises an amino acid sequence having at least 70% identity to the sequence set forth in SEQ ID NO: 83. In some embodiments, the EEEV

[0025] 12673398.1 replicase comprises the amino acid sequence set forth in SEQ ID NO: 83. In some embodiments, the EEEV replicase comprises an amino acid sequence having at least 70% identity to the sequence set forth in SEQ ID NO: 117. In some embodiments, the EEEV replicase comprises the amino acid sequence set forth in SEQ ID NO: 117.

[0026] In some embodiments, the first RNA polynucleotide comprises the nucleotide sequence set forth in SEQ ID NO: 49. In some embodiments, the first RNA polynucleotide comprises the nucleotide sequence set forth in SEQ ID NO: 116.

[0027] In some embodiments, the second RNA polynucleotide comprises a nucleotide sequence of any one of SEQ ID NO: 80-82.

[0028] In some aspects, the instant disclosure relates to compositions comprising a deoxyribonucleic acid (DNA) polynucleotide that can be transcribed to produce the first RNA polynucleotide of a taRNA provided herein. In some embodiments, the compositions further comprise an additional DNA polynucleotide that can be transcribed to produce the second RNA polynucleotide of a taRNA provided herein.

[0029] In some aspects, the instant disclosure relates to cells comprising a taRNA provided herein.

[0030] In some aspects, the instant disclosure relates to methods of expressing a payload in a cell, the method comprising: transfecting a cell with a taRNA provided herein or contacting the cell with a composition provided herein.

[0031] In some aspects, the instant disclosure relates to methods of expressing a payload in a subject, the method comprising: administering to a subject a taRNA provided herein or a composition provided herein. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0032] In some aspects, the instant disclosure provides a fusion protein comprising, from N-terminal to C-terminal: an Eastern Equine Encephalitis Virus (EEEV) replicase, a linker, and an immune modulating protein (IMP). In some embodiments, the IMP is encephalomyocarditis virus (EMCV) leader protease (Lpro). In some embodiments, the linker is a 2A peptide. In some embodiments, the EEEV replicase is a wildtype EEEV replicase. In some embodiments, the wildtype EEEV replicase comprises the amino acid sequence set forth in SEQ ID NO: 83. In some embodiments, the EEEV replicase is an Opal-R EEEV Replicase. In some embodiments, the wildtype EEEV replicase comprises the amino acid sequence set forth in SEQ ID NO: 117.

[0033] In some aspects, the instant disclosure provides a fusion protein comprising, from N-terminal to C-terminal: a first immune modulating protein (IMP), a first linker, an Eastern

[0034] 12673398.1 Equine Encephalitis Virus (EEEV) replicase, a second linker, and a second IMP. In some embodiments, the first IMP is Influenza Virus A / Puerto Rico / 8 / 34 nonstructural protein 1 (PR8 NS1) or Toscana Virus (TOSV) nonstructural protein (NSs). In some embodiments, the second IMP is EMCV Lpro. In some embodiments, the EEEV replicase is a wildtype EEEV replicase. In some embodiments, the wildtype EEEV replicase comprises the amino acid sequence set forth in SEQ ID NO: 83. In some embodiments, the EEEV replicase is an Opal-R EEEV Replicase. In some embodiments, the wildtype EEEV replicase comprises the amino acid sequence set forth in SEQ ID NO: 117.

[0035] In some aspects, the instant disclosure relates to deoxyribonucleic acids (DNA) comprising nucleic acids encoding a fusion protein provided herein.

[0036] In some aspects, the instant disclosure relates to ribonucleic acid (RNA) comprising nucleic acids encoding a fusion protein provided herein.

[0037] In some aspects, the instant disclosure relates to a method of amplifying a ribonucleic acid (RNA) polynucleotide comprising or encoding a payload in a subject in trans without inducing substantial cytotoxicity in the liver of the subject, the method comprising administering to the subject a / / zw / .s-amplifying ribonucleic acid (RNA) (taRNA), the taRNA comprising: a first RNA polynucleotide comprising: a nucleic acid encoding an Eastern Equine Encephalitis Virus (EEEV) replicase, and a nucleic acid encoding an immune modulating protein (IMP); and a second RNA polynucleotide comprising: a nucleic acid encoding a payload, and a conserved sequence element (CSE) cognate to the EEEV replicase.

[0038] In some embodiments, the administering comprises intramuscular injection. In some embodiments, the administering comprises intravenous injection.

[0039] In some embodiments, the payload is an antigen. In some embodiments, is a nucleic acid payload.

[0040] In some embodiments, the nucleic acid encoding an IMP encodes a first IMP and a second IMP. In some embodiments, the nucleic acid encoding an IMP encodes encephalomyocarditis virus (EMCV) leader protease (Lpro).

[0041] In some embodiments, the first RNA polynucleotide is a first RNA polynucleotide (e.g., replicase construct) provided herein.

[0042] In some embodiments, administering the taRNA comprises administering a taRNA provided herein.

[0043] 12673398.1 BRIEF DESCRIPTION OF DRAWINGS

[0044] FIGs. 1A-1B show luciferase (Flue) expression in adult wildtype mice (C57BL / 6) after intramuscular (IM) injection of compositions of ALC-315 LNPs and 5 pg of either mRNA or taRNA with replicase constructs encoding SFV replicase and trRNAs encoding luciferase (mRNA-LNP and taRNA-LNP, respectively). FIG. 1A shows representative images of 2D fluorescence imaging in live animals. FIG. IB shows quantification of luciferase activity (p / s = photons / second) in the whole body, the hind leg (injection site) and the liver; each circle represents a separate mouse.

[0045] FIGs. 2A-2C show effects of intramuscular (IM) vs intravenous (IV) administration on SEAP expression for compositions of ALC-315 LNPs and 5 pg mRNA or taRNA with replicase constructs encoding SFV replicase and trRNA encoding SEAP (mRNA-LNP, taRNA-LNP). FIG. 2A shows in vivo expression of SEAP in adult wildtype mice (C57BL / 6) injected with mRNA-LNP or taRNA-LNP via intramuscular (IM) or intravenous (IV) administration.

[0046] Expression is shown as relative luminance units (RLU) * hours (area under curve (AUC)). FIG.

[0047] 2B shows changes to body weight (% body weight) in mice injected with mRNA-LNP, taRNA-LNP, or saline via IM vs IV across 168 hours. FIG. 2C shows expression of IP- 10 (pg / mL) at 6 hours, 24 hours, or 72 hours after injection (IM or IV) with mRNA-LNP, taRNA-LNP, or saline.

[0048] FIGs. 3A-3B show cytokine expression and liver enzyme levels in the blood of wildtype mice (C57BL / 6) administered lipid nanoparticles (LNPs) comprising mRNAs or taRNAs encoding luciferase (Flue) (mRNA-LNP, taRNA-LNP), with or without immune modulating proteins (IMPs). FIG. 3A shows fold-expression of cytokines in wildtype mice at 24 hours post IM administration of saline or compositions of LNPs formulated with Ipg of: mRNA encoding luciferase (mRNA Flue); taRNA having a replicase construct encoding a wildtype Semliki Forest Virus (SFV) replicase and a trRNA with an oeSTR 5' UTR and encoding E3L-IRES-luciferase luciferase (oeSTR Flue) (SFV + oeSTR Flue); taRNA having a replicase construct encoding a GAA mutant SFV replicase and a trRNA with an oeSTR 5' UTR and encoding luciferase (oeSTR Flue) (GAA SFV + oeSTR Flue); taRNA having a replicase construct encoding a wildtype SFV replicase and a trRNA with a wildtype SINV 5' UTR and encoding E3L-IRES-luciferase (E3L-IRES-Fluc) (SFV + E3L-IRES-Fluc); taRNA having a replicase construct encoding a wildtype Eastern Equine Encephalitis Virus (EEEV) replicase and a trRNA with an oeSTR 5' UTR and encoding E3L-IRES-luciferase (oeE3L-IRES-Fluc) (EEEV + oeE3L-IRES-Fluc); taRNA having a replicase construct encoding an RLE mutant SFV replicase and a trRNA with an oeSTR 5' UTR and encoding luciferase (oeSTR Flue) (RLE SFV + oeSTR 12673398.1 - 1-

[0049] Flue). Expression of tumor necrosis factor (TNF)-alpha (TNF-a), interferon gamma (IFNg), interferon beta (IFNb), keratinocyte chemoattractant (KC) / human growth regulated oncogene (GRO) (KC / GRO), monocyte chemoattractant protein- 1 (MCP-1), interferon gamma induced protein 10 (IP-10), macrophage inflammatory protein 1 alpha (MIP-la; also known as CCL3), and interleukin 6 (IL6) is shown as fold change relative to saline. FIG. 3B shows levels of two liver enzymes, aspartate aminotransferase (AST) and alanine aminotransferase (ALT), at 24 hours after administration of mRNA-LNPs or taRNA-LNPs as in FIG. 1 A.

[0050] FIG. 4 shows expression of luciferase in the spleen wildtype (C57BL / 6) mice or interferon A receptor (IFNAR) knockout (IFNARz) mice at 24 hours post IV administration of saline or compositions of LNPs formulated with: Ipg of mRNA encoding luciferase (Flue mRNA); or Ipg or 5 pg of taRNA having a replicase construct encoding a wildtype Eastern Equine Encephalitis Virus (EEEV) replicase and a trRNA with an oeSTR 5' UTR and encoding E3L-IRES-luciferase (oeE3L-IRES-Fluc) (EEEV + oeE3L-IRES-Fluc).

[0051] FIG. 5 shows expression of SEAP in human B J fibroblasts 24 hours post transfection with taRNA having: a replicase construct encoding a wildtype Eastern Equine Encephalitis Virus (EEEV) replicase, a mutant (“Opal-R”) EEEV replicase, or a wildtype Semliki Forest Virus (SFV) replicase; and a trRNA with an oeSTR 5' UTR and encoding E3L-CVB3-SEAP, PR8 NS1-CVB3-SEAP, E3L-P2A-PR8 NS1-CVB3-SEAP, SOCS1-P2A-PR8 NS1-CVB3-SEAP, E3L-P2A-F1L-CVB3-SEAP, E3L-P2A-F1L-CVB3-SEAP, or SEAP-T2A-E3L-P2A-SOCS1.

[0052] FIGs. 6A-6B are schematics of illustrative trRNA constructs with a payload-coding nucleic acid sequence (PCS) and one immune modulating protein (IMP) coding nucleic acid sequence. FIG. 6A is a schematic of an illustrative “IMP-linker-PCS” trRNA comprising, from 5' to 3', a 5' CSE, an IMP-coding sequence, a linker, a PCS, and a 3' CSE. FIG. 6B is a schematic of an illustrative “PCS-linker-IMP” trRNA comprising, from 5' to 3', a 5' CSE, a PCS, a linker, an IMP-coding sequence, and a 3' CSE.

[0053] FIGs. 7A-7B are schematics of illustrative trRNA constructs with a payload-coding nucleic acid sequence (PCS) and two immune modulating protein (IMP)-coding nucleic acid sequence in different configurations. FIG. 7A shows an illustrative “IMP-linker-IMP-linker-PCS” trRNA comprising, from 5' to 3', a 5' conserved sequence element (CSE), a first IMP coding sequence, a first linker, a second IMP coding sequence, a second linker, a PCS, and a 3' CSE. FIG. 7B shows an illustrative “PCS-linker-IMP -linker-IMP” trRNAs comprising, from 5'

[0054] 12673398.1 to 3': a 5' CSE a PCS, a first linker, a first IMP coding sequence, a second linker, a second IMP coding sequence, and a 3' CSE.

[0055] FIGs. 8A-8B are schematics of illustrative trRNA constructs with a payload-coding nucleic acid sequence (PCS) and three immune modulating protein (IMP) coding nucleic acid sequence in different configurations. FIG. 8A shows an illustrative “IMP-linker-IMP-linker-IMP-linker-PCS” trRNA comprising, from 5' to 3', a 5' conserved sequence element (CSE), a first IMP coding sequence, a first linker, a second IMP coding sequence, a second linker, a third IMP coding sequence, a third linker, a PCS, and a 3' CSE. FIG. 8B shows an illustrative “PCS-linker-IMP-linker-IMP-linker-IMP” trRNA comprising, from 5' to 3': a 5' conserved sequence element (CSE), a PCS, a first linker, a first IMP coding sequence, a second linker, a second IMP coding sequence, a third linker, a third IMP coding sequence, and a 3' CSE (top).

[0056] FIGs. 9A-9D are schematics of EEEV replicase-IMP fusion proteins. FIG. 9A is a schematic of an “EEEV replicase-3 / 4J-protein” fusion protein, including an immune modulating protein (IMP) inserted between a third domain (nsP3) and fourth domain (nsP4) of an EEEV replicase. FIG. 9B is a schematic of a “EEEV replicase-linker-IMP” fusion protein, including an EEEV replicase joined at the N-terminus to an IMP by a linker (L). FIG. 9C is a schematic of an “IMP-linker EEEV replicase” fusion protein, including an EEEV replicase joined at the C-terminus to an IMP by a linker (L). FIG. 9D is a schematic of an “IMP-linker-EEEV replicase-linker-IMP” fusion protein, including an EEEV replicase joined at the C-terminus to a first IMP (IMP1) by a first linker (LI) and joined at the N-terminus to a second IMP (IMP2) by a second linker (L2).

[0057] FIGs. 10A-10D are schematics of replicase constructs encoding EEEV replicase-IMP fusion proteins. FIG. 10A shows an illustrative replicase construct comprising, from 5' to 3', a 5' UTR, a nucleic acid encoding an EEEV replicase-3 / 4J-IMP fusion protein (as in FIG. 9A), and a 3' UTR. FIG. 10B shows an illustrative replicase construct including, from 5' to 3',a 5' UTR, a nucleic acid encoding an EEEV replicase-linker-IMP fusion protein (as in FIG. 9B), and a 3' UTR. FIG. IOC shows an illustrative replicase construct including, from 5' to 3', a 5' UTR, a nucleic acid encoding an IMP-linker-EEEV replicase fusion protein (as in FIG. 9C), and a 3' UTR. FIG. 10D shows an illustrative replicase construct including, from 5' to 3', a 5' UTR, a nucleic acid encoding an IMP-linker-EEEV replicase-linker-IMP fusion protein (as in FIG. 9D), and a 3' UTR.

[0058] 12673398.1 DETAILED DESCRIPTION

[0059] Provided herein, in some aspects, are / ra / / .s-amplifying ribonucleic acids (RNA) (taRNA) comprising a first RNA polynucleotide comprising a nucleic acid encoding an Eastern Equine Encephalitis Virus (EEEV) replicase; and a second RNA polynucleotide comprising a nucleic acid payload or a nucleic acid encoding a payload, wherein the first RNA polynucleotide and / or the second RNA polynucleotide further comprise a nucleic acid encoding an immune modulating protein (IMP).

[0060] Several alphaviral replicases have been shown to be useful for amplifying RNA (e.g., as part of saRNA) at desirable levels of payload-expression. However, wildtype replicases with high rates of amplification of RNA (e.g., Semliki Forest Virus (SFV) replicases) are often associated with cytotoxic responses in hosts which are not observed with non-amplifying mRNA (Comes, J. D., et al. Trends in Biotechnology, 2023 Nov 1;41(11): 1417-1429; Chang YH, et al. Journal of Controlled Release. 2021 Oct 10;338:694-704.). Indeed, as shown in the section entitled “Examples,” administration of taRNA to a subject (e.g., IM administration) can result in different cytokine induction and transfection / expression patterns across various organ systems compared to mRNA. For example, intramuscular (IM) administration of taRNA leads to increased muscle transfection, decreased liver transfection, increased liver enzyme levels, and unique cytokine expression relative to IM administration of mRNA. Intravenous administration (IV) administration of taRNA is poorly expressive, but induces similar cytokine expression as IM administration. Without wishing to be bound by theory, taRNA-induced cytokine expression is thought to be due, in part, to accumulation and activity of replicase constructs in the liver, and to specific host immune responses induced by taRNA components. The inventors have identified certain combinations of taRNA components which reduce cytotoxicity and immunogenicity attributed to alphaviral replicases.

[0061] Provided herein, in some aspects, are compositions and methods useful for amplifying RNA polynucleotides in a subject in trans (i.e., taRNAs) without inducing substantial cytotoxicity in the liver of the subject. In other aspects, provided herein are compositions and methods useful for modulating immune responses to taRNA in a subject. In still other aspects, provided herein are compositions and methods useful for increasing expression of a payload encoded by a taRNA in a subject.

[0062] A “ / ra / / .s-amplifying RNA,” hereinafter referred to as “taRNA,” comprises a first and second RNA polynucleotide, wherein the first RNA polynucleotide encodes a replicase, the second RNA polynucleotide comprises a nucleic acid payload or nucleic acids encoding a

[0063] 12673398.1 payload, and wherein the second RNA polynucleotide can be replicated by the encoded replicase in trans. A “polynucleotide” refers to a polymer of nucleotides. A polynucleotide is generally composed of nucleotides that are naturally found in DNA or RNA (e.g.,

[0064] adenosine / deoxy adenosine (A), uridine (U) / deoxythymidine (T), guanosine / deoxy guanosine (G), cytidine / deoxy cytidine (C)) and joined by phosphodiester bonds. However, the term polynucleotide may also refer to polynucleotides comprising nucleotides or nucleotide analogs containing chemically or biologically modified bases, modified backbones, etc., whether or not these modifications are found in naturally occurring nucleic acids; indeed, such molecules may be preferred for certain applications. In some sequences described herein, T / U is used to denote a particular nucleotide may be a T or U depending on whether the polynucleotide is an RNA polynucleotide (U) or a DNA polynucleotide (T). The first and second RNA polynucleotides of a taRNA are separate polynucleotides (i.e., separate molecules which are not a single continuous strand of RNA). The terms “replicase construct” and “ trans replicon” (trRNA) construct are used synonymously herein to refer to the first and second RNA polynucleotides of a taRNA, respectively. As used herein, a “construct” refers to an artificial (i.e., not naturally occurring) polynucleotide.

[0065] Replicase Constructs

[0066] A “replicase construct” (i.e., the first RNA polynucleotide) refers to an mRNA that comprises a nucleic acid encoding a replicase and does not comprise a nucleic acid encoding a payload that is operably linked to a CSE cognate to the replicase encoded by the replicase construct.. In some embodiments, a replicase construct is a non-replicating mRNA. As used herein, the term “non-replicating mRNA” refers to an mRNA which is processed for translation into a gene product or else degraded, and which does not self-replicate. Once introduced to an environment comprising translational machinery (such as a cell), replicase constructs can be translated to generate the encoded replicase.

[0067] A “replicase” is an RNA-dependent RNA polymerase (RdRp) capable of transcribing (i.e., reading) an RNA template to produce an RNA (e.g., trRNA). A replicase construct of the instant disclosure will be understood to encode a replicase derived from an Eastern Equine Encephalitis Virus (EEEV). EEEV is an alphavirus belonging to the Togaviridae family and comprising a single-stranded RNA genome encoding at least nsPl, nsP2, nsP3, nsP4, El, E2, E3, 6K / TF and capsid proteins. In some embodiments, a taRNA comprises a replicase construct encoding an EEEV replicase. An EEEV replicase may be a wildtype EEEV replicase (e.g., SEQ

[0068] 12673398.1 ID NO: 83) or a variant thereof (e.g., a replicase derived from EEEV). Typically, an EEEV replicase comprises a complex formed by the non-structural proteins nsPl, nsP2, nsP3, and nsP4. Thus, a nucleic acid encoding a functional, full-length alphaviral replicase (also referred to herein as a “replicase-coding sequence”) is understood to encode at least nsPl, nsP2, nsP3, nsP4, and variants thereof.

[0069] In some embodiments, a wildtype EEEV replicase comprises EEEV nsPl / nsP2. In some embodiments, a wildtype EEEV nspl / nsP2 comprises the amino acid sequence set forth in SEQ ID NO: 86. In some embodiments, a wildtype EEEV replicase comprises EEEV nsP3. Wildtype EEEV nsP3 may be present in at least two variants (e.g., “version 1” and “version 2” of Table 4), based on naturally occurring differences in translational readthrough of an internal stop codon of EEEV (as discussed herein) and cleavage thereafter. In some embodiments, a wildtype EEEV nsP3 comprises the amino acid sequence set forth in SEQ ID NO: 87 or SEQ ID NO: 88. In some embodiments, a wildtype EEEV nsP4 comprises the amino acid sequence set forth in SEQ ID NO: 89. In some embodiments, a wildtype EEEV replicase is a full-length wildtype EEEV replicase, such that it comprises EEEV nsPl-nsP2-nsP3-nsP4.

[0070] A wildtype EEEV replicase is typically encoded by a nucleic acid sequence comprising an internal stop codon towards the C-terminal of nsP3 and upstream of nsP4 of a full-length wildtype EEEV replicase (e.g., a full-length wildtype EEEV replicase comprising the sequence set forth in SEQ ID NO: 83). This feature is common to many wildtype alphavirus replicase-encoding nucleotide sequences (Strauss, et al. “The alphaviruses: gene expression, replication, and evolution.” Microbiological reviews 58.3 (1994): 491-562.). These internal stop codons are typically “leaky” stop codons, such as an “Opal” stop codon (i.e., UGA) which suppress most, but not all, translation of the full nucleic acid sequences in which they are contained, resulting in excess expression of truncated replicase (i.e., nsPl-nsP2-nsP3) and low levels of expression of full-length replicase (i.e., nsPl-nsP2-nsP3-nsP4).

[0071] A wildtype EEEV replicase can be understood to be encoded by a nucleotide sequence having an internal stop codon (e.g., an Opal stop codon) within the nucleotide sequence encoding nsP3 and upstream of to the nucleotide sequence encoding and nsP4. In some embodiments, a wildtype EEEV replicase is encoded by the nucleotide sequence set forth in SEQ ID NO: 49. In some embodiments, the internal stop codon of a nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO: 49 is at a position corresponding to nucleotides 5638-5640 of SEQ ID NO: 49.

[0072] 12673398.1 In some embodiments, a full-length wildtype EEEV replicase comprises or consists of the sequence set forth in SEQ ID NO: 83. In some embodiments, a full-length wildtype EEEV replicase is encoded by a nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO: 49. In some embodiments, a full-length wildtype EEEV replicase comprising the amino acid sequence set forth in SEQ ID NO: 83 is encoded by a nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO: 49. In some embodiments, the internal stop codon of a nucleic acid encoding a wildtype EEEV replicase corresponds to a location between positions N1879 and R1880 of an amino acid sequence of a full-length wildtype EEEV replicase, wherein the positions are numbered according to the amino acid sequence set forth in SEQ ID NO: 83; an illustrative example of this is represented in Table 3 as a in SEQ ID NO: 83.

[0073] In some embodiments, the internal stop codon of a nucleic acid encoding a wildtype EEEV replicase results in a wildtype EEEV replicase that is truncated at a location in nsP3, such that nsP4 is not expressed. In some embodiments, nsP4 of a wildtype EEEV replicase comprises the amino acid sequence set forth in SEQ ID NO: 89. In some embodiments, the internal stop codon of a nucleic acid encoding a wildtype EEEV replicase results in a wildtype EEEV replicase lacking the amino acid sequence set forth in SEQ ID NO: 89.

[0074] The location of the truncation in nsP3, relative to a full-length wildtype EEEV replicase, can vary. In some embodiments, the truncated wildtype EEEV replicase comprises nsPl-nsP2-nsP3, wherein nsPl-nsP2 comprise the amino acid sequence set forth in SEQ ID NO: 86, and nsP3 comprises the amino acid sequence set forth in SEQ ID NO: 87. In some embodiments, a truncated wildtype EEEV replicase comprises nsPl-nsP2-nsP3, wherein nsPl-nsP2 comprise the amino acid sequence set forth in SEQ ID NO: 86, and nsP3 comprises the sequence set forth in SEQ ID NO: 88. In some embodiments, a truncated wildtype EEEV replicase comprises nsPl-nsP2-nsP3 and comprises the amino acid sequence set forth in SEQ ID NO: 118. In some embodiments, a truncated wildtype EEEV replicase lacks the amino acid sequence set forth in SEQ ID NO: 119 relative to a full-length wildtype EEV replicase (e.g., relative to SEQ ID NO: 83). In some embodiments, a polynucleotide comprises a first polynucleic acid encoding an amino acid sequence having at least 95% identity to SEQ ID NO: 118, a second polynucleic acid encoding an amino acid sequence having at least 95% identity to SEQ ID NO: 119, and an internal stop codon (e.g., an opal stop codon) between the first polynucleic acid and the second polynucleic acid. In some embodiments, a polynucleotide comprises a first polynucleic acid encoding an amino acid sequence of SEQ ID NO: 118, a second polynucleic acid encoding an

[0075] 12673398.1 amino acid sequence of SEQ ID NO: 119, and an internal stop codon (e.g., an opal stop codon) between the first polynucleic acid and the second polynucleic acid.

[0076] Once expressed, alphaviral replicase may interact with a RNA polynucleotide comprising one or more CSEs which are cognate to the replicase and generate mirrored copies of the RNA polynucleotide, which can be subsequently translated (e.g., by a host cell). Replicase constructs of a taRNA necessarily do not comprise a CSE; thus, once the replicase of a replicase construct is translated, the encoded replicase cannot replicate the replicase construct. A replicase that is “cognate” to a CSE refers to a replicase that is the capable of transcribing a portion of a polynucleotide comprising the CSE (e.g., capable of transcribing a polynucleotide comprising or encoding a payload). A replicase construct and trRNA construct are considered “compatible” or “cognate” when a trRNA comprises a CSE to which the replicase encoded by the replicase construct can bind, such that the trans replicon is replicated.

[0077] As shown in the section entitled “Examples,” administration of taRNAs to a subject may induce certain cytotoxic and / or immunogenic responses which are not present after mRNA administration and which may affect expression of taRNA-encoded payloads (see Example 1). These effects have largely been thought to be a result of either RNA amplification (e.g., due to large copy number of mRNA in cells) and / or recognition of components and / or amplification activity of replicases by host immune factors to trigger innate immune responses (IIRs) by hosts. As shown in the section entitled “Examples,” the inventors have determined that activity of certain alphaviral replicases, but not the presence of replicases in a cell nor amount of trRNA expression, is in large part responsible for immune responses by hosts see Example 2). The inventors have also determined that many of these effects are at least partly attenuated by use of EEEV replicases, for example, in combination with co-expression of an immune modulating protein (IMP). Accordingly, in preferred embodiments, a replicase construct of a taRNA described herein encodes an EEEV (i.e., an EEEV replicase). In some embodiments, a replicase construct of a taRNA encodes a wildtype EEEV replicase. In some embodiments, a replicase construct of a taRNA encodes an EEEV replicase variant.

[0078] In some embodiments, an EEEV replicase variant is encoded by a nucleotide sequence comprising one or more substitutions relative to a reference (e.g., wildtype) EEEV replicase-coding nucleotide sequence. In some embodiments, an EEEV replicase variant is encoded by a nucleic acid sequence comprising a readthrough substitution at a position corresponding to an internal stop codon of a reference EEEV replicase-coding nucleotide sequence (e.g., a wildtype EEEV replicase-coding nucleotide sequence). As used herein, the term “readthrough

[0079] 12673398.1 substitution” refers to a substitution of one or more nucleotides in an internal stop codon of a reference EEEV replicase-coding nucleotide sequence, such that the internal stop codon of the reference EEEV replicase-coding nucleotide sequence is mutated into an amino acid-encoding codon. A readthrough substitution may be useful for, in some aspects, increasing expression of a replicase.

[0080] Notably, several studies have shown that readthrough substitution alphaviral replicase-encoding sequence have unpredictable effects- for example, while certain readthrough substitutions in Chikungunya virus (CHIKV) replicases may reduce inflammatory effects in hosts during viral infection (Jones, J. E., et al., “Disruption of the Opal Stop Codon Attenuates Chikungunya Virus-Induced Arthritis and Pathology.” MBio. (2017): 10-1128.), readthrough substitutions in Sindbis virus (SINV) replicases result in accumulation of non-functional, immature replicases (Li, G. P. & Rice, C. M. “Mutagenesis of the in-frame opal termination codon preceding nsP4 of Sindbis virus: studies of translational readthrough and its effect on virus replication.” Journal of Virology. (1989). 63:1326-1337.) or increase morbidity and mortality in hosts (Suthar MS, et al., “Identification of adult mouse neurovirulence determinants of the Sindbis virus strain AR86.” Journal of Virology. (2005) 79:4219-4228.). As described in the instant application and demonstrated in the Examples included herein, the inventors have surprisingly discovered that increased expression of EEEV replicases (e.g., by readthrough substitutions in EEEV replicase-coding sequences) increases EEEV replicase-mediated payload expression while still attenuating host immune responses to taRNA.

[0081] In some embodiments, the readthrough substitution comprises one or more point mutations in the internal stop codon. In some embodiments, the readthrough substitution comprises two or more point mutations in the internal stop codon. In some embodiments, the readthrough substitution comprises a replacement of the internal stop codon with an amino-acid encoding codon. In some embodiments, an EEEV replicase variant is encoded by a nucleotide sequence comprising a readthrough substitution relative to a reference EEEV replicase-coding nucleotide sequence having an internal opal stop codon, wherein the internal opal stop codon is mutated into an amino acid-encoding codon. In some embodiments, the readthrough substitution comprises a point mutation of the internal opal stop codon that mutates the internal opal stop codon into a codon encoding arginine (R). In some embodiments, the readthrough substitution comprises a replacement of the internal opal stop codon with a codon encoding an arginine (R). An EEEV replicase-coding nucleotide sequence encoding a readthrough substitution that

[0082] 12673398.1 mutates an internal Opal stop codon into a codon encoding arginine will be understood to encode an “Opal-R” EEEV replicase.

[0083] In some embodiments, an EEEV replicase variant is encoded by a nucleotide sequence comprising a readthrough substitution relative to SEQ ID NO: 49. In some embodiments, an EEEV replicase variant is encoded by a nucleotide sequence comprising a readthrough substitution relative to SEQ ID NO: 49, wherein the readthrough substitution comprises replacing an internal stop codon with a codon encoding an arginine (R). In some embodiments, an EEEV replicase variant is encoded by a nucleotide sequence comprising a readthrough substitution relative to positions 5638-5640 (UGA) of SEQ ID NO: 49. In some embodiments, an EEEV replicase variant is encoded by a nucleotide sequence comprising a readthrough substitution relative to positions 5638-5640 of SEQ ID NO: 49, wherein the readthrough substitution comprises replacing the nucleotides at positions 5638-5640 with a codon encoding an arginine (R). In some embodiments, the readthrough substitution comprises one or more point mutations at positions corresponding to U5638 and / or A5640 of SEQ ID NO: 49. In some embodiments, the readthrough substitution comprises a substitution at position corresponding to U5638 of SEQ ID NO: 49. In some embodiments, the readthrough substitution comprises a substitution at position corresponding to A5640 of SEQ ID NO: 49. In some embodiments, the readthrough substitution comprises a U5638C mutation. In some embodiments, the readthrough substitution comprises a U5638C mutation and a A5640G mutation. In some embodiments, the readthrough substitution comprises a A5640U mutation. In some embodiments, the readthrough substitution comprises a A5640C mutation. In some embodiments, the readthrough substitution comprises a substitution of UGA5638-5640 to CGA, CGG, CGU, or CGC. In some embodiments, an Opal-R EEEV replicase variant is encoded by a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequence set forth in SEQ ID NO: 116. In some embodiments, an Opal-R EEEV replicase variant is encoded by a nucleotide sequence set forth in SEQ ID NO: 116.

[0084] In some embodiments, an EEEV replicase is an EEEV replicase variant (i.e., not a wildtype EEEV replicase). In some embodiments, an EEEV replicase variant comprises 1, 2, 3, or 4 additional mutations (e.g., substitution, insertion) relative to a reference EEEV replicase amino acid sequence. In some embodiments, the reference EEEV replicase amino acid sequence is a wildtype EEEV replicase amino acid sequence (e.g., SEQ ID NO: 83). In some embodiments, an EEEV replicase variant comprises an amino acid sequence comprising an

[0085] 12673398.1 insertion of an arginine (R) relative to a reference EEEV replicase amino acid sequence. In some embodiments, an EEEV variant comprises an insertion of an R between nsP3 and nsP4 of the reference EEEV replicase. In some embodiments, an EEEV replicase variant comprises an insertion of an R at position corresponding to residue 1880 of SEQ ID NO: 83, also referred to herein as an “Opal-R” EEEV replicase. In some embodiments, an Opal-R EEEV replicase comprises an amino acid sequence having at least at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 117. In some embodiments, an Opal-R EEEV variant comprises the sequence set forth in SEQ ID NO: 117. In some embodiments, a replicase construct encoding an EEEV replicase comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequence set forth in SEQ ID NO: 49. In some embodiments, a replicase construct encoding an EEEV replicase comprises the nucleotide sequence set forth in SEQ ID NO: 49. In some embodiments, the replicase construct comprises a nucleic acid sequence encoding an Opal-R EEEV replicase. In some embodiments, a replicase construct encoding an Opal-R EEEV replicase comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequence set forth in SEQ ID NO: 116. In some embodiments, a replicase construct encoding an Opal-R EEEV replicase comprises the nucleotide sequence set forth in SEQ ID NO: 116.

[0086] In some embodiments, a replicase construct comprises a nucleic acid sequence encoding a EEEV replicase having at least at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 83. In some embodiments, a replicase construct comprises a nucleic acid sequence encoding a wildtype EEEV replicase having the amino acid sequence of SEQ ID NO: 83. In some embodiments, a replicase construct comprises a nucleic acid sequence encoding an EEEV replicase variant having the amino acid sequence of SEQ ID NO: 83 with 1, 2, 3, or 4 additional mutations (e.g., substitution mutations). In some embodiments, a replicase construct comprises a nucleic acid sequence encoding an EEEV replicase variant having an insertion of an arginine between nsP3 and nsP4. In some embodiments, a replicase construct comprises a nucleic acid sequence encoding an Opal-R EEEV replicase. In some embodiments, a replicase construct comprises a nucleic acid sequence encoding an Opal-R EEEV replicase having at least at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino

[0087] 12673398.1 acid sequence of SEQ ID NO: 117. In some embodiments, a replicase construct comprises a nucleic acid sequence encoding an Opal-R EEEV replicase having the amino acid sequence of SEQ ID NO: 117. In some embodiments, a EEEV variant or replicase derived from EEEV has replicase activity.

[0088] In some embodiments, a replicase derived from an EEEV is capable of binding to a trRNA comprising a CSE from an EEEV. In some embodiments, a compatible replicase construct and CSE are derived from different alphaviruses. In some embodiments, a replicase derived from an EEEV is capable of binding to a trRNA comprising a CSE from a SINV. In some embodiments, a compatible replicase construct and CSE are derived from the same alphavirus. In some embodiments, a replicase and CSE from the same species are cognate; for example, an EEEV replicase is expected to be cognate to a CSE derived from an EEEV.

[0089] Trans Replicon (trRNA) Constructs

[0090] A “trans replicon” (trRNA) refers to a polynucleotide (e.g., RNA polynucleotide) comprising a nucleic acid encoding a payload operably linked to a conserved sequence element (CSE), wherein the polynucleotide does not encode a replicase cognate to the CSE. trRNAs are capable of being replicated by cognate replicases. In some embodiments, the trRNA comprises a 5' UTR and / or a 3' UTR. In some embodiments, the trRNA comprises a nucleic acid sequence encoding an IMP.

[0091] Payloads

[0092] trRNAs comprise at least a payload-encoding sequence and one or more CSEs. A trRNA does not comprise the replicase that can amplify the trRNA. Instead, the simultaneous presence of a trRNA in a cell and the presence of a cognate replicase can result in amplification of the trRNA and its encoded payload. As used herein, a “payload” refers to one or more gene products of interest for delivery to or expression by an organism. A payload may be a functional nucleic acid (e.g., RNA), a protein, a peptide or protein fragment, or a fusion protein.

[0093] In some embodiments, a payload is a selectable marker. As used herein, a “selectable marker” is a peptide or protein that can be used to screen cells by artificial selection. Nonlimiting examples of selectable markers include antibiotic resistance proteins (e.g., ampicillin, puromycin) and negative selection markers (e.g., thymidine kinase).

[0094] In some embodiments, a payload is a reporter. A “reporter” is a peptide or protein which alters the appearance of a cell such that cells can be visually or optically screened for presence or absence of the peptide or protein. In some embodiments, a reporter is an enzyme which alters 12673398.1 the appearance of a cell, such as beta-galactosidase. In some embodiments, a reporter is a peptide or peptide fragment (e.g., secreted embryonic alkaline phosphatase (SEAP)) which can be detected in combination with additional reagents (e.g., assay-specific media). In some embodiments, a reporter is a fluorophore, such as, but not limited to, green fluorescent protein (GFP), red fluorescent protein (RFP), blue fluorescent protein (BFP), yellow fluorescent protein (YFP), or any derivative thereof.

[0095] In some embodiments, a payload is “therapeutic payload,” here referring to a gene product useful for treating or preventing a disease or disorder. A “gene” refers to a nucleic acid (e.g., DNA, RNA) encoding a polypeptide (e.g., a vaccine antigen or a replicase). In some embodiments, a gene is a naturally occurring gene. In some embodiments, a gene is a transgene (e.g., derived from a different organism). In some embodiments, a therapeutic payload knocks down, knocks in, increases, inhibits, or otherwise modulates gene expression. In some embodiments, a therapeutic payload replaces or edits an endogenous gene or gene product. In some embodiments, a therapeutic payload is a functional RNA; non-limiting examples of include short hairpin RNA (shRNA), microRNA (miRNA), artificial microRNA (amiRNA), small interfering RNA (siRNA), or circular RNA (circRNA).

[0096] In some embodiments, a therapeutic payload is a protein. A therapeutic protein may replace or interfere with activity of deficient or absent endogenous proteins; augment activity of existing metabolic or synthetic pathways; provide a novel function or activity; or interfere with the activity of a pathogen or toxic molecule. Non-limiting examples of therapeutic proteins suitable for use as payloads include membrane proteins, membrane-associated proteins, secreted proteins, intracellular proteins, antigens, antibodies, or fragments thereof.

[0097] In some embodiments, a therapeutic protein is an antigen. An “antigen” refers to a protein or protein fragment of a pathogen. In some embodiments, an antigen is a protein or peptide that can be recognized by the immune system of a host organism. In some embodiments, an antigen, when introduced into a host, induces an immune response in the host (e.g., antibody production against the antigen). In some embodiments, an antigen is a nucleic acid (e.g., aptamer), polysaccharide, polypeptide, oligosaccharide, lipid, particulate antigen, or cancer antigen (e.g., tumor antigen). Preferably, an antigen is a protein or a fragment thereof (e.g., a surface protein). In some embodiments, an antigen is a protein derived from a pathogen. In some embodiments, the antigen is a viral antigen or bacterial antigen.

[0098] 12673398.1 Conserved Sequence Elements (CSEs)

[0099] trRNAs comprise one or more conserved sequence elements (CSEs). A “conserved sequence element” (CSE) refers to a recognition site for an alphavirus replicase. Typically, a CSE functions as a core promoter or enhancer for initiation of replication of a downstream sequence, such that a 5'-CSE may initiate synthesis of a plus-strand and a 3'-CSE may initiate synthesis of a minus-strand. An RNA polynucleotide may comprise one or more 5'-CSEs and / or 3'-CSEs. CSEs may be comprised in a UTR, for example a 5'-UTR and / or a 3' UTR. In some embodiments, a CSE forms one or more secondary structure(s), such as one or more stem-loops. Non-limiting examples of CSEs include CSE1, CSE2, CSE3, CSE4, and variants or derivatives thereof. In some embodiments, the CSE is a CSE derived from SFV, SINV, VEEV, or CHIKV alphavirus. CSEs are known in the art, e.g., as described in Hyde JL, Virus Res. 405 Aug 3;45:99-107.

[0100] Untranslated Regions (UTRs)

[0101] In some embodiments, a trRNA and / or replicase construct comprise one or more untranslated regions (UTRs). UTRs may act as stabilizing elements and / or provide regulation of transcription of a gene (e.g., a nucleic acid encoding an antigen). Typically, UTRs are found upstream and / or downstream of a gene or transgene. A UTR located directly upstream of a start codon and operably linked to a gene is referred to herein as a 5'-UTR. As a skilled artisan will understand, 5 '-UTRs may comprise sequence elements which play roles in regulation of expression (e.g., Kozak sequences) or structural elements which alter stability of the molecule (e.g., 5' cap structures). A UTR located directly downstream of a stop codon operably linked to a gene or transgene is referred to herein as a 3'-UTR. 3'-UTRs may comprise structural elements which alter the stability of a construct and / or provide transcriptional control, including, but not limited to AU-rich elements and polyA tails. A variety of 5'-UTRs and a 3'-UTRs are known to those of ordinary skill in the art. UTRs may be naturally occurring or synthetic.

[0102] In some embodiments, a trRNA comprises a UTR. In some embodiments, a trRNA and / or replicase construct comprise a UTR derived from an alphavirus. In some embodiments, an alphavirus UTR comprises a CSE. In some embodiments, a trRNA comprises a 5'-UTR of an alphavirus (e.g., an alphavirus 5'-UTR). In some embodiments, a trRNA comprises a 3'-UTR of an alphavirus (e.g., an alphavirus 3'-UTR). Alphavirus 5'-UTR and alphavirus 3'-UTR sequences are described in the art, e.g., by Hyde JL et al., Virus Res. 2015 Aug 3;45:99-107. In some embodiments, a trRNA comprises a 5'-UTR and / or 3'-UTR derived from a Semliki Forest 12673398.1 Virus (SFV), hereinafter referred to as a “SFV-UTR”. In some embodiments, the RNA polynucleotide comprises a 5'-UTR and / or 3'-UTR derived from a Sindbis virus (SINV), hereinafter referred to as a “SINV-UTR”. An illustrative wildtype 5'-SINV-UTR is provided in SEQ ID NO: 42. In some embodiments, an alphavirus 5'-UTR comprises one or more mutations relative to a wildtype alphavirus 5'-UTR. In some embodiments, an alphavirus 3'-UTR comprises one or more mutations relative to a wildtype alphavirus 3'-UTR. In some embodiments, the RNA polynucleotide comprises a 5'-UTR and 3'-UTR from the same virus. Non-limiting examples include an RNA polynucleotide comprising a 5'-UTR derived from a SINV (5 '-SINV-UTR), and a 3'-UTR derived from a SINV (3'-SINV-UTR) or the RNA polynucleotide comprising a 5'-UTR derived from a SFV (5'-SFV-UTR), and a 3'-UTR derived from a SFV (3'-SFV-UTR). In some embodiments, the RNA polynucleotide comprises a 5'-UTR and 3'-UTR from different alphaviruses.

[0103] In some embodiments, a trRNA comprises one or more CSEs, wherein the CSEs are present in one or more UTRs. In some embodiments, a trRNA comprises a 5'-UTR having one or more CSEs. In some embodiments, a trRNA comprises a 3'-UTR having one or more CSEs. In some embodiments, a trRNA comprises a 5'-UTR having one or more CSEs, a nucleic acid encoding a payload, and a 3'-UTR having one or more CSEs. In some embodiments, a trRNA comprises a 5'-UTR having one or more CSEs, a nucleic acid encoding a payload, and a 3'-UTR having one or more CSEs. In some embodiments, a trRNA comprises UTRs having one or more CSEs, wherein the UTRs are derived from one or more alphaviruses. A skilled artisan will appreciate that UTRs derived from alphaviruses comprise one or more CSEs unless stated otherwise. In some embodiments, a trRNA comprises a 5'-UTR derived from a first alphavirus and a 3'-UTR derived from a second alphavirus. In some embodiments, a trRNA comprises a 3'-UTR comprising one or more repeat sequence elements (RSE).

[0104] In some embodiments, a trRNA comprises a 5'-UTR having an “oeSTR” extension. An oeSTR extension is a sequence of 9 nucleotides in length (AGAAGAUGG) inserted into the 5' terminal of a 5' UTR. The term “5' terminal of a 5'-UTR” refers to nucleotides at the 5' end of the 5'-UTR. The 5' terminal of the wildtype 5' SINV UTR comprises six conserved nucleotides AUCGGC. The 5' extension described herein is inserted between the AU and CGGC (e.g., AUAGAAGAUGGCGGC (SEQ ID NO: 114), as shown in SEQ ID NO: 45).

[0105] In some embodiments, a replicase construct comprises a 5'-UTR and / or a 3'-UTR. In some embodiments, a replicase construct comprises a 5'-UTR. In some embodiments, a replicase construct comprises a 3'-UTR. In some embodiments, a replicase construct comprises a 5'-UTR

[0106] 12673398.1 and a 3'-UTR. In some embodiments, a replicase construct comprises a 5'-UTR derived from human alpha-globin (5'-HAG-UTR). An illustrative wildtype 5'-HAG-UTR is provided in SEQ ID NO: 42. In some embodiments, a replicase construct comprises a 3'-UTR derived from human alpha-globin (3'-HAG-UTR). An illustrative wildtype 3'-HAG-UTR is provided in SEQ ID NO: 46. In some embodiments, a replicase construct comprises a 5'-HAG-UTR and a 3'-HAG-UTR. In some embodiments, a replicase construct comprises a 5'-HAG-UTR, an EEEV replicase-encoding sequence, and a 3'-HAG-UTR (5'-HAG-UTR-EEEV replicase-3'-HAG-UTR).

[0107] Immune Modulating Proteins

[0108] In some embodiments, a taRNA comprises a first RNA polynucleotide (i.e., replicase construct) encoding an EEEV replicase, a second RNA polynucleotide comprising a nucleic acid encoding a payload, and comprises a nucleic acid (e.g., comprised in a replicase construct and / or a trRNA of a taRNA) encoding one or more immune modulating proteins. As used herein, the term “immune modulating protein (IMP),” refers to a protein, peptide or protein fragment, or fusion protein, which interferes with a mammalian innate immune response (IIR) pathway.

[0109] Innate immune response (HR) pathways comprise numerous signaling pathways by which cells recognize, activate, and ultimately respond to non-specific danger molecules in the cytosol or in extracellular space. HRs comprise cellular and host organism responses to nonspecific danger molecules. Danger molecules, also known as alarmins and danger signals, are molecular warning signals which are not highly specific to a particular antigen, but which generally signal infection by pathogens, cell injury / damage, or cell death (e.g., by necrosis / necroptosis). Danger molecules include, but are not limited to, pathogen-associated molecular patterns (PAMPs), damage-associated molecular patterns (DAMPs), non-self-nucleic acids, and foreign substances. A“non-self’ nucleic acid refers to any nucleic acid which is not part of a given host cell, for example, a nucleic acid of a pathogen (e.g., virus, bacteria), a synthetic nucleic acid, or certain nucleic acids of other cells of the same organism.

[0110] A “pathway,” as used herein, is a molecular process by which a cell detects and responds to a signal. A pathway comprises at least a receptor of a signal and an effector of a response to the signal, which may be comprised in one or more molecules. Molecular signals, also referred to herein as “ligands,” bind and activate protein-based receptors. Once a receptor interacts with a signal, one or more molecular events may occur which enable a response to the signal. Nonlimiting examples of responses to a signal include changes to gene expression, changes to cell

[0111] 12673398.1 cycle stage, degradation / sequestering of a molecule, changes to membrane properties (e.g., voltage, permeability), movement of the cell, recruitment of immune cells and / or molecules to a particular location.

[0112] In some embodiments, IIRs are inflammatory or apoptotic. Inflammatory IIR pathways comprise several mechanisms by which non-specific immune cells quickly damage, destroy, or otherwise neutralize pathogens, molecular danger signals (e.g., non-self-nucleic acids), and injured host tissue. Inflammatory responses are typically rapid and self-limiting, and thus require both pro-inflammatory pathways (i.e., pathways which trigger an inflammatory response) and anti-inflammatory pathways (i.e., pathways which suppress or eliminate an inflammatory response. Notably, inflammatory responses differ from adaptive immune responses, which comprise mechanisms by which pathogen-specific lymphocytes precisely target a particular pathogen. Adaptive immune responses are highly effective and specific (i.e., do not target normal, healthy host tissue), but are a much slower response than inflammatory responses.

[0113] Apoptosis is a type of programmed cell death which occurs in response to intrinsic or extrinsic signaling pathways. Apoptosis can occur in response to detection of danger molecules (e.g., non-self-nucleic acids). During viral infection, for example, programmed death of an infected cell can greatly reduce release of progeny virus. Apoptosis can also suppress inflammatory responses by releasing anti-inflammatory molecules.

[0114] An IMP interferes with an IIR pathway when the presence of the IMP affects the induction or activity of the pathway such that the outcome of the pathway (e.g., inflammatory response, apoptotic response) is reduced, suppressed, prevented, or reversed compared to the outcome of the pathway in the absence of the IMP. Similarly, an IMP “interferes” with a particular molecule when the presence of the IMP reduces the quantity, availability, or function of the molecule relative to the same in the absence of the IMP. For example, an IMP can interfere with the response of an IIR pathway such that the immune response of a cell to a non-self-nucleic acid is reduced, suppressed, prevented, or reversed compared to the response of the cell to the non-self-nucleic acid in the absence of the IMP. The ability of an IMP to interfere with a molecule of pathway may be direct (e.g., by direct contact with a molecule) or indirect (e.g., by contact with a first pathway which regulates a second pathway). An IMP may interfere with an IIR pathway by directly antagonizing a molecule associated with the pathway and / or by competing with a molecule associated with the pathway.

[0115] In some embodiments, an IMP is an antagonist of an IIR pathway-associated molecule. An “antagonist,” as used herein, refers to a molecule which blocks or dampens binding activity

[0116] 12673398.1 of a protein (e.g., enzyme, receptor); non-limiting examples of antagonists include competitive antagonists, allosteric antagonists, partial agonists, and inverse agonists. A “competitive antagonist” is a molecule which can bind to the active site of a target protein, and which competes with endogenous ligands, such that other molecules compete with the competitive antagonist to bind the same target protein. An “allosteric antagonist” is a molecule which binds to an allosteric site of a target protein and indirectly block binding of endogenous ligands to the active site (e.g., by preventing a conformational change required for activation, or by inducing a conformational change of the active site). A “partial agonist” is a molecule which partially activate (i.e., activate to a reduced level compared to an endogenous ligand) a target protein, such that activity of the target protein is reduced. An “inverse agonist” is a molecule which binds to a target protein and alters its natural activity, for example, by triggering an alternative pathway or reversing its activity. Antagonists may be reversible or irreversible.

[0117] In some embodiments, an IMP competes with an IIR pathway-associated protein. An IMP can be understood to “compete” with a wildtype protein when it perturbs the normal activity of the protein by interacting with ligands of the wildtype protein, such that the ligands are less available for binding to the wildtype protein. For example, an IMP may be a dominant negative mutant (e.g., variant) of a protein. Dominant negative mutants, also referred to herein as “dn” proteins, are competitive mutants which comprise genetic mutations that render the protein partially functional or non-functional compared to a wildtype version of the same protein. Dominant negative mutants can bind ligands of the wildtype protein but are less effective (or ineffective) in propagating pathways associated with the wildtype protein.

[0118] Antiviral Factors

[0119] In some embodiments, an IMP antagonizes a mammalian antiviral factor. “Antiviral factors” are molecules which mediate IIR pathways related to the suppression of viral infection, elimination of viral proteins, degradation of non-self-nucleic acids, and / or clearance of viral infections from a host. Non-limiting examples of antiviral factors include proteins which recognize viral components (e.g., nucleic acids, viral proteins), molecules involved in antiviral IIR signaling, transcription factors which mediate synthesis of antiviral-associated genes, and proteins which target, sequester, or otherwise interfere with viral reproduction, infection, or stability. Mammalian antiviral factors are those which are natively expressed in mammalian cells.

[0120] 12673398.1 In some embodiments, an IMP interferes with recognition of non-self-nucleic acids by IIR-associated proteins. Recognition of danger molecules generally comprises detection by pattern recognition receptors (PRRs). Pattern recognition receptors (PRRs) are germline-encoded proteins which can detect danger molecules in both the extracellular and intracellular space and which are localized in the cell membrane or in the cytoplasm. PRRs typically fall under five categories of receptors: RIG-I-like receptors (RLRs), Toll-like receptors (TLRs), nucleotide oligomerization domain (NOD)-like receptors (NLRs), C-type lectin receptors (CLRs), absent in melanoma-2 (AIM2)-like receptors (ALRs), and cyclic GMP-AMP synthase (cGAS); however, some scavenger receptors (SRs) have also been classed as PRRs. APRR which can detect a non-self-nucleic acid (such as an RLR, TLR, ALR, NLR, cGAS) would, within the context of this disclosure, be considered an antiviral factor. Importantly, while non-self-nucleic acids are not always viral nucleic acids, responses to non-self-nucleic acids generally overlap with antiviral IIR responses. Indeed, antiviral IIR responses have been identified as contributing significantly to instability of mRNA therapeutics and regulation immunogenicity and / or inflammation in response to the same (Wang, Y. Z. (2021). Molecular Cancer, 20(1), 33.). In some embodiments, an IMP interferes with antiviral PRR mediated signaling. An IMP can be considered to interfere with PRR mediated signaling if it interferes with the synthesis, activity, or function of the PRR, adaptor molecules for the PRR, or downstream molecules activated by the PRR or its adaptor molecules.

[0121] In some embodiments, an IMP interferes with (e.g., antagonizes) a retinoic acidinducible gene I (RIG-I)-like receptor (RLR) mediated signaling. Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) are a family of cytosolic proteins characterized by a central ATPase containing a DExD / H box helicase domain and, optionally, C-terminal repressor domains (RD) and / or N-terminal caspase Recruitment domain (CARD) (Dixit, E., & Kagan, J. (2013). Advances in Immunology, 117, 99-125.). RLRs are RNA sensors which detect immunostimulatory (e.g., non-self) nucleic acids to mediate induction of host responses. RLRs are key molecules in IIRs to viral infection and are also thought to mediate responses to synthetic RNAs, such as mRNA vaccines (Yong, H., & Luo, D. (2018). Frontiers in Immunology, 9, 379456.). Non-limiting examples of RLRs include: RIG-1, melanoma differentiation-associated protein (MDA) 5 (MDA5), and laboratory of genetics and physiology (LGP) 2 (LGP2). In some embodiments, an IMP antagonizes an RLR. In some embodiments, an IMP antagonizes RIG-1. In some embodiments, an IMP antagonizes MDA5. In some embodiments, an IMP antagonizes LGP2.

[0122] 12673398.1 In some embodiments, an IMP interferes with (e.g., antagonizes) Toll-like receptor (TLR) mediated signaling. Toll-like receptors (TLRs) are a family of type I transmembrane proteins having N-terminal ectodomains with leucine-rich repeats, a transmembrane domain, and a cytosolic TIR domain. TLRs are activated by various extracellular stimuli, including, but not limited to, viral, bacteria, fungi, and endogenous ligands (Fitzgerald, K. A., & Kagan, J. C.

[0123] (2020). Cell, 180(6), 1044-1066). TLRs recognize extracellular ligands (e.g., nucleic acids) which have reached endosomes via endocytosis. Non-limiting examples of TLRs include: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, and TLR10 in humans, as well as TLR11, TLR12, and TLR13 in other mammals. TLRs localized in the plasma membrane (e.g., TLR1, TLR2, TLR4, TLR 5, and TLR 6) typically detect microbes and extracellular molecules, while TLRs localized in endosomal membranes (e.g., TLR3, TLR7, TLR8, TLR9, TLR13) typically detect nucleic acids (e.g., viral nucleic acids). In particular, TLR3, TR7, and TLR8 are associated with IIRs against non-self RNA. In some embodiments, IMPs interfere with TLR3 mediated signaling pathways. In some embodiments, IMPs interfere with TLR7 mediated signaling pathways. In some embodiments, IMPs interfere with TLR8 mediated signaling pathways.

[0124] In some embodiments, an IMP interferes with (e.g., antagonizes) nucleotide oligomerization domain (NOD)-like receptor (NLR) mediated signaling. Nucleotide oligomerization domain (NOD)-like receptors (NLRs) are a family of cytosolic proteins characterized by the presence of a conserved NOD that recognizes danger molecules in the cytosol, as well as a variable amino-terminal effector-binding domains (EBDs) and a carboxyl-terminal ligand-recognition domain (LRD). NLRs are activated by pathogenic ligands and play a key role in initiating pathogen resistance and apoptosis (Inohara, N., et al. (2005). Annual Reviews Biochemistry, 74, 355-383.). Non-limiting examples of NLRs include: PYRIN-containing Apafl-like protein (PYPAF) 7 (PYPAF7; also known as NALP12, NLRP12, Monach 1, and RNO2), NACHT-LRR-and-pyrin domain-containing protein 2 (NALP2; aka PYPAF2, NLRP3), NODI (also known as CARD4), NOD2 (also known as CARD15), NOD3, NOD5 (also known as NALP14), NOD6 (also known as NALP9), NOD8 (NALP10, PYNOD), NOD9, NOD12 (also known as PYPAF3, NALP7), NOD14 (also known as NALP13), NOD16 (also known as NALP8), NOD17 (also known as PYPAF6, NALP11), Caspase- 1 / Interleukin- 1 converting enzyme (ICE)-protease Activating Factor (IPAF; also known as CARD12, CLAN), Cryopyrin (also known as NALP3, PYPAF 1, CIAS1), neuronal apoptosis inhibitor protein (NAIP; also known as BIRC1); nucleotide-binding oligomerization domain-like receptor family,

[0125] 12673398.1 pyrin domain-containing (NALP) 1 (NALP1; also known as NAC, DEFCAP, CARD7);

[0126] Apoptotic protease activating factor (APAF)-l (APAF-1); Pyrin-and-Nacht domain (PAN) 2 (PAN2; also known as PYPAF4, NALP4); Maternal Antigen That Embryos Require (Mater; also known as PYPAF8, NALP5); and Class II Major Histocompatibility Complex Transactivator (CIITA). NLRP3 and NOD2 have been especially recognized for their role in the recognition of RNA. In some embodiments, an IMP antagonizes an NLR. In some embodiments, an IMP antagonizes NLRP3. In some embodiments, an IMP antagonizes NOD2.

[0127] In some embodiments, an IMP interferes with (e.g., antagonizes) absent in melanoma-2 (AIM2)-like receptor (ALR) mediated signaling. Absent in melanoma 2 (AIM2)-like receptors (ALRs) are a family of receptors having a pyrin signaling domain and a DNA-binding hematopoietic-expression, interferon-inducible nature, and nuclear-localization (HIN) domain. ALRs. ALRs detect cytosolic DNA, including foreign and genomic DNA, and initiate inflammatory responses (Lugrin, J., & F., M. (2017). Immunological Reviews, 281(1), 99-114.). Non-limiting examples of ALRs include: AIM2, Interferon inducible protein 16 (IFI16); Pyrin and HIN domain Family Member 1 (PYHIN1); and myeloid cell nuclear differentiation antigen (MNDA). AIM2 and IFI16 are known to be particularly important for DNA sensing. In some embodiments, an IMP antagonizes an ALR. In some embodiments, an IMP antagonizes AIM2. In some embodiments, an IMP antagonizes IFI16.

[0128] In some embodiments, an IMP interferes with (e.g., antagonizes) cGAS-mediated signaling. cGAS is a cytosolic protein which can bind non-self-nucleic acids and catalyze 2’-6'-cyclic AMP-GMP (cGAMP) for further signaling; though typically, cGAS binds DNA, it has been increasingly implicated in RNA binding (Ma, Y, et al. (2021). Frontiers in Immunology, 12, 741599.). In some embodiments, an IMP antagonizes cGAS.

[0129] Antiviral factors also comprise RNA binding proteins (RBPs). Broadly, RNA-binding proteins (RBPs) are proteins which are capable of binding double stranded RNA (dsRNA) and / or single stranded RNA (ssRNA); the term includes PRRs which directly bind RNA, but also proteins which are otherwise capable of recognizing non-self RNAs. Non-limiting examples of HR-associated RBPs include: protein kinase R (PKR), 2’-8' oligoadenylate synthase (OAS), ribonuclease latent (RNaseL), adenosine deaminase acting on RNAs (ADARs), Dicer, Drosha, protein activator of PKR (PACT), transactivation response RBP (TRBP), superkiller viralicidic activity 2-like (SKIV2L), heterogeneous nuclear ribonucleoprotein (hnRNP) M (hnRNP M), hnRNP ULI, tristetraprolin (TTP), human antigen R (HuR), T-cell restricted intracellular

[0130] 12673398.1 antigen- 1 (TIA-1), hnRNP A2B1, GTPase-activating protein SH3 domain-binding protein 1 (G3BP1), AT-rich interactive domain-containing protein 5a (Arid5a), methyltransferase like 3 (Mettl3), serine and arginine-rich splicing factor 1 (SRSF1), zinc finger antiviral proteins (ZAPs), and polypyrimidine tract binding protein 1 (PTBP1). In some embodiments, an IMP interferes with RBP-mediated signaling. In some embodiments, an IMP antagonizes an RBP In some embodiments, an IMP interferes with PKR-mediated signaling. PKR is an antiviral RBP which can bind double-stranded RNA (dsRNA). Binding of dsRNAby PKR triggers dimerization of PKR, after which it can induce various antiviral IIRs. Expression and activation of PKR is mediated by additional activity of IIR pathways. PKR-mediated signaling is associated with inhibition of translation machinery, activation of apoptosis, proinflammatory responses, and cell cycle regulation. In some embodiments, an IMP is an antagonist of PKR. In some embodiments, an IMP is a dominant negative PKR (dnPKR). In some embodiments, dnPKR comprises the sequence set forth in SEQ ID NO: 90.

[0131] In some embodiments, an IMP interferes with ZAP -mediated signaling. ZAPs are CCCH-type zinc finger proteins which function as host restriction factors against non-self RNAs by recognition of CpG motifs. ZAP binds to RNA and recruits additional factors which degrade the RNA. ZAP can also inhibit translation of viral RNA by interactions with eukaryotic initiation factors (elFs). Expression of ZAP can be initiated as part of IIR pathways, but some versions of ZAP also exist constitutively. In some embodiments, an IMP is an antagonist of ZAP. In some embodiments, an IMP is a dominant negative ZAP (dnZAP). In some embodiments, an IMP is dnZAP with an additional mutation. In some embodiments, an IMP is dnZAP with a cysteine to arginine (C to R) mutation at position 88 (ZAPC88R). In some embodiments, an IMP is a rat dnZAPC88R (rZAPC88R). In some embodiments, rZAPC88R comprises the sequence set forth in SEQ ID NO: 91.

[0132] Antiviral factors further comprise adaptor protein which of an antiviral IIR pathway. Adaptor proteins are IIR-associated proteins which can integrate signals from antiviral PRRs or RBPs to activate downstream signaling proteins (e.g., via signaling cascades) and, ultimately, contribute to regulation of IIRs. Adaptor proteins generally mediate protein-protein interactions (e.g., between a PRR and a downstream signaling protein) and lack independent intrinsic enzymatic activity. Non-limiting examples of antiviral adaptor proteins include myeloid differentiation primary response 88 (MyD88), Toll / IL-1 receptor / resistance (TIR) domaincontaining adaptor inducing interferon-P (TRIF, also known as TICAM1), TRIF-related adaptor molecule (TRAM), mitochondrial viral signaling protein (MAVS), microprotein in antiviral

[0133] 12673398.1 immunity 1 (MA VII), apoptosis-associated speck-like protein containing a caspase recruiting domain (CARD) (ASC), stimulator of interferon genes (STING), inflammasomes, and PANoptosomes. In some embodiments, an IMP interferes with synthesis or activity of an antiviral adaptor protein. In some embodiments, an IMP antagonizes an antiviral adaptor protein.

[0134] In some embodiments, an IMP interferes with MyD88-mediated signaling. MyD88 is an authorized adaptor protein for TLR and interleukin-1 (IL-1) receptors (IL-1R). MyD88 links TLRs and / or IL-lRs to IL-lR-associated kinase (IRAK) family members, which include IRAKI, IRAK4, IRAK2, and IRAKM. MyD88-mediated signaling is associated with activation of inflammatory responses, such as production of pro-inflammatory cytokines (e.g., tumor necrosis factor (TNF), IL-6, IL-1), and type I interferons (IFN). In some embodiments, an IMP antagonizes MyD88.

[0135] In some embodiments, an IMP interferes with TRIF-mediated signaling. TRIF is an adaptor protein for TLRs and contributes to both MyD88-dependent and MyD88-independent pathways. TRIF pathways link TLRs to IRAK family members, tumor necrosis factor receptor (TNFR) associated factor (TRAF) family members, receptor-interacting proteins (RIPs), and inflammasomes. TRIF-mediated signaling is associated with activation of inflammatory responses, such as production of type-I IFN-P and analogs thereof, and initiation of apoptosis and necroptosis. In some embodiments, an IMP antagonizes TRIF.

[0136] In some embodiments, an IMP interferes with TRAM-mediated signaling. TRAM is an adaptor protein for TLR4 and contributes to both TRIF -dependent and MyD88-dependent signaling pathways. TRAM-mediated signaling is associated with pro-inflammatory responses, such as production of pro-inflammatory cytokines and type I IFN. In some embodiments, an IMP antagonizes TRAM.

[0137] In some embodiments, an IMP interferes with MAVS-mediated signaling. MAVS is a mitochondria-associated adaptor molecule for RLRs. Once activated, MAVS forms prion-like aggregates on the outer membrane of mitochondria, through which downstream signals are propagated. MAVS-mediated signaling is associated with immune and inflammatory responses, such as production of type I IFNs and cytokines (e.g., TNF-a and interleukins). In some embodiments, an IMP antagonizes MAVS. In some embodiments, an IMP is a dominant negative MAVS (dnMAVS). In some embodiments, dnMAVS comprises the sequence set forth in SEQ ID NO: 92. In some embodiments, an IMP facilitates signaling by inhibitors of MAVS. In some embodiments, an IMP facilitates MAVI1 -mediated signaling. MAVI1 is an endoplasmic-reticulum associated negative regulator of RLR-MAVS signaling, which attenuates

[0138] 12673398.1 MAVS activity. MAVI1 inhibits MAVS aggregation and reduces type I IFN signaling activation. MAVI1 is down-regulated during inflammatory responses (e.g., in response to viral infection), and is up-regulated to deactivate immune responses. In some embodiments, an IMP is MAVI1. In some embodiments, an IMP over expresses MAVI1. In some embodiments, MAVI1 comprises the sequence set forth in SEQ ID NO: 93.

[0139] In some embodiments, an IMP interferes with STING-mediated signaling. STING is an endoplasmic reticulum-associated adaptor molecule for cGAS. Once activated, STING forms a complex with TANK-binding kinase 1 (TBK1), which initiates pathways for production of type I interferon and cytokines. In some embodiments, IMPs antagonize STING. In some embodiments, IMPs antagonizes STING.

[0140] In some embodiments, an IMP interferes with inflammasome-mediated signaling.

[0141] Inflammasomes are multi-protein complexes formed by PRRs in the cytoplasm and which, when activated, trigger activation of caspase-1. Inflammasomes are comprised of NLRs or ALRs, and adapter protein apoptosis-associated speck-like protein containing a CARD (ASC). Several inflammasomes are known; non-limiting examples include the NLRP1 inflammasome, the NLRP3 inflammasome, the Caspase- 11 inflammasome, the NAIP-NLRC4 inflammasome, and the AIM2 inflammasome. In some embodiments, an IMP antagonizes an inflammasome.

[0142] In some embodiments, IMPs interfere with PANoptosome-mediated signaling.

[0143] PANoptosomes are complexes formed by numerous pyroptotic, apoptotic, and necroptotic (PANoptotic molecules), and are mainly composed of PRRs, sensory proteins (e.g., Z-DNA binding proteins), adapter proteins with caspase recruiting domains (e.g., apoptosis-associated speck-like protein), and proteins with catalytic effects (e.g., receptor interacting protein kinases, caspases). At least four types of PANoptosomes are known: ZBPl-PANoptosome (ZBP1, NLRP3, ASC, caspase-1, caspase-6, caspase-8, RIPK1, RIPK3), AIM2-PANoptosome (AIM2, Pyrin, ZBP1, ASC, caspase-1, caspase-8, FADD, RIPK1, and RIPK3), RIPKl-PANoptosome (RIPK1, RIPK3, NLRP3, ASC, caspase-1, and caspase-8), andNLRP12- PANoptosome (NLRP12, ASC, caspase-8, and RIPK3). In some embodiments, an IMP antagonizes a PANoptosome.

[0144] Mammalian antiviral factors also comprise transcription factors activated as part of antiviral IIR pathways. Transcription factors are sequence-specific proteins which regulate the rate of transcription of endogenous genes by binding to regions of DNA, such as enhancers or promoter regions. An IMP may interfere with transcription factors by for example, modulating the binding of transcription factors to DNA, reducing synthesis of transcription factors,

[0145] 12673398.1 suppressing nuclear localization of transcription factors, or downregulating their activation. Though the precise mechanisms through which IIR pathways ultimately produce immune and / or inflammatory responses vary, IIR pathways generally converge on the transcription factors nuclear factor kappa-light chain-enhancer of activated B cells (NF-KB) and interferon regulatory factors (IRFs).

[0146] In some embodiments, IMPs interfere with regulation of NF-KB and / or transcription of NF-KB target genes. NF-KB is a rapid-acting, inducible transcription factor involved in producing inflammatory cytokine responses to danger signals. Illustrative NF-KBS include NF-KB 1, NF-KB2, RelA, RelB, and c-Rel. In the absence of danger signals, NF-KBS are sequestered in the cytosol by inhibitor of NF-KB (IKB). Detection of certain danger signals by IIR-associated proteins, such as TLRs, can lead to the phosphorylation of IKB inhibitors, which release NF-KB into the cytosol. Once translocated to the nucleus, NF-KB can bind to DNAto regulate transcription of NF-KB target genes, which include several chemokines, cytokines, cell cycle regulators, anti-apoptotic factors, and adhesion molecules. Importantly, NF-KB can induce transcription of genes which promote inflammatory responses, as well as genes which limit the duration and magnitude of inflammatory responses; thus, NF-KB activity can regulate the onset, duration, and offset of inflammation by differential induction of NF-KB target genes. Nonlimiting examples of NF-KB target genes include first apoptosis signal (Fas), B-cell lymphoma 2 (BCL-2), cellular FLICE-like inhibitory protein (c-FLIP), caspases, inhibitors of apoptosis (IAPS), BCL-2 related gene expressed in fetal liver (BFL-1), Survivin, BCL-2-like protein 1 (BCL-2L1), plasminogen activator inhibitor 2 (PAI2), Cyclin, interleukin (IL)-l, IL-2, IL-6, IL-8, IL-12, IL-18, tumor necrosis factor alpha (TNFa), monocyte chemoattractant protein 1 (MCP-1), chemokine (C-C motif) ligand (CCL) 5 (CCL5; also known as RANTES), macrophage inflammatory protein 2 (MIP-2), chemokine (C-X-C motif) ligand (CXCL) 1 (CXCL1), CXCL10, intercellular adhesion molecule (ICAM) 1 (ICAM-1), vascular cell adhesion molecule (VCAM) 1 (VCAM-1), endothelial cell adhesion molecule (ECAM) 1 (ECAM-1), matrix metalloproteinase (MMPs) and Selectin.

[0147] In some embodiments, IMPs interfere with regulation of IRFs and / or transcription of IRF target genes. IRFs are a family of transcription factors that regulate expression of pro-inflammatory responses, apoptosis, and immune responses to danger signals. In the absence of danger signals, IRFs are localized in the cytoplasm in an inactive, monomeric form. Detection of certain danger signals by IIR-associated proteins, such as RLRs, lead to post-translational modifications of IRFs which allow for protein-protein interactions. IRFs can then homodimerize

[0148] 12673398.1 - SI-

[0149] with other IRFs or heterodimerize with other molecules, after which IRFs can translocate to the nucleus and bind to IRF-target genes. Non-limiting examples of IIR-associated IRFs include IRF1, IRF2, IRF3, IRF4 (also known as PIP and ICSAT), IRF5, IRF6, IRF7, IRF8 (also known as ISCBP) and IRF9 (also known as p48 and ISG3y). Several IRFs function as transcription factors for type I interferons (e.g., IFNa, IFNP) and interferon stimulated genes (ISGs). Some IRFs are activated by MAVS, STING, and TRIF; several IRFs can also indirectly activate NF-KB, for example, via TNFa signaling.

[0150] Mammalian antiviral factors further include molecules which mediate immune and / or inflammatory responses. Ultimately, activation of IIR pathways lead to the production and regulation of cytokines and apoptotic factors which mediate inflammatory responses and apoptosis. Cytokines are cell-signaling, secreted proteins which promote immune responses to infection, inflammation, trauma, and disease. Non-limiting examples of cytokines include interferons (IFNs), interleukins (IL), and tumor necrosis factors (TNFs). In some embodiments, IMPs interfere with transcription of pro-inflammatory genes. In some embodiments, IMPs induce transcription of anti-inflammatory genes. In some embodiments, IMPs interfere with cytokine production. Apoptotic factors are proteins which promote or inhibit apoptosis. Nonlimiting examples of apoptotic factors include caspases, Bel -2 proteins, and TNFs. In some embodiments, IMPs interfere with (e.g., suppress) pro-apoptotic signaling pathways. In some embodiments, IMPs promote anti-apoptotic signaling pathways.

[0151] In some embodiments, IMPs interfere with interferon (IFN) transcription, synthesis, or signaling. Interferons (IFNs) are molecules which interfere with viral replication. Of particular importance to IIRs against non-self-nucleic acids are the type I IFNs, IFNa and IFNP, and the type III IFN, IFNI. IFN production in cells can be initiated by PRR activity, after which IFN can act as a signal for further gene transcription (e.g., of interferon stimulated genes (ISGs)) and for responses by neighboring cells. Type I IFN synthesis and activity can be promoted by various IIR pathways, for example, via the activity of RLRs, TLRs, MyD88, TRIF, TRAM, MAVS, and PKR. Synthesis and activity of type I IFNs can also be downregulated by various cytokine signaling inhibitors, such as suppressors of cytokine signaling (SOCS) family members and IKBS. Suppressor of cytokine signaling (SOCS) proteins are negative-feedback inhibitors of cytokine signaling pathways and are activated via JAK / STAT pathways. SOCS proteins include cytokine inducible SH2-containing protein (CISH), SOCS1, SOCS2, SOCS3, SOCS4, SOCS5, SOCS6, and SOCS7. In some embodiments, IMPs interfere with IFNa production or activity. In some embodiments, IMPs interfere with IFNP production or activity. In some embodiments,

[0152] 12673398.1 IMPs interfere with IFNI production or activity. In some embodiments, an IMP is a SOCS protein. In some embodiments, an IMP is SOCS1. In some embodiments, SOCS1 comprises the sequence set forth in SEQ ID NO: 94. In some embodiments, an IMP is SOCS3. In some embodiments, SOCS3 comprises the sequence set forth in SEQ ID NO: 95.

[0153] In some embodiments, IMPs interfere with interleukin (IL) transcription, synthesis, or signaling. Interleukins (ILs) are molecules which are secreted by white blood cells and are critical for immune function. Several interleukins are known (e.g., IL-1, IL-2, IL-3, IL-4, efc.), each having regulatory and / or signaling functions in immune responses and / or inflammatory responses. In some embodiments, an IMP is an antagonist of IL.

[0154] In some embodiments, IMPs interfere with a tumor necrosis factors (TNF) transcription, synthesis, or signaling. Tumor necrosis factors (TNFs) are molecules mediate inflammatory and apoptotic responses. TNF signaling is mediated by two receptors: TNF receptor 1 (TNFR1) and TNFR2. TNFR1 -mediated signaling is generally pro-inflammatory and apoptotic. TNFR2-mediated signaling is generally anti-inflammatory and pro-cell growth. TNF-a, a TNF which is produced by several IIR pathways, can bind to both TNFR1 and TNFR2 to differentially regulate immune responses. First apoptosis signal (Fas) ligand is a member of the TNF family which can interact with Fas receptors. Binding of Fas ligand to Fas receptors initiates an apoptosis pathway. In some embodiments, IMPs interfere with TNFR function or activity. In some embodiments, IMPs interfere with TNF transcription, synthesis, or signaling. In some embodiments, IMPs interfere with Fas ligand transcription, synthesis, or signaling. In some embodiments, an IMP is a TNF antagonist. In some embodiments, an IMP is a Fas receptor antagonist.

[0155] In some embodiments, IMPs interfere with caspase transcription, synthesis, or signaling. Caspases are cysteine-aspartic proteases which can initiate apoptosis, execute apoptosis, and / or promote inflammation. Caspase synthesis is tightly regulated and can occur as a result of HR pathway initiation. Caspases enter an active form when they dimerize and form multi-protein complexes with various functions. In some embodiments, IMPs interfere with caspase synthesis. In some embodiments, IMPs interfere with caspase dimerization. In some embodiments, IMPs interfere with caspase complex functions. In some embodiments, an IMP is a caspase antagonist.

[0156] In some embodiments, IMPs interfere with Bcl-2 protein transcription, synthesis, or signaling. B-cell lymphoma 2 (Bcl-2) proteins are regulatory proteins which regulate apoptosis. Bcl-2 is a receptor localized on the outer membrane of mitochondria and can promote or inhibit

[0157] 12673398.1 apoptosis. In some embodiments, IMPs interfere with Bcl-2 synthesis or function. In some embodiments, an IMP is a Bcl-2 antagonist.

[0158] Viral Immune Evasion Proteins

[0159] In some embodiments, an IMP is a viral immune evasion protein (VIEP). Viral immune evasion proteins (VIEPs) are viral proteins which interfere with one or more antiviral HR pathways and ultimately reduce the magnitude of an immune response (e.g., inflammation) against the virus or extend the time before which an HR against the virus is initiated. VIEPs are expressed by numerous viruses, for example, vaccinia virus (VACV), Orf virus, herpes simplex virus (HSV), influenza A viruses (IAV), and Toscana virus (TOSV). VIEPs may exert their effect through various mechanisms. For example, a VIEP may interfere with (e.g., antagonize) PRRs, RBPs, adaptor molecules, transcription factors, cytokines, and regulators thereof which are associated with antiviral IIR pathways.

[0160] In some embodiments, an IMP is a VIEP from vaccinia virus (VACV). VACV is an enveloped virus of the Poxviridae family and comprises a linear, double-stranded DNA genome. Because VACV replicates in the cytoplasm of a host cell and not the nucleus, it possesses a large genome which encodes enzymes and proteins related to DNA replication and transcription, as well as VIEPs which endow the virus with resistance to HRs, for example, F1L, K3L, E3L, and B18R (Perdiguero, B., & Esteban, M. (2009). Journal of Interferon and Cytokine Research, 29(9), 581-598.). In some embodiments, an IMP is F1L. F1L is a VACV protein which has been shown to interfere with NLR-mediated signaling. VACV is a homolog of Bcl-2, and can inhibit NLRP1 by directly binding to it. In some embodiments, F1L comprises the sequence set forth in SEQ ID NO: 96. In some embodiments, an IMP is K3L. K3L is a VACV protein with homology to an elF, eIF2a. eIF2a is a substrate of PKR and is important for initiation of translation of many proteins, including cytokines. K3L is a partial agonist of PKR, and competitively inhibits activation of endogenous eIF2a. In some embodiments, an IMP is RNA-binding protein E3 (E3L). E3L is a dsRNA binding protein which has been shown to interfere with PKR, OAS, ADAR, IRF3, IRF7, and NF-KB-mediated signaling, as well as the expression of IFNs and ISGs. E3L is also capable of binding directly to PKR to prevent its activation of elFs. In some embodiments, E3L comprises the sequence set forth in SEQ ID NO: 97. In some embodiments, an IMP is soluble IFN-a / p receptor B18 (B18R). B18R is a glycoprotein with some similarity to cellular IFNa and IFNP receptors; thus, B18R competes with IFNa and IFNP receptors to bind IFNa and IFNP, and thereby interferes with IFN related signaling. In some embodiments, B18R

[0161] 12673398.1 comprises the sequence set forth in SEQ ID NO: 98. In some embodiments, an IMP is NIL. NIL may contain BH3-like binding domains to bind BH3 to exert control over induced apoptosis in infected cells. In some embodiments, NIL comprises the sequence set forth in SEQ ID NO: 106.

[0162] In some embodiments, an IMP is a VIEP from HSV. HSV is an enveloped virus comprising a large, double-stranded linear DNA genome. HSVs include HSV 1 and HSV2. Several DNA sequences, dsRNA intermediates, and viral proteins of HSV are recognized by PRRs (including TLRs, RLRs, NLRs, ALRs, and cGAS). IIR pathways which respond to HSV involve several molecules, such as MyD88, STING, and LGP2. HSV encodes several VIEPs, including US1, US11, and ISC34.5 (Zhu, H., & Zheng, C. (2020). Microbiology and Molecular Biology Reviews, 84(4), 10-1128. doi:10.1128 / MMBR.00099-20; Zhang, M. L., & Yan, Y. (2015). The Journal of Immunology, 194(7), 3102-3115.). In some embodiments, an IMP is US1. US1 is an HSV immediate early protein which has been found to suppress production of IFN-P through inhibition of IRF3. Specifically, US1 binds to DNA binding domains of IRF3 to prevent its association with the IFN-P promoter. In some embodiments, US1 comprises the sequence set forth in SEQ ID NO: 99. In some embodiments, an IMP is US11. US11 is an HSV RNA-binding protein which interrupts RLR-mediated antiviral IIR pathways. US11 is an antagonist of RIG-I and MDA5 and binds directly to the C-terminal domains of both receptors. In some embodiments, US11 comprises the sequence set forth in SEQ ID NO: 100. In some embodiments, an IMP is infected cell protein 34.5 (ISC34.5, also known as ICP34.5). ISC34.5 is an HSV protein which acts on eIF-2A, and which has been shown to target STING by preventing its translocation from the endoplasmic reticulum, thereby inhibiting downstream production of cytokines and propagation of antiviral IIRs. In some embodiments, ISC34.5 comprises the sequence set forth in SEQ ID NO: 101. In some embodiments, an IMP is infected cell protein 0 (ICP0). ICP0 is an HSV protein and ubiquitination (Ub) ligase which targets proteins in numerous cell pathways, such as SUMOylation pathways, cell cycle checkpoint factors, DNA repair pathways, TLR-associated proteins, and cytokine signaling pathways to promote HSV lytic replication and propagation. In some embodiments, ICP0 comprises the sequence set forth in SEQ ID NO: 102.

[0163] In some embodiments, an IMP is a VIEP from influenza A (IAV). IAV is a negativesense, single-stranded segmented, enveloped RNA virus of the family Orthomyxoviridae. IAVS subtypes are categorized by the type of surface protein in its envelope: hemagglutinin (H) and neuraminidase (N). IAVs are recognized by TLRs, RLRs, and NLRs. One critical IAV VIEP is

[0164] 12673398.1 non- structural protein 1 (NS1) (Van de Sandt, C., Kreijtz, J., & Rimmelzwaan, G. (2012).

[0165] Viruses, 4(9), 1438-1476.). In some embodiments, an IMP is an IAVNS1. IAVNS1 blocks recognition of RIG-I mediated signaling through various means, such as antagonizing RIG-I, inhibiting oligomerization of TRIM25, inhibiting IRF-3, inhibiting NF-KB, and interfering with mRNA export machinery. In some embodiments, an IMP is NS1 from influenza A / Puerto Rico / 8 / 34 (PR8 NS1). In some embodiments, PR8 NS1 comprises the sequence set forth in SEQ ID NO: 103.

[0166] In some embodiments, an IMP is a VIEP from Toscana virus (TOSV). Toscana virus an enveloped RNA arbovirus of the family Phenuiviridae. In some embodiments, an IMP is a non-structural (NSs) protein of TOSV. The NSs proteins of TOSV have been associated with inhibition of IIRs in cells (Gori-Savellini, G. V. (2013). Journal of Virology, 87(12), 6660-6667; Kalveram, B., et al. (2013). Journal of virology, 87(7), 3710-3718.). TOSV NSs have been shown to downregulate PKR and inhibit the induction of IFN production by RLR-mediated HR pathways. In some embodiments, TOSV NSs comprises the sequence set forth in SEQ ID NO: 104.

[0167] In some embodiments, an IMP is a VIEP from encephalomyocarditis virus (EMCV). EMCV is a single-stranded, non-enveloped virus of the family Picornaviridae. In some embodiments, an IMP is an Lpro protein of EMCV. The leader protease (Lpro) protein of EMCV has been associated with interfering with IRF pathways and ultimately inhibiting the induction of IFN production. In some embodiments, EMCV Lpro comprises the sequence set forth in SEQ ID NO: 105.

[0168] taRNA Constructs Encoding IMPs

[0169] In some embodiments, a taRNA comprises an RNA polynucleotide comprising a nucleic acid encoding an IMP. In some embodiments, a first RNA polynucleotide (e.g., a replicase construct) of a taRNA comprises a nucleic acid encoding an IMP. In some embodiments, a second RNA polynucleotide (e.g., a trRNA) of a taRNA comprises a nucleic acid encoding an IMP. In some embodiments a first RNA polynucleotide of a taRNA and a second RNA polynucleotide of a taRNA each comprise a nucleic acid encoding an IMP.

[0170] In some embodiments, a taRNA comprises an RNA polynucleotide (e.g., a replicase construct, a trRNA) comprising a nucleic acid encoding multiple IMPs, for example, a first IMP and a second IMP. In some embodiments, a taRNA comprises an RNA polynucleotide comprising nucleic acids encoding two or more IMPs. In some embodiments, a taRNA

[0171] 12673398.1 comprises an RNA polynucleotide comprising nucleic acids encoding three or more IMPs. In some embodiments, a taRNA comprises an RNA polynucleotide comprising nucleic acids encoding four or more IMPs. In some embodiments, a taRNA comprises an RNA polynucleotide comprising nucleic acids encoding five or more IMPs. In some embodiments, a taRNA comprises an RNA polynucleotide comprising nucleic acids encoding six or more IMPs. In some embodiments, a taRNA comprises an RNA polynucleotide comprising nucleic acids encoding one, two, three, four, five, or six IMPs. In some embodiments, the multiple IMPs are different IMPs.

[0172] Schematics of illustrative trRNAs comprising nucleic acids encoding payloads or nucleic acid payloads and nucleic acids encoding IMPs as described herein are shown in FIGs. 6A-6B, 7A-7B, and 8A-8B. Schematics of illustrative replicase constructs encoding EEEV replicases and IMPs are shown in FIGs. 10A-10D.

[0173] Linkers

[0174] In some embodiments, a taRNA comprises two or more IMPs joined by linkers. In some embodiments, a trRNA comprises an IMP joined with a payload by a linker. For example, the trRNAfrom 8' to 6' may comprise a nucleic acid encoding an IMP, a nucleic acid encoding a linker or a nucleic acid linker, and a nucleic acid encoding a payload. In another example, the trRNAfrom 8' to 6' may comprise, a nucleic acid encoding a payload, a nucleic acid encoding a linker or a nucleic acid linker, and a nucleic acid encoding an IMP. In some embodiments, a replicase construct comprises an IMP joined with EEEV replicase by a linker. For example, the replicase construct from 8' to 6' may comprise a nucleic acid encoding an EEEV replicase, a nucleic acid encoding a linker or a nucleic acid linker, and a nucleic acid encoding an IMP. As another example, the replicase construct from 8' to 6' may comprise a nucleic acid encoding an IMP, a nucleic acid encoding a linker or a nucleic acid linker, and a nucleic acid encoding an EEEV replicase. As used herein, the term “linker” refers to sequences which allow for the concatenation of one or more nucleic acids encoding a gene product, for example, in a multi ci str onic vector. Linkers may be nucleic acid linkers, for example, nucleic acid elements which can induce cap-independent translation and internal promoters, or peptide linkers, such as ribosomal skip proteins. Proteins (e.g., IMPs) are considered to be “joined” by a linker if the nucleic acids encoding the proteins and the nucleic acid linkers or nucleic acids encoding a linker are comprised within a continuous polynucleotide.

[0175] 12673398.1 In some embodiments, a linker is an internal ribosome entry site (IRES) element. As used herein, the term “IRES element” refers to a cis RNA sequence region which allows for the initiation of translation of a sequence in a cap-independent manner. The terms “IRES element,” “IRES,” “IRES sequence,” and “IRES region” may be used interchangeably. The term “capindependent” refers to a mechanism for initiation of translation of an RNA polynucleotide which does not require a 8' cap (e.g., Cap-0, Cap-1, Cap-2); cap-independent mechanisms for initiating translation are found in many viral RNA and in some eukaryotic mRNA. In contrast, “cap-dependent” mechanisms require a 8' cap (e.g., Cap-0) which recruits a series of initiation factors and catalyzes the formation of a functional ribosome to initiate translation by a ribosome; cap-dependent mechanisms for initiating translation are found in most eukaryotic mRNA. When used as linkers in an RNA polynucleotide, IRES elements can initiate the translation of downstream nucleic acids (i.e., coding sequence). Though rates of translation of a downstream nucleic acid can vary between different IRES elements and / or promoters, IRES elements may be useful for expressing multiple gene products off of a single polynucleotide, as translation can be reinitiated at each IRES element. IRES elements also allow for expression of multiple proteins without further alteration of their amino acid sequence (e.g., as with some peptide-based linkers). IRES elements are often observed in viruses, though virus-like endogenous IRES elements in eukaryotes have been observed. Illustrative viral and eukaryotic IRES sequences can be found on IRESbase (available on the worldwide web at reprod.njmu.edu.cn / cgi-bin / iresbase / index.php). In some embodiments, a linker is a viral IRES element. Non-limiting examples of viral IRES elements include IRES elements of Taura syndrome virus, Triatoma virus, Theiler’s encephalomyelitis virus, simian Virus 40, Solenopsis invicta virus 1, Rhopalosiphum padi virus, Reticuloendotheliosis virus, human poliovirus 1, Plautia stall intestine virus, Kashmir bee virus, Manhattan Parechovirus (MPV), Human rhinovirus (iHRV), Homalodisca coagulata virus-1, Human Immunodeficiency Virus type 1, Homalodisca coagulata virus-1, Himetobi P virus, Hepatitis C virus, Hepatitis A virus, Hepatitis GB virus, foot and mouth disease virus, Human enterovirus 71 (EV71), Equine rhinitis virus, Ectropis obliqua picoma-like virus, Encephalomyocarditis virus (EMCV), Drosophila C Virus, Crucifer tobamo virus, Cricket paralysis virus, Bovine viral diarrhea virus 1, Black Queen Cell Virus, Aphid lethal paralysis virus, Avian encephalomyelitis virus, Acute bee paralysis virus, Hibiscus chlorotic ringspot virus, Classical swine fever virus, Porcine kobuvirus (PKV), Human FGF2, Human SFTPA1, Human AML 1 / RUNX1, Drosophila antennapedia, Human AQP4, Human

[0176] 12673398.1 AT1R, Human BAG-1, Human BCL2, Human BiP, Human c-IAPl, Human c-myc, Human eIF4G, Mouse NDST4L, Human LEF1, Mouse HIF1 alpha, Human n.myc, Mouse Gtx, Human p27kipl, Human PDGF2 / c-sis, Human p53, Human Pim-1, Mouse Rbm3, Drosophila reaper, Canine Scamper, Drosophila Ubx, Salivirus, Cosavirus, Parechovirus, Human UNR, Mouse UtrA, Human VEGF-A, Human XIAP, Drosophila hairless, S. cerevisiae TFIID, S. cerevisiae YAP1, Human c-src, Human FGF-1, Simian picomavirus, Turnip crinkle virus, Coxsackievirus B3 (CVB3), Coxsackievirus A (CVB 1 / 2), tortoise rafivirus (TraV), and Poliovirus (PV). In some embodiments, a linker is an IRES from CVB3. In some embodiments, an IRES from CVB3 comprises the sequence set forth in SEQ ID NO: 69. In some embodiments, a linker is an IRES from EMCV. In some embodiments, an IRES from EMCV comprises the sequence set forth in SEQ ID NO: 70. In some embodiments, a linker is an IRES from PKV In some embodiments, an IRES from PKV comprises the sequence set forth in SEQ ID NO: 71. In some embodiments, a linker is an IRES from MPV. In some embodiments, an IRES from MPV comprises the sequence set forth in SEQ ID NO: 72. In some embodiments, a linker is an IRES from TraV. In some embodiments, an IRES from TRAV comprises the sequence set forth in SEQ ID NO: 73.

[0177] In some embodiments, a linker is a ribosomal skip peptide. Ribosomal skip peptides, also known as “self-cleaving peptides,” are peptides which induce ribosomal skipping during translation of an RNA polynucleotide, such that the ribosome fails to covalently bond an amino acid to the nascent chain, then resumes translation downstream, such that multiple, separate proteins are produced. Sequences encoding ribosomal skip peptides tend to be significantly shorter than most IRES elements, and so may be particularly useful when constraints on polynucleotide length are important. Ribosomal skip peptides are typically viral peptides such as 2A peptides. 2A peptides are peptides of about 18-22 amino acids in length which can induce ribosomal skipping. Non-limiting examples of 2A peptides include 2A peptides from: thosea asigna virus 2 A (T2A), porcine teschovirus-1 2 A (P2A), equine rhinitis A virus 2 A (E2A), and foot-and-mouth disease virus 18 (F2A). In some embodiments, a linker is a 2A peptide. In some embodiments, a linker is a T2A peptide. In some embodiments, a T2A peptide comprises the sequence set forth in SEQ ID NO: 107. In some embodiments, a T2A peptide is encoded by the sequence set forth in SEQ ID NO: 74. In some embodiments, a linker is a P2A peptide. In some embodiments, a P2A peptide comprises the sequence set forth in SEQ ID NO: 108. In some embodiments, a P2A peptide is encoded by the sequence set forth in SEQ ID NO: 75. In some embodiments, a linker is an E2A peptide. In some embodiments, an E2A peptide comprises the

[0178] 12673398.1 sequence set forth in SEQ ID NO: 109. In some embodiments, an E2A peptide is encoded by the sequence set forth in SEQ ID NO: 76.

[0179] Though expression of multiple proteins linked by 2A sequences is generally even, the addition of a 2A peptide alone adds additional amino acids onto each protein flanking it, which may be undesirable for certain proteins which do not tolerate terminal modifications. 2A sequences can also be combined with furin recognition sites to allow for removal of these 2A residues from a translated protein. Furin, also known as Paired basic Amino acid Cleaving Enzyme (PACE), is a protease capable of cleaving proteins at furin recognition sites. Furin-2A comprises a furin cleavage site (FCS) upstream of a given 2A peptide (e.g., T2A, P2A). In some embodiments, a linker is a Furin-2A peptide. In some embodiments, a linker is a Furin-T2A peptide. In some embodiments, a Furin-T2A peptide comprises the sequence set forth in SEQ ID NO: 110. In some embodiments, a Furin-T2A peptide is encoded by the sequence set forth in SEQ ID NO: 77. In some embodiments, a linker is a Furin-P2A peptide. In some embodiments, a Furin-P2A peptide comprises the sequence set forth in SEQ ID NO: 111. In some embodiments, a Furin-P2A peptide is encoded by the sequence set forth in SEQ ID NO: 78.

[0180] In some embodiments, a linker is an alphaviral subgenomic promoter. An alphaviral subgenomic promoter is a nucleic acid upstream (8' ) of a nucleic acid encoding a payload, to which it is operably linked. Subgenomic promoters (SGP) comprise recognition and binding sites for RNA polymerase (e.g., RNA-dependent RNA polymerase), and may also comprise recognition sites or binding sites for other transcription factors. An alphaviral (SGP) is a promoter which is native to an alphavirus, and which can regulate transcription of nucleic acids to which it is operably linked. An alphaviral subgenomic promoter may be a promoter which, in wildtype alphaviruses, regulates transcription of a structural protein or a non-structural protein. SGPs of alphaviruses are typically found at the junction between nsP4 and structural (STR) coding regions of the alphaviral genome. These SGPs are described by the position spanned relative to the subgenomic start site; for example, an SGP spanning 100 nucleotides, starting 50nt upstream of the start site and ending 50nt downstream of the start site would be described as-50 / 50. In some embodiments, an SGP is a Chikungunya virus (CHIKV) SGP. In some embodiments, a CHIKV SGP is CHIKV (-75 / 69). In some embodiments, an SGP is an Eastern equine encephalitis virus (EEV) SGP. In some embodiments, a EEV SGP is EEV (-84 / 58). In some embodiments, an SGP is a Ross River virus (RRV) SGP. In some embodiments, an RRV SGP is RRV (-75 / 48). In some embodiments, an SGP is an SFV SGP. In some embodiments, a SFV SGP is SFV (-37 / 51); SFV (-50 / 0); SFV (-50 / 51), SFV (-100 / 0), SFV (-100 / 51), SFV (- 12673398.1 150 / 0), SFV (-150 / 51), SFV (-200 / 0), or SFV (-200 / 51). In some embodiments, an SGP is an SINV SGP. In some embodiments, an SINV SGP is SINV (SINV-75 / 49). In some embodiments, an SGP is a western equine encephalitis virus (WEEV) SGP. In some embodiments, an WEEV SGP is WEEV (-84 / 37).

[0181] In some embodiments, a linker is an alphaviral replicase junction region. The term “junction” refers to a region between the C-terminal amino acid of the first domain of a protein and the N-terminal amino acid of the second domain of a protein. A protein “domain” refers to a distinct functional or structural subunit of a protein or protein complex; for example, an EEEV replicase protein is a protein complex comprising EEEV nsPl, EEEV nsP2, EEEV nsP3, and EEEV nsP4, each of which may be referred to herein as a domain of a replicase. For example, a junction between EEEV nsP3 and EEEV nsP4 domains is referred to herein as “3 / 4J”. In some embodiments, an EEEV 3 / 4J comprises the sequence set forth in SEQ ID NO: 112 or SEQ ID NO: 113. In some embodiments, an EEEV 3 / 4J is encoded by the sequence set forth in SEQ ID NO: 79. In some embodiments, a 3 / 4J linker is joined to an FCS.

[0182] In some embodiments, EEEV nsPl and EEEV nsP2 are in a fusion protein that comprises an amino acid sequence having 95% similarity to the sequence set forth in SEQ ID NO: 86. In some embodiments, EEEV nsPl and EEEV nsP2 are in a fusion protein that comprises the amino acid sequence set forth in SEQ ID NO: 86. In some embodiments, EEEV nsP3 comprises an amino acid sequence having 95% similarity to the sequence set forth in SEQ ID NO: 87 or SEQ ID NO: 88. In some embodiments, EEEV nsP3 comprises the amino acid sequence set forth in SEQ ID NO: 87 or SEQ ID NO: 88. In some embodiments, EEEV nsP4 comprises an amino acid sequence having 95% similarity to the sequence set forth in SEQ ID NO: 89. In some embodiments, EEEV nsP4 comprises the amino acid sequence set forth in SEQ ID NO: 89.

[0183] EEEV Replicase-IMP Fusion Proteins

[0184] Described herein, in some aspects, are RNA polynucleotides encoding an EEEV replicase (e.g., a wildtype EEEV replicase, an Opal-R EEEV replicase, an EEEV replicase variant) and an IMP. In some embodiments, an RNA polynucleotide encoding an EEEV replicase (e.g., a wildtype EEEV replicase, an Opal-R EEEV replicase, an EEEV replicase variant) and an IMP encodes an EEEV-IMP fusion protein. The term “fusion protein,” also known as a chimeric protein, refers to any protein comprising at least two domains wherein each

[0185] 12673398.1 domain is derived from a different protein, and wherein the at least two domains are joined at a fusion junction. Domains of a fusion protein may be joined directly by a covalent bond, or by a linker. Though domains of a fusion protein are encoded by the same polynucleotide, these domains may be cleaved or otherwise separated, for example, during translation or via post-translational modification. Generally, EEEV replicase-IMP fusion proteins comprising a linker comprise a 2A peptide and do not comprise an IRES. In some embodiments, an EEEV replicase-IMP fusion protein comprises an EEEV replicase (e.g., a wildtype EEEV replicase, an Opal-R EEEV replicase, an EEEV replicase variant) which is fused to an VAC V B 18R, VACV E3L, VACVF1L, VACV N1L, HSVISC34.5, HSVUS1, HSVUS11, influenza NS1 (e.g., PR8 NS1), TOSVNS, dnMAVS, MAVI1, dnPKR, SOCS, dnZAP, and / or EMCV Lpro.

[0186] In some aspects, an EEEV-IMP fusion protein comprises a replicase protein wherein an IMP is inserted between domains of the EEEV replicase, for example, after the 3 / 4J and between the nsP3 and nsP4 domains, but before the nsP4, such that the EEEV-IMP fusion protein comprises EEEV nsPl-nsP2-nsP3-3 / 4J-IMP-nsP4. An “EEEV nsPl-nsP2-nsP3-3 / 4J-IMP-nsP4” will be understood to comprise the nsPl, nsP2, nsP3, and nsP4 corresponding to EEEV. In some embodiments, a nsPl-nsP2-nsP3-3 / 4J-IMP-nsP4 EEEV-IMP fusion protein is encoded by a replicase construct comprising, from 5' to 3': a 5' UTR, a nucleic acid encoding EEEV nsPl, a nucleic acid encoding EEEV nsP2, a nucleic acid encoding EEEV nsP3, a nucleic acid encoding an IMP, a nucleic acid encoding EEEV nsP4, and a 3' UTR. In some embodiments, a nsPl-nsP2-nsP3-3 / 4J-IMP-nsP4 EEEV replicase-IMP fusion protein further comprises a 3 / 4J duplicate. A 3 / 4J duplicate, as referred to herein, comprises a 3 / 4J which is inserted into an EEEV replicase protein, such that the replicase protein comprises two 3 / 4 Js. In some embodiments, an IMP is flanked between a 3 / 4J and a 3 / 4J duplicate and comprises: EEEV nsP 1 -nsP2-nsP3 -3 / 4 J-IMP-3 / 4 J-nsP4.

[0187] In some embodiments, an EEEV replicase-IMP fusion protein comprises an EEEV replicase which is fused to an IMP on either or both terminals (i.e., not between domains of the replicase). In some embodiments, an IMP is fused to the replicase using a linker (e.g., linked to the C-terminal and / or N-terminal of the replicase).

[0188] In some embodiments, a replicase-IMP fusion protein comprises an IMP fused to the C-terminal of a replicase protein. In some embodiments, an EEEV replicase-IMP fusion protein comprises IMP-nsPl-nsP2-nsP3-nsP4. In some embodiments, an EEEV replicase-IMP fusion protein comprises IMP-linker-nsPl-nsP2-nsP3-nsP4. In some embodiments, an IMP-linker-nsPl-nsP2-nsP3-nsP4 EEEV replicase-IMP fusion protein is encoded by a replicase construct

[0189] 12673398.1 comprising, from 5' to 3': a 5' UTR, a nucleic acid encoding an IMP, a nucleic acid encoding a linker or a nucleic acid linker, a nucleic acid encoding EEEV nsPl, a nucleic acid encoding EEEV nsP2, a nucleic acid encoding EEEV nsP3-nsP4, and a 3' UTR.

[0190] In some embodiments, a replicase-IMP fusion protein comprises an IMP fused to the N-terminal of a replicase protein. In some embodiments, an EEEV replicase-IMP fusion protein comprises EEEV nsPl-nsP2-nsP3-nsP4-IMP. In some embodiments, an EEEV replicase-IMP fusion protein comprises EEEV nsPl-nsP2-nsP3-nsP4-linker-IMP. In some embodiments, an EEEV-IMP fusion protein is encoded by a replicase construct comprising, from 5' to 3': a 5' UTR, a nucleic acid encoding EEEV nsPl, a nucleic acid encoding EEEV nsP2, a nucleic acid encoding EEEV nsP3-nsP4, a nucleic acid encoding a linker or a nucleic acid linker, a nucleic acid encoding an IMP, and a 3' UTR.

[0191] In some embodiments, a replicase-IMP fusion protein comprises a first IMP fused to the C -terminal of a replicase protein and a second IMP fused to the N-terminal of a replicase protein. In some embodiments, an EEEV replicase-IMP fusion protein comprises IMP-nsPl-nsP2-nsP3-nsP4-IMP. In some embodiments, the first IMP and the second IMP are the same IMP. In some embodiments, the first IMP and the second IMP are different IMPs. In some embodiments, an EEEV replicase-IMP fusion protein comprises IMP 1 -linker l-nsPl-nsP2-nsP3 -nsP4-linker2-IMP2, wherein IMP1 and IMP2 are both IMPs and linker 1 and linker2 are both linkers; IMP1 and IMP2 may be the same IMP, or may be different IMPs, and linker 1 and linker2 may be the same linker, or may be different linkers. In some embodiments, an IMP-linker-nsPl-nsP2-nsP3-nsP4-linker-IMP EEEV replicase-IMP fusion protein is encoded by a replicase construct comprising, from 5' to 3': a 5' UTR, a nucleic acid encoding a first IMP, a nucleic acid encoding a first linker, a nucleic acid encoding EEEV replicase (e.g., a nucleic acid encoding EEEV nsPl, a nucleic acid encoding EEEV nsP2, and a nucleic acid encoding EEEV nsP3-nsP4), a nucleic acid encoding a second linker, a nucleic acid encoding a second IMP, and a 3' UTR.

[0192] In some embodiments, an EEEV replicase-IMP fusion protein comprises EEEV nsPl-nsP2-nsP3-nsP4-3 / 4J-IMP. In some embodiments, an EEEV replicase-IMP fusion protein comprises IMP-3 / 4J-nsPl-nsP2-nsP3-nsP4. In some embodiments, an EEEV replicase-IMP fusion protein comprises IMP-nsPl-nsP2-nsP3-nsP4-3 / 4J-IMP.

[0193] Schematics of illustrative replicase constructs encoding EEEV replicases and IMPs are shown in FIGs. 9A-9D.

[0194] 12673398.1 In some embodiments, an EEEV replicase-IMP fusion protein comprises an EEEV replicase (e.g., EEEV nsPl, EEEV nsP2, EEEV nsP3, and EEEV nsP4) and a PR8 NS1. In some embodiments, an EEEV replicase-IMP fusion protein comprises PR8 NS1 inserted between domains of the EEEV replicase. In some embodiments, PR8 NS1 is inserted after the 3 / 4J and between the nsP3 and nsP4 domains of the EEEV replicase. In some embodiments, the EEEV replicase-IMP fusion protein comprises EEEV nsPl-nsP2-nsP3-3 / 4J-PR8 NSl-nsP4. In some embodiments, an EEEV replicase-IMP fusion protein comprises an EEEV nsPl-nsP2 fusion protein comprising the amino acid sequence set forth in SEQ ID NO: 86; an EEEV nsP3 domain comprising the amino acid sequence set forth in SEQ ID NO: 87 or 88; an EEEV 3 / 4J comprising the amino acid sequence set forth in SEQ ID NO: 112 or SEQ ID NO: 113; a PR8 NS1 comprising the amino acid sequence set forth in SEQ ID NO: 103; and an EEEV nsP4 domain comprising the amino acid sequence set forth in SEQ ID NO: 89. In some embodiments, an EEEV replicase-IMP fusion protein consists essentially of an EEEV nsPl-nsP2 fusion protein comprising the amino acid sequence set forth in SEQ ID NO 86; an EEEV nsP3 domain comprising the amino acid sequence set forth in SEQ ID NO 87 or 88; an EEEV 3 / 4J comprising the amino acid sequence set forth in SEQ ID NO 112 or SEQ ID NO: 113; a PR8 NS1 comprising the amino acid sequence set forth in SEQ ID NO: 103; and an EEEV nsP4 domain comprising the amino acid sequence set forth in SEQ ID NO: 89.

[0195] In some embodiments, the EEEV replicase-IMP fusion protein comprises PR8 NS1 flanked by 3 / 4J and a 3 / 4J duplicate. In some embodiments, an EEEV replicase-IMP fusion protein comprises an EEEV nsPl-nsP2 fusion protein comprising the amino acid sequence set forth in SEQ ID NO: 86; an EEEV nsP3 domain comprising the amino acid sequence set forth in SEQ ID NO: 87 or 88; a first EEEV 3 / 4J comprising the amino acid sequence set forth in SEQ ID NO: 112 or SEQ ID NO: 113; a PR8 NS1 comprising the amino acid sequence set forth in SEQ ID NO: 103; a second EEEV 3 / 4J comprising the amino acid sequence set forth in SEQ ID NO: 112 or SEQ ID NO: 113; and an EEEV nsP4 domain comprising the amino acid sequence set forth in SEQ ID NO: 89. In some embodiments, an EEEV replicase-IMP fusion protein consists essentially of an EEEV nsPl-nsP2 fusion protein comprising the amino acid sequence set forth in SEQ ID NO: 86; an EEEV nsP3 domain comprising the amino acid sequence set forth in SEQ ID NO: 87 or 88; a first EEEV 3 / 4J comprising the amino acid sequence set forth in SEQ ID NO: 112 or SEQ ID NO: 113; a PR8 NS1 comprising the amino acid sequence set forth in SEQ ID NO: 103; a second EEEV 3 / 4 J comprising the amino acid sequence set forth in

[0196] 12673398.1 SEQ ID NO: 112 or SEQ ID NO: 113; and an EEEV nsP4 domain comprising the amino acid sequence set forth in SEQ ID NO: 89.

[0197] In some embodiments, an EEEV replicase-IMP fusion protein comprises PR8 NS1 fused to a terminal of an EEEV replicase (e.g., linked to the C-terminal and / or N-terminal of the EEEV replicase). In some embodiments, an EEEV replicase-IMP fusion protein comprises a replicase joined to PR8 NS1 via a linker (e.g., a 2A linker). In some embodiments, an EEEV replicase-IMP fusion protein is an EEEV nsPl-nsP2-nsP3-nsP4-linker-PR8 NS1 replicase-IMP fusion protein. In some embodiments, an EEEV replicase-IMP fusion protein is a PR8 NS1-linker-EEEV nsPl-nsP2-nsP3-nsP4 replicase-IMP fusion protein. In some embodiments, the linker is a 2A peptide. In some embodiments, an EEEV replicase-IMP fusion protein comprises a PR8 NS1 comprising the amino acid sequence set forth in SEQ ID NO: 103; a T2A linker comprising the amino acid sequence set forth in SEQ ID NO: 107, and an EEEV replicase comprising the amino acid sequence set forth in SEQ ID NO: 83 or 117. In some embodiments, an EEEV replicase-IMP fusion protein consists essentially of a PR8 NS1 comprising the amino acid sequence set forth in SEQ ID NO: 103; a T2A linker comprising the amino acid sequence set forth in SEQ ID NO: 107, and an EEEV replicase comprising the amino acid sequence set forth in SEQ ID NO: 83 or 117.

[0198] In some embodiments, an EEEV replicase-IMP fusion protein is a PR8 NS1-T2A-EEEV nsPl-nsP2-nsP3-nsP4 replicase-IMP fusion protein. In some embodiments, an EEEV replicase-IMP fusion protein comprises, from N-terminal to C-terminal, a PR8 NS1 comprising the amino acid sequence set forth in SEQ ID NO: 103; a T2A linker comprising the amino acid sequence set forth in SEQ ID NO: 107, and an EEEV replicase comprising the amino acid sequence set forth in SEQ ID NO: 83 or 117. In some embodiments, an EEEV replicase-IMP fusion protein is EEEV nsPl-nsP2-nsP3-nsP4-T2A-PR8 NS1 replicase-IMP fusion protein. In some embodiments, an EEEV replicase-IMP fusion protein comprises, from N-terminal to C-terminal, an EEEV replicase comprising the amino acid sequence set forth in SEQ ID NO: 83 or 117; a T2A linker comprising the amino acid sequence set forth in SEQ ID NO: 107; and a PR8 NS1 comprising the amino acid sequence set forth in SEQ ID NO: 103.

[0199] In some embodiments, an EEEV replicase-IMP fusion protein comprises an EEEV replicase (e.g., EEEV nsPl, EEEV nsP2, EEEV nsP3, and EEEV nsP4) and a TOSVNSs. In some embodiments, an EEEV replicase-IMP fusion protein comprises TOSVNSs inserted between domains of the EEEV replicase. In some embodiments, the EEEV replicase-IMP fusion protein comprises EEEV nsPl-nsP2-nsP3-3 / 4J-TOSVNSs-nsP4. In some embodiments, an

[0200] 12673398.1 EEEV replicase-IMP fusion protein comprises an EEEV nsPl-nsP2 fusion protein comprising the amino acid sequence set forth in SEQ ID NO: 86; an EEEV nsP3 domain comprising the amino acid sequence set forth in SEQ ID NO: 87 or 88; an EEEV 3 / 4J comprising the amino acid sequence set forth in SEQ ID NO: 112 or SEQ ID NO: 113; a TOSV NSs comprising the amino acid sequence set forth in SEQ ID NO: 104; and an EEEV nsP4 domain comprising the amino acid sequence set forth in SEQ ID NO: 89. In some embodiments, an EEEV replicase-IMP fusion protein consists essentially of an EEEV nsPl-nsP2 fusion protein comprising the amino acid sequence set forth in SEQ ID NO: 86; an EEEV nsP3 domain comprising the amino acid sequence set forth in SEQ ID NO: 87 or 88; an EEEV 3 / 4J comprising the amino acid sequence set forth in SEQ ID NO: 112 or SEQ ID NO: 113; a TOSV NSs comprising the amino acid sequence set forth in SEQ ID NO: 104; and an EEEV nsP4 domain comprising the amino acid sequence set forth in SEQ ID NO: 89.

[0201] In some embodiments, the EEEV replicase-IMP fusion protein comprises TOSV NSs flanked by 3 / 4J and a 3 / 4J duplicate. In some embodiments, the EEEV replicase-IMP fusion protein comprises TOSV NSs flanked by 3 / 4J and a 3 / 4J duplicate. In some embodiments, an EEEV replicase-IMP fusion protein comprises an EEEV nsPl-nsP2 fusion protein comprising the amino acid sequence set forth in SEQ ID NO: 86; an EEEV nsP3 domain comprising the amino acid sequence set forth in SEQ ID NO: 87 or 88; a first EEEV 3 / 4J comprising the amino acid sequence set forth in SEQ ID NO: 112 or SEQ ID NO: 113; a TOSV NSs comprising the amino acid sequence set forth in SEQ ID NO: 104; a second EEEV 3 / 4J comprising the amino acid sequence set forth in SEQ ID NO: 112 or SEQ ID NO: 113; and an EEEV nsP4 domain comprising the amino acid sequence set forth in SEQ ID NO: 89. In some embodiments, an EEEV replicase-IMP fusion protein consists essentially of an EEEV nsPl-nsP2 fusion protein comprising the amino acid sequence set forth in SEQ ID NO: 86; an EEEV nsP3 domain comprising the amino acid sequence set forth in SEQ ID NO: 87 or 88; a first EEEV 3 / 4J comprising the amino acid sequence set forth in SEQ ID NO: 112 or SEQ ID NO: 113; a TOSV NSs comprising the amino acid sequence set forth in SEQ ID NO: 104; a second EEEV 3 / 4J comprising the amino acid sequence set forth in SEQ ID NO: 112 or SEQ ID NO: 113; and an EEEV nsP4 domain comprising the amino acid sequence set forth in SEQ ID NO: 89.

[0202] In some embodiments, an EEEV replicase-IMP fusion protein comprises TOSV NSs fused to a terminal of an EEEV replicase (e.g., linked to the C-terminal and / or N-terminal of the EEEV replicase). In some embodiments, an EEEV replicase-IMP fusion protein comprises a replicase joined to TOSV NSs via a linker (e.g., a 2A linker). In some embodiments, an EEEV

[0203] 12673398.1 replicase-IMP fusion protein is an EEEV nsPl-nsP2-nsP3-nsP4-linker-TOSV NSs replicase-IMP fusion protein. In some embodiments, an EEEV replicase-IMP fusion protein is a TOSV NSs-linker-EEEV nsPl-nsP2-nsP3-nsP4 replicase-IMP fusion protein. In some embodiments, the linker is a 2A peptide. In some embodiments, an EEEV replicase-IMP fusion protein comprises: a TOSV NSs comprising the amino acid sequence set forth in SEQ ID NO: 104; a T2A linker comprising the amino acid sequence set forth in SEQ ID NO: 107, and an EEEV replicase comprising the amino acid sequence set forth in SEQ ID NO: 83 or 117. In some embodiments, an EEEV replicase-IMP fusion protein consists essentially of: a TOSV NSs comprising the amino acid sequence set forth in SEQ ID NO: 104; a T2A linker comprising the amino acid sequence set forth in SEQ ID NO: 107, and an EEEV replicase comprising the amino acid sequence set forth in SEQ ID NO: 83 or 117.

[0204] In some embodiments, an EEEV replicase-IMP fusion protein is a TOSVNSs-T2A-EEEV nsPl-nsP2-nsP3-nsP4 replicase-IMP fusion protein. In some embodiments, an EEEV replicase-IMP fusion protein comprises, from N-terminal to C-terminal, a TOSV NSs comprising the amino acid sequence set forth in SEQ ID NO: 104; a T2A linker comprising the amino acid sequence set forth in SEQ ID NO: 107, and an EEEV replicase comprising the amino acid sequence set forth in SEQ ID NO: 83 or 117. In some embodiments, an EEEV replicase-IMP fusion protein is EEEV nsPl-nsP2-nsP3-nsP4-T2A-TOSV NSs replicase-IMP fusion protein. In some embodiments, an EEEV replicase-IMP fusion protein comprises, from N-terminal to C-terminal, an EEEV replicase comprising the amino acid sequence set forth in SEQ ID NO: 83 or 117; a T2A linker comprising the amino acid sequence set forth in SEQ ID NO: 107; and a TOSV NSs comprising the amino acid sequence set forth in SEQ ID NO: 104.

[0205] In some embodiments, an EEEV replicase-IMP fusion protein comprises an EEEV replicase (e.g., EEEV nsPl, EEEV nsP2, EEEV nsP3, and EEEV nsP4) and an EMCV Lpro. In some embodiments, an EEEV replicase-IMP fusion protein comprises EMCV Lpro inserted between domains of the EEEV replicase. In some embodiments, the EEEV replicase-IMP fusion protein comprises EEEV nsPl-nsP2-nsP3-3 / 4J-EMCV Lpro-nsP4. In some embodiments, an EEEV replicase-IMP fusion protein comprises an EEEV nsPl-nsP2 fusion protein comprising the amino acid sequence set forth in SEQ ID NO: 86; an EEEV nsP3 domain comprising the amino acid sequence set forth in SEQ ID NO: 87 or 88; an EEEV 3 / 4J comprising the amino acid sequence set forth in SEQ ID NO: 112 or SEQ ID NO: 113; an EMCV Lpro comprising the amino acid sequence set forth in SEQ ID NO: 105; and an EEEV nsP4 domain comprising the amino acid sequence set forth in SEQ ID NO: 89. In some embodiments, an EEEV replicase-

[0206] 12673398.1 IMP fusion protein consists essentially of an EEEV nsPl-nsP2 fusion protein comprising the amino acid sequence set forth in SEQ ID NO: 86; an EEEV nsP3 domain comprising the amino acid sequence set forth in SEQ ID NO: 87 or 88; an EEEV 3 / 4J comprising the amino acid sequence set forth in SEQ ID NO: 112 or SEQ ID NO: 113; an EMCV Lpro comprising the amino acid sequence set forth in SEQ ID NO: 105; and an EEEV nsP4 domain comprising the amino acid sequence set forth in SEQ ID NO: 89.

[0207] In some embodiments, the EEEV replicase-IMP fusion protein comprises EMCV Lpro flanked by 3 / 4J and a 3 / 4J duplicate. In some embodiments, the EEEV replicase-IMP fusion protein comprises EMCV Lpro flanked by 3 / 4J and a 3 / 4J duplicate. In some embodiments, an EEEV replicase-IMP fusion protein comprises an EEEV nsPl-nsP2 fusion protein comprising the amino acid sequence set forth in SEQ ID NO: 86; an EEEV nsP3 domain comprising the amino acid sequence set forth in SEQ ID NO: 87 or 88; a first EEEV 3 / 4J comprising the amino acid sequence set forth in SEQ ID NO: 112 or SEQ ID NO: 113; an EMCV Lpro comprising the amino acid sequence set forth in SEQ ID NO: 105; a second EEEV 3 / 4J comprising the amino acid sequence set forth in SEQ ID NO: 112 or SEQ ID NO: 113; and an EEEV nsP4 domain comprising the amino acid sequence set forth in SEQ ID NO: 89. In some embodiments, an EEEV replicase-IMP fusion protein consists essentially of an EEEV nsPl-nsP2 fusion protein comprising the amino acid sequence set forth in SEQ ID NO: 86; an EEEV nsP3 domain comprising the amino acid sequence set forth in SEQ ID NO: 87 or 88; a first EEEV 3 / 4J comprising the amino acid sequence set forth in SEQ ID NO: 112 or SEQ ID NO: 113; an EMCV Lpro comprising the amino acid sequence set forth in SEQ ID NO: 105; a second EEEV 3 / 4J comprising the amino acid sequence set forth in SEQ ID NO: 112 or SEQ ID NO: 113; and an EEEV nsP4 domain comprising the amino acid sequence set forth in SEQ ID NO: 89.

[0208] In some embodiments, an EEEV replicase-IMP fusion protein comprises an EMCV Lpro fused to a terminal of an EEEV replicase (e.g., linked to the C-terminal and / or N-terminal of the EEEV replicase). In some embodiments, an EEEV replicase-IMP fusion protein comprises a replicase joined to EMCV Lpro via a linker (e.g., a 2A linker). In some embodiments, an EEEV replicase-IMP fusion protein is an EEEV nsPl-nsP2-nsP3-nsP4-linker-EMCV Lpro replicase-IMP fusion protein. In some embodiments, an EEEV replicase-IMP fusion protein is a EMCV Lpro-linker-EEEV nsPl-nsP2-nsP3-nsP4 replicase-IMP fusion protein. In some embodiments, the linker is a 2A peptide. In some embodiments, an EEEV replicase-IMP fusion protein comprises: an EMCV Lpro comprising the amino acid sequence set forth in SEQ ID NO: 105; a T2A linker comprising the amino acid sequence set forth in SEQ ID NO: 107, and an EEEV

[0209] 12673398.1 replicase comprising the amino acid sequence set forth in SEQ ID NO: 83 or 117. In some embodiments, an EEEV replicase-IMP fusion protein consists essentially of: an EMCV Lpro comprising the amino acid sequence set forth in SEQ ID NO: 105; a T2A linker comprising the amino acid sequence set forth in SEQ ID NO: 107, and an EEEV replicase comprising the amino acid sequence set forth in SEQ ID NO: 83 or 117.

[0210] In some embodiments, an EEEV replicase-IMP fusion protein is an EMCV Lpro-T2A-EEEV nsPl-nsP2-nsP3-nsP4 replicase-IMP fusion protein. In some embodiments, an EEEV replicase-IMP fusion protein comprises, from N-terminal to C-terminal, an EMCV Lpro comprising the amino acid sequence set forth in SEQ ID NO: 105; a T2A linker comprising the amino acid sequence set forth in SEQ ID NO: 107, and an EEEV replicase comprising the amino acid sequence set forth in SEQ ID NO: 83 or 117. In some embodiments, an EEEV replicase-IMP fusion protein is EEEV nsPl-nsP2-nsP3-nsP4-T2A-EMCV Lpro replicase-IMP fusion protein. In some embodiments, an EEEV replicase-IMP fusion protein comprises, from N-terminal to C-terminal, an EEEV replicase comprising the amino acid sequence set forth in SEQ ID NO: 83 or 117; a T2A linker comprising the amino acid sequence set forth in SEQ ID NO: 107; and an EMCV Lpro comprising the amino acid sequence set forth in SEQ ID NO: 105.

[0211] In some embodiments, an EEEV replicase-IMP fusion protein comprises an EEEV replicase, PR8 NS1, and TOSVNSs. In some embodiments, an EEEV replicase-IMP fusion protein comprises PR8 NS1 fused to a first terminal of the EEEV replicase and TOSV NSs fused to a second terminal of the EEEV replicase. In some embodiments, an EEEV replicase-IMP fusion protein comprises PR8 NS1 fused to the C-terminal of the EEEV replicase and TOSV NSs fused to the N-terminal of the EEEV replicase. In some embodiments, an EEEV replicase-IMP fusion protein comprises PR8 NS1 fused to the N-terminal of the EEEV replicase and TOSV NSs fused to the C-terminal of the EEEV replicase. In some embodiments, an EEEV replicase-IMP fusion protein comprises an EEEV replicase joined to PR8 NS1 via a first linker and TOSV NSs via a second linker. In some embodiments, an EEEV replicase-IMP fusion protein comprises PR8 NS1 joined to the C-terminal of the EEEV replicase via a first linker and TOSV NSs joined to the N-terminal of the EEEV replicase via a second linker. In some embodiments, an EEEV replicase-IMP fusion protein comprises PR8 NS1 joined to the N-terminal of the EEEV replicase via a first linker and TOSV NSs joined to the C-terminal of the EEEV replicase via a second linker. In some embodiments, the first linker and second linker are different types of linkers (e.g., the first linker is an IRES element, the second linker is a 2A peptide). In some embodiments, the first linker and second linker are the same type of linker

[0212] 12673398.1 (e.g., a 2 A peptide linker). In some embodiments, the first linker and second linker are the same linker (e.g., the first linker and the second linker are both T2A linkers). In some embodiments, an EEEV replicase-IMP fusion protein comprises, a TOSV NSs comprising the amino acid sequence set forth in SEQ ID NO: 104; a first T2A linker comprising the amino acid sequence set forth in SEQ ID NO: 107; an EEEV replicase comprising the amino acid sequence set forth in SEQ ID NO: 83 or 117; a second T2A linker comprising the amino acid sequence set forth in SEQ ID NO: 107; and a PR8 NS1 comprising the amino acid sequence set forth in SEQ ID NO:103.

[0213] In some embodiments, an EEEV replicase-IMP fusion protein comprises, from N-terminal to C-terminal: PR8 NS 1 -linker 1 -EEEV nsPl-nsP2-nsP3-nsP4-linker2-TOSVNSs; wherein linkerl is a first linker and linker2 is a second linker. In some embodiments, an EEEV replicase-IMP fusion protein comprises, from N-terminal to C-terminal: TOSVNSs-linkerl-EEEV nsPl-nsP2-nsP3-nsP4-linker2-PR8 NS1; wherein linkerl is a first linker and linker2 is a second linker. In some embodiments, an EEEV replicase-IMP fusion protein comprises, from N-terminal to C-terminal: PR8 NS1-T2A-EEEV nsPl-nsP2-nsP3-nsP4-T2A-TOSVNSs. In some embodiments, an EEEV replicase-IMP fusion protein comprises, from N-terminal to C-terminal: TOSV NSs-T2A-EEEV nsPl-nsP2-nsP3-nsP4-T2A-PR8 NS1. In some embodiments, an EEEV replicase-IMP fusion protein comprises, from N-terminal to C-terminal, a TOSV NSs comprising the amino acid sequence set forth in SEQ ID NO: 104; a first T2A linker comprising the amino acid sequence set forth in SEQ ID NO: 107; an EEEV replicase comprising the amino acid sequence set forth in SEQ ID NO: 83 or 117; a second T2A linker comprising the amino acid sequence set forth in SEQ ID NO: 107; and a PR8 NS1 comprising the amino acid sequence set forth in SEQ ID NO: 103. In some embodiments, an EEEV replicase-IMP fusion protein comprises, from N-terminal to C-terminal, a PR8 NS1 comprising the amino acid sequence set forth in SEQ ID NO: 103; a first T2A linker comprising the amino acid sequence set forth in SEQ ID NO: 107; an EEEV replicase comprising the amino acid sequence set forth in SEQ ID NO: 83 or 117; a second T2A linker comprising the amino acid sequence set forth in SEQ ID NO: 107; and a TOSV NSs comprising the amino acid sequence set forth in SEQ ID NO: 104.

[0214] In some embodiments, an EEEV replicase-IMP fusion protein comprises an EEEV replicase, PR8 NS1, and EMCV Lpro. In some embodiments, an EEEV replicase-IMP fusion protein comprises PR8 NS1 fused to a first terminal of the EEEV replicase and EMCV Lpro fused to a second terminal of the EEEV replicase. In some embodiments, an EEEV replicase-IMP fusion protein comprises PR8 NS1 fused to the C-terminal of the EEEV replicase and

[0215] 12673398.1 - SO-

[0216] EMCV Lpro fused to the N-terminal of the EEEV replicase. In some embodiments, an EEEV replicase-IMP fusion protein comprises PR8 NS1 fused to the N-terminal of the EEEV replicase and EMCV Lpro fused to the C-terminal of the EEEV replicase. In some embodiments, an EEEV replicase-IMP fusion protein comprises an EEEV replicase joined to PR8 NS1 via a first linker and EMCV Lpro via a second linker. In some embodiments, an EEEV replicase-IMP fusion protein comprises PR8 NS1 joined to the C-terminal of the EEEV replicase via a first linker and EMCV Lpro joined to the N-terminal of the EEEV replicase via a second linker. In some embodiments, an EEEV replicase-IMP fusion protein comprises PR8 NS1 joined to the N-terminal of the EEEV replicase via a first linker and EMCV Lpro joined to the C-terminal of the EEEV replicase via a second linker. In some embodiments, the first linker and second linker are different types of linkers (e.g., the first linker is an IRES element, the second linker is a 2A peptide). In some embodiments, the first linker and second linker are the same type of linker (e.g., a 2 A peptide linker). In some embodiments, the first linker and second linker are the same linker (e.g., the first linker and the second linker are both T2A linkers). In some embodiments, an EEEV replicase-IMP fusion protein comprises, an EMCV Lpro comprising the amino acid sequence set forth in SEQ ID NO: 105; a first T2A linker comprising the amino acid sequence set forth in SEQ ID NO: 107; an EEEV replicase comprising the amino acid sequence set forth in SEQ ID NO: 83 or 117; a second T2A linker comprising the amino acid sequence set forth in SEQ ID NO: 107; and a PR8 NS1 comprising the amino acid sequence set forth in SEQ ID NO:103.

[0217] In some embodiments, an EEEV replicase-IMP fusion protein comprises, from N-terminal to C-terminal: PR8 NS 1 -linker 1 -EEEV nsPl-nsP2-nsP3-nsP4-linker2-EMCV-Lpro; wherein linkerl is a first linker and linker2 is a second linker. In some embodiments, an EEEV replicase-IMP fusion protein comprises, from N-terminal to C-terminal: EMCV-Lpro-linkerl-EEEV nsPl-nsP2-nsP3-nsP4-linker2-PR8 NS1; wherein linkerl is a first linker and linker2 is a second linker. In some embodiments, an EEEV replicase-IMP fusion protein comprises, from N-terminal to C-terminal: PR8 NS1-T2A-EEEV nsPl-nsP2-nsP3-nsP4-T2A-EMCV-Lpro. In some embodiments, an EEEV replicase-IMP fusion protein comprises, from N-terminal to C-terminal: EMCV-Lpro-T2A-EEEV nsPl-nsP2-nsP3-nsP4-T2A-PR8 NS1. In some embodiments, an EEEV replicase-IMP fusion protein comprises, from N-terminal to C-terminal, an EMCV Lpro comprising the amino acid sequence set forth in SEQ ID NO: 105; a first T2A linker comprising the amino acid sequence set forth in SEQ ID NO: 107; an EEEV replicase comprising the amino acid sequence set forth in SEQ ID NO: 83 or 117; a second T2A linker comprising the amino

[0218] 12673398 1 - SI-

[0219] acid sequence set forth in SEQ ID NO: 107; and a PR8 NS1 comprising the amino acid sequence set forth in SEQ ID NO: 103. In some embodiments, an EEEV replicase-IMP fusion protein comprises, from N-terminal to C-terminal, a PR8 NS1 comprising the amino acid sequence set forth in SEQ ID NO: 103; a first T2A linker comprising the amino acid sequence set forth in SEQ ID NO: 107; an EEEV replicase comprising the amino acid sequence set forth in SEQ ID NO: 83 or 117; a second T2A linker comprising the amino acid sequence set forth in SEQ ID NO: 107; and an EMCV Lpro comprising the amino acid sequence set forth in SEQ ID NO: 105.

[0220] In some embodiments, an EEEV replicase-IMP fusion protein comprises an EEEV replicase, TOSV NSs, and EMCV Lpro. In some embodiments, an EEEV replicase-IMP fusion protein comprises TOSV NSs fused to a first terminal of the EEEV replicase and EMCV Lpro fused to a second terminal of the EEEV replicase. In some embodiments, an EEEV replicase-IMP fusion protein comprises TOSV NSs fused to the C-terminal of the EEEV replicase and EMCV Lpro fused to the N-terminal of the EEEV replicase. In some embodiments, an EEEV replicase-IMP fusion protein comprises TOSV NSs fused to the N-terminal of the EEEV replicase and EMCV Lpro fused to the C-terminal of the EEEV replicase. In some embodiments, an EEEV replicase-IMP fusion protein comprises an EEEV replicase joined to TOSV NSs via a first linker and EMCV Lpro via a second linker. In some embodiments, an EEEV replicase-IMP fusion protein comprises TOSV NSs joined to the C-terminal of the EEEV replicase via a first linker and EMCV Lpro joined to the N-terminal of the EEEV replicase via a second linker. In some embodiments, an EEEV replicase-IMP fusion protein comprises TOSV NSs joined to the N-terminal of the EEEV replicase via a first linker and EMCV Lpro joined to the C-terminal of the EEEV replicase via a second linker. In some embodiments, the first linker and second linker are different types of linkers (e.g., the first linker is an IRES element, the second linker is a 2A peptide). In some embodiments, the first linker and second linker are the same type of linker (e.g., a 2A peptide linker). In some embodiments, the first linker and second linker are the same linker (e.g., the first linker and the second linker are both T2A linkers). In some embodiments, an EEEV replicase-IMP fusion protein comprises, a EMCV Lpro comprising the amino acid sequence set forth in SEQ ID NO: 105; a first T2A linker comprising the amino acid sequence set forth in SEQ ID NO: 107; an EEEV replicase comprising the amino acid sequence set forth in SEQ ID NO: 83 or 117; a second T2A linker comprising the amino acid sequence set forth in SEQ ID NO: 107; and a TOSV NSs comprising the amino acid sequence set forth in SEQ ID NO: 104.

[0221] 12673398.1 In some embodiments, an EEEV replicase-IMP fusion protein comprises, from N-terminal to C-terminal: TOSVNSs-linkerl-EEEV nsPl-nsP2-nsP3-nsP4-linker2-EMCV-Lpro; wherein linkerl is a first linker and linker2 is a second linker. In some embodiments, an EEEV replicase-IMP fusion protein comprises, from N-terminal to C-terminal: EMCV-Lpro-linkerl-EEEV nsPl-nsP2-nsP3-nsP4-linker2-TOSVNSs; wherein linkerl is a first linker and linker2 is a second linker. In some embodiments, an EEEV replicase-IMP fusion protein comprises, from N-terminal to C-terminal: TOSV NSs-T2A-EEEV nsPl-nsP2-nsP3-nsP4-T2A-EMCV-Lpro. In some embodiments, an EEEV replicase-IMP fusion protein comprises, from N-terminal to C-terminal: EMCV-Lpro-T2A-EEEV nsPl-nsP2-nsP3-nsP4-T2A-TOSVNSs. In some embodiments, an EEEV replicase-IMP fusion protein comprises, from N-terminal to C-terminal, an EMCV Lpro comprising the amino acid sequence set forth in SEQ ID NO: 105; a first T2A linker comprising the amino acid sequence set forth in SEQ ID NO: 107; an EEEV replicase comprising the amino acid sequence set forth in SEQ ID NO: 83 or 117; a second T2A linker comprising the amino acid sequence set forth in SEQ ID NO: 107; and a TOSV NSs comprising the amino acid sequence set forth in SEQ ID NO: 104. In some embodiments, an EEEV replicase-IMP fusion protein comprises, from N-terminal to C-terminal, a TOSV NSs comprising the amino acid sequence set forth in SEQ ID NO: 104; a first T2A linker comprising the amino acid sequence set forth in SEQ ID NO: 107; an EEEV replicase comprising the amino acid sequence set forth in SEQ ID NO: 83 or 117; a second T2A linker comprising the amino acid sequence set forth in SEQ ID NO: 107; and an EMCV Lpro comprising the amino acid sequence set forth in SEQ ID NO: 105.

[0222] Methods of Use

[0223] Provided herein, in some aspects, are methods for amplifying RNA polynucleotides in a subject in trans without inducing substantial cytotoxicity in the liver of the subject. In some embodiments, the method comprises administering to a subject a taRNA described herein. In some embodiments, the method comprises administering to a subject a taRNA comprising a first RNA polynucleotide (e.g., a replicase construct) comprising a nucleic acid encoding an EEEV replicase, and a nucleic acid encoding an IMP; and a second RNA polynucleotide (e.g., a trRNA) comprising a nucleic acid payload or a nucleic acid encoding a payload, and a CSE cognate to the EEEV replicase. In some embodiments, the method comprises administering to a subject a taRNA comprising a first RNA polynucleotide (e.g., a replicase construct) comprising a nucleic acid encoding an EEEV replicase; and a second RNA polynucleotide (e.g., a trRNA)

[0224] 12673398.1 comprising a nucleic acid payload or a nucleic acid encoding a payload, a nucleic acid encoding an IMP, and a CSE cognate to the EEEV replicase. In some embodiments, the method comprises administering to a subject a taRNA comprising: a first RNA polynucleotide (e.g., a replicase construct) comprising a nucleic acid encoding an EEEV replicase and a nucleic acid encoding a first IMP; and a second RNA polynucleotide (e.g., a trRNA) comprising a nucleic acid payload or a nucleic acid encoding a payload, a nucleic acid encoding a second IMP, and a CSE cognate to the EEEV replicase.

[0225] In some embodiments, administration of the taRNA to the subject reduces cytotoxicity in a subject. The term “cytotoxic” or “cytotoxicity” as used herein, refers to an effect of an agent or activity which injures and / or kills a cell. Measures of cytotoxicity may include, for example, necrosis, cell membrane integrity, cell arrest, apoptosis, cellular metabolism, mitochondrial function, oxidative stress, cytoplasmic shrinkage, and nuclear condensation. Cytotoxicity can be measured in vitro or in vivo by a number of methods known in the art. Examples of suitable assays for determining general cytotoxicity in vitro are described by Tabernilla, A., et al. “In Vitro Liver Toxicity Testing of Chemicals: A Pragmatic Approach.” International Journal of Molecular Sciences 22(9) (2021): 5038, Adan, A, et al. “Cell proliferation and cytotoxicity assays.” Current pharmaceutical biotechnology 17.14 (2016): 1213-1221 and Istifli, E. S., et al. “ division, cytotoxicity, and the assays used in the detection of cytotoxicity.” Cytotoxicitydefinition, identification, and cytotoxic compounds 1 (2019).

[0226] In some embodiments, a method provided herein reduces cytotoxicity in the liver (i.e., hepatotoxicity) of a subject. Cytotoxicity in the liver, also known as hepatotoxicity, can be measured by assessing liver-specific markers of injury, for example, elevation of liver enzymes in the blood of a subject above or below a normal baseline (see Lala et al. “Liver function tests.” StatPearls (2023)). Examples of liver enzymes which may be indicative of hepatotoxicity include alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), albumin and total protein, gamma-glutamyl transferase (GGT), total bilirubin (TB), and L-lactate dehydrogenase (LD). Normal baseline values of liver enzymes may vary depending on subject characteristics and history, but baseline levels and deviations thereon can be determined by trained medical professionals.

[0227] “Substantial cytotoxicity in the liver,” can be determined using one or more assay for liver cytotoxicity (e.g., as described herein). In some embodiments, substantial cytotoxicity in the liver is indicated by higher-than-normal levels of one or more liver enzymes in the subject (e.g., higher than normal liver enzymes described above). In some embodiments, substantial

[0228] 12673398.1 cytotoxicity in the liver is indicated by at least 1.5 times least 2 times (e.g., at least 3 times, at least 4 times, at least 5 times, or at least 10 times) higher than normal levels of one or more liver enzymes in the subject. In some embodiments, an aspartate aminotransferase (AST) level in a subject exceeding 33 units / liter of blood (e.g., exceeding 50 units / liter of blood, exceeding 100 units / liter of blood, exceeding 250 units / liter of blood, exceeding 500 units / liter of blood, or exceeding 100 units / liter of blood) indicates substantial cytotoxicity in the liver. In some embodiments, an alanine aminotransferase (ALT) level in a subject exceeding 56 units / liter of blood (e.g., exceeding 100 units / liter of blood, exceeding 250 units / liter of blood, exceeding 500 units / liter of blood, or exceeding 100 units / liter of blood) indicates substantial cytotoxicity in the liver. In some embodiments, a bilirubin level in a subject exceeding 1.3 mg / deciliter of blood (e.g., exceeding 2 mg / deciliter of blood, 5 mg / deciliter of blood, 7.5 mg / deciliter of blood, 10 mg / deciliter of blood, 15 mg / deciliter of blood, 20 mg / deciliter of blood, or 30 mg / deciliter of blood) indicates substantial cytotoxicity in the liver.

[0229] In some embodiments, hepatotoxicity of a taRNA comprising a replicase construct encoding an EEEV replicase is reduced in a subject compared to a taRNA encoding a different replicase (e.g., SFV replicase). In some embodiments, hepatotoxicity of a taRNA is reduced in a subject to whom the taRNA has been administered by at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% compared to a subject to whom a reference taRNA is administered (e.g., a taRNA encoding SFV replicase). In some embodiments, the hepatotoxicity is determined by measuring ALT, AST, ALP, albumin and total protein, GGT, TB, LD, or a combination thereof.

[0230] Also provided herein, in some aspects, are methods for amplifying RNA polynucleotides in a subject in trans without inducing substantial immunogenicity in a subject. In some embodiments, the method comprises administering to the subject a taRNA comprising: a first RNA polynucleotide (e.g., a replicase construct) comprising a nucleic acid encoding an EEEV replicase, and a nucleic acid encoding an IMP; and a second RNA polynucleotide (e.g., a trRNA) comprising a nucleic acid payload or a nucleic acid encoding a payload, and a CSE cognate to the EEEV replicase. In some embodiments, reducing the immunogenicity of the taRNA comprises reducing expression of one or more interferon (IFN), chemokine, and / or interleukin that are associated with an immune response of the cell (e.g., the non-target cell) and / or the subject to the taRNA as compared to a reference taRNA (e.g., a taRNA encoding SFV

[0231] 12673398.1 replicase). In some embodiments, reducing the immunogenicity of the taRNA comprises reducing expression of one or more IFNs as compared to a reference taRNA (e.g., a taRNA encoding SFV replicase). In some embodiments, the interferon is IFN-a, IFN-P, and / or IFN-y. In some embodiments, reducing the immunogenicity comprises significantly decreasing expression of IFN-a as compared to a reference taRNA (e.g., a taRNA encoding SFV replicase). In some embodiments, reducing the immunogenicity comprises significantly decreasing expression of IFN- P as compared to a reference taRNA (e.g., a taRNA encoding SFV replicase). In some embodiments, reducing the immunogenicity does not comprise significantly decreasing expression of IFN-y as compared to a reference taRNA (e.g., a taRNA encoding SFV replicase).

[0232] In some embodiments, reducing the immunogenicity of the taRNA comprises reducing expression of one or more chemokines as compared to a reference taRNA (e.g., a taRNA encoding SFV replicase). In some embodiments, the chemokine is keratinocyte chemoattractant (KC) / human growth-regulated oncogene (GRO) chemokine, monocyte chemoattractant protein-1 (MCP-1), interferon gamma-induced protein 10 (IP- 10), or macrophage inflammatory protein la (MIP-la). In some embodiments, the chemokine is an interleukin. In some embodiments, the interleukin is IL-6. IL-6, also known as B-cell stimulatory factor-2 (BSF-2) and interferon beta-2, is critical for differentiation of B cells and several other cell types, and acute phase reactants in hepatocytes.

[0233] In some embodiments, immunogenicity of a taRNA described herein (e.g., a taRNA comprising a replicase construct encoding an EEEV replicase, a trRNA encoding a payload, and a nucleic acid encoding an IMP) is reduced in a subject. In some embodiments, immunogenicity of a taRNA is reduced in a subject to whom the taRNA has been administered by at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% compared to a subject to whom a reference taRNA is administered.

[0234] The skilled artisan will recognize that reduction of cytotoxicity and immunogenicity may, in some embodiments, also increase expression of a payload encoded by a taRNA. Host immune responses to non-self-nucleic acids typically result in degradation or instability of the non-self-nucleic acids in a cell. By reducing cytotoxicity (e.g., hepatotoxicity) and / or immunogenicity of a taRNA described herein (e.g., using methods described herein), expression of the payload of said taRNA may be increased relative to expression of a reference taRNA. In some embodiments, expression of a taRNA is increased in a subject to whom the taRNA has

[0235] 12673398.1 been administered by at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 150-fold, or at least 200-fold compared to a subject to whom a reference taRNA is administered.

[0236] In some embodiments, this disclosure describes a cell comprising one or more of any of the taRNAs described herein. In some aspects, the taRNAs described herein may be used to express a payload in a mammalian cell. Mammalian cells may be derived from any mammal, including, but not limited to, mice, hamsters, pigs, cows, sheep, goats, horses, and primates, including humans. In some embodiments, the taRNA described herein are used to express payloads in isolated cells from established cell lines generally known in the art, such as 3T3, A549, BHK21, C127, CHO, HeLa, HEK, HT-1080, Huh7, Jurkat, NSO, PER. C6, Sp2 / 0, Vero, and derivatives thereof. In some embodiments, the taRNA described herein are used to express payloads in cells isolated from a subject (e.g., immune cells).

[0237] In some embodiments, the taRNAs described herein are transfected into a cell. The term “transfection” refers to the process by which cells uptake foreign polynucleotides into the cytoplasm, in the absence of viral vectors. Non-limiting examples of transfection include electroporation, heat shock, liposome-mediated delivery, nanoparticle-mediated delivery, microinjections, sonoporation, photoporation, magnetofection, hydroporation, biolistics, continuous infusion, impalefection, and any technique known to those of ordinary skill in the art. In some embodiments, a cell transfected with a taRNA is transfected with the replicase construct and trans replicon construct concurrently (i.e., at the same time). In some embodiments, a cell transfected with a taRNA is transfected with the replicase construct and trans replicon construct sequentially; for example, the replicase construct may be transfected at a first time point and a trans replicon construct transfected at a second, later, time point. Once one or more foreign polynucleotides have entered the cytoplasm of a cell, the polynucleotides may be expressed by the cell. In some embodiments, a cell transfected with a taRNA expresses the replicase encoded by the replicase construct. In some embodiments, a cell transfected with a taRNA expresses (i.e., translates) the payload encoded by the trans replicon construct or its replicants.

[0238] In some aspects, the taRNAs described herein may be used to express a payload in a cell (e.g., a cell of subject). taRNAs described herein may be administered to a subject using any method known to those of ordinary skill in the art, such as injection (e.g., intravenous, intraocular, intravitreal, intramuscular, intradermal, intracardiac, intraperitoneal, and subcutaneous). In some embodiments, the taRNAs described herein are formulated as a

[0239] 12673398.1 pharmaceutical composition. A “pharmaceutical composition” refers to a composition comprising taRNAs formulated with one or more pharmaceutically acceptable excipients. In some embodiments, taRNAs are formulated in a carrier (e.g., lipidoid, liposome, lipid nanoparticle, polymer, lipoplex, ligand). In some embodiments, one or more cells transfected with taRNAs may be administered to the subject.

[0240] In some embodiments, taRNAs are administered to mammalian subjects. Non-limiting examples of mammalian subjects include mice, hamsters, pigs, cows, sheep, goats, horses, and primates (e.g., humans, non-human primates).

[0241] In some embodiments, taRNAs are administered to human subjects.

[0242] In some embodiments, taRNAs are expressed in a cell-free system, for example, using in vitro transcription.

[0243] 12673398.1 ILLUSTRATIVE SEQUENCES

[0244] Table 1. Illustrative 5' UTR Sequences

[0245] UTR Rational Name SEQID NO: 5' HAG UTR

[0246] DNA AGGAGAATAAACTAGTATTCTTCTGGTCACAGACTCAGAGAGAACCCGCCACC 1 Sequence

[0247] RNA AGGAGAAUAAACUAGUAUU CUU CU GGU CACAGACU CAGAGAGAAC C C GC GAG C 42 Sequence

[0248] 5' SINVUTR

[0249] DNA ATTGACGGCGTAGTACACACTATTGAATCAAACAGCCGACCAATTGCACTACCATCAC 2 Sequence AAC GGAGAAGC CAGT AGT AAAC GT AGAC GT AGAC C C C CAGAGT CCGTTTGTC GT GCAA CTGCAAAAAAGCTTCCCGCAATTTGAGGTAGTAGCACAGGAGGTGACTCCAAATCACC ATCCTAATCCCAGAGCATTTTCGCATCTGGGCAGTAAACTAATCGAGCTGGAGGTTCC TACCACAGCGACGATCTTGGACATAGGCAGCGCACCGGCTCGTAGAACGATAAACCCC TAGTGCCACC RNA AUU GAG GGC GUAGUACACACUAUU GAAU CAAACAGC C GAG CAAUU GCACUAC GAU GAG 43 Sequence AAC G GAGAAG C C AGU AGU AAAC GUAGACGUAGACCCCCAGAGUCCGUUUGUCGUG C AA CUGCAAAAAAGCUUCCCGCAAUUUGAGGUAGUAGCACAGGAGGUGACUCCAAAUCACC AUCCUAAUCCCAGAGCAUUUUCGCAUCUGGGCAGUAAACUAAUCGAGCUGGAGGUUCC UACCACAGCGACGAUCUUGGACAUAGGCAGCGCACCGGCUCGUAGAACGAUAAACCCC UAGUGCCACC

[0250] U3A 5' SINVUTR

[0251] DNA ATAGACGGCGTAGTACACACTATTGAATCAAACAGCCGACCAATTGCACTACCATCAC 3 Sequence AAC GGAGAAGC CAGT AGT AAAC GT AGAC GT AGAC C C C CAGAGT CCGTTTGTC GT GCAA CTGCAAAAAAGCTTCCCGCAATTTGAGGTAGTAGCACAGGAGGTGACTCCAAATCACC ATCCTAATCCCAGAGCATTTTCGCATCTGGGCAGTAAACTAATCGAGCTGGAGGTTCC TACCACAGCGACGATCTTGGACATAGGCAGCGCACCGGCTCGTAGAACGATAAACCCC TAGTGCCACC RNA AU AGAC GGC GUAGUACACACUAUU GAAU C AAAC AG C C GAC CAAUU GCACUAC C AU C AC 44 Sequence AAC G GAGAAG C C AGU AGU AAAC GUAGACGUAGACCCCCAGAGUCCGUUUGUCGUG C AA CUGCAAAAAAGCUUCCCGCAAUUUGAGGUAGUAGCACAGGAGGUGACUCCAAAUCACC AUCCUAAUCCCAGAGCAUUUUCGCAUCUGGGCAGUAAACUAAUCGAGCUGGAGGUUCC UACCACAGCGACGAUCUUGGACAUAGGCAGCGCACCGGCUCGUAGAACGAUAAACCCC UAGUGCCACC

[0252] oeSTR5' SINVUTR

[0253] DNA ATAGAAGATGGCGGCGTAGTACACACTATTGAATCAAACAGCCGACCAATTGCACTAC 4 Sequence CAT CACAAC GGAGAAGC CAGT AGT AAAC GT AGAC GT AGAC C C C CAGAGT CCGTTTGTC GTGCAACTGCAAAAAAGCTTCCCGCAATTTGAGGTAGTAGCACAGGAGGTGACTCCAA ATCACCATCCTAATCCCAGAGCATTTTCGCATCTGGGCAGTAAACTAATCGAGCTGGA GGTTCCTACCACAGCGACGATCTTGGACATAGGCAGCGCACCGGCTCGTAGAACGATA AACCCCTAGTGCCACC RNA AUAGAAGAUGGCGGCGUAGUACACACUAUUGAAUCAAACAGCCGACCAAUUGCACUAC 45 Sequence C AU CACAAC G GAGAAG C C AGU AGU AAAC GUAGACGUAGACCCCCAGAGUCCGUUUGUC GUGCAACUGCAAAAAAGCUUCCCGCAAUUUGAGGUAGUAGCACAGGAGGUGACUCCAA AUCACCAUCCUAAUCCCAGAGCAUUUUCGCAUCUGGGCAGUAAACUAAUCGAGCUGGA GGUUCCUACCACAGCGACGAUCUUGGACAUAGGCAGCGCACCGGCUCGUAGAACGAUA

[0254]

[0255] AACCCCUAGUGCCACC

[0256] Table 2, Illustrative 3' UTR Sequences

[0257] UTR Rational Name SEQID NO: 3' HAG UTR

[0258] DNA GCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTC 5

[0259]

[0260] Sequence CCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC

[0261] 12673398.1 RNA GCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUC 46 Sequence CCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC

[0262] 3' SINVUTR

[0263] DNA CTCGAGGCGGCCGCCACGCAGCGTCTGCATAACTTTTATTATTTCTTTTATTAATCA 6 Sequence ACAAAAT T T T GT T T T T AACAT T T C

[0264] RNA CUCGAGGCGGCCGCCACGCAGCGUCUGCAUAACUUUUAUUAUUUCUUUUAUUAAUCA 47 Sequence ACAAAAUUUUGUUUUUAACAUUUC

[0265] 3' SFVUTR

[0266] DNA CTCGAGGCGGCCGCAGGAGCTTAATTCGACGAATAATTGGATTTTTATTTTATTTTG 7 Sequence CAATTGGTTTTTAATATTTCCA

[0267] RNA CUCGAGGCGGCCGCAGGAGCUUAAUUCGACGAAUAAUUGGAUUUUUAUUUUAUUUUG 48

[0268]

[0269] Sequence CAAUU GGUUUUUAAUAUUU C GA

[0270] Table 3, Illustrative Replicase Sequences

[0271] Replicase Rational Name SEQID NO: Wildtype Eastern Equine Encephalitis (EEEV) Replicase

[0272] DNA ATGGAGAAAGTACACGTAGACTTGGACGCCGACAGCCCCTTCGTGAAGTCCCTGCAGAG 8 Sequence GTGCTTCCCCCACTTCGAGATCGAGGCCACCCAGGTGACCGACAACGACCACGCCAACG CCAGGGCCTTCAGCCACCTGGCCACCAAGCTGATCGAGGGCGAGGTGGACACCGACCAG GTGATCCTGGACATCGGCAGCGCCCCCGTGAGGCACACCCACAGCAAGCACAAGTACCA CTGCATCTGCCCCATGAAGTCCGCCGAGGACCCCGACAGGCTGTACAGGTACGCCGACA AGCTGAGAAAGAGCGACGTGACCGACAAGTGCATCGCCAGCAAGGCCGCCGACCTGCTG ACCGTGATGAGCACCCCCGACGCCGAGACACCCAGCCTGTGCATGCACACCGACAGCAC CTGCAGGTACCACGGCAGCGTGGCCGTGTACCAGGACGTGTACGCCGTGCACGCCCCCA CCAGCATCTACTACCAGGCCCTGAAGGGCGTGAGAACCATCTACTGGATCGGCTTCGAC ACCACCCCCTTCATGTACAAGAACATGGCCGGCGCCTACCCCACCTACAACACCAACTG GGCCGACGAGAGCGTGCTGGAGGCCAGGAACATCGGCCTGGGCAGCAGCGACCTGCACG AGAAGTCCTTCGGCAAGGTGAGCATCATGAGGAAGAAGAAGCTGCAGCCCACCAACAAG GTGATCTTCAGCGTGGGCAGCACCATCTACACCGAGGAGAGGACCCTGCTGAGGAGCTG GCACCTGCCCAACGTGTTCCACCTGAAGGGCAAGACCAGCTTCACCGGCAGGTGCAACA CCATCGTGAGCTGCGAGGGCTACGTGGTGAAGAAGATCACCCTGAGCCCCGGCATCTAC GGCAAGGTGGACAACCTGGCCAGCACCATGCACAGGGAGGGCTTCCTGAGCTGCAAGGT GACCGACACCCTGAGGGGCGAGAGAGTGAGCTTCCCCGTGTGCACCTACGTGCCCGCCA CCCTGTGCGACCAGATGACCGGCATCCTGGCCACCGACGTGAGCGTGGACGACGCCCAG AAGCTGCTGGTGGGCCTGAACCAGAGGATCGTGGTGAACGGCAGGACCCAGAGAAACAC CAACACCATGCAGAACTACCTGCTGCCCGTGGTGGCCCAGGCCTTCAGCAGGTGGGCCA GGGAGCACAGGGCCGACCTGGAGGACGAGAAGGGCCTGGGCGTGAGAGAGAGAAGCCTG GTGATGGGCTGCTGCTGGGCCTTCAAGACCCACAAGATCACCAGCATCTACAAGAGGCC CGGCACCCAGACCATCAAGAAGGTGCCCGCCGTGTTCAACAGCTTCGTGATCCCCCAGC CCACCAGCTACGGCCTGGACATCGGCCTGAGGAGGAGAATCAAGATGCTGTTCGACGCC AAGAAGGCCCCCGCCCCCATCATCACCGAGGCCGACGTGGCCCACCTGAAGGGCCTGCA GGACGAGGCCGAGGCCGTGGCCGAGGCCGAGGCCGTGAGGGCCGCCCTGCCCCCCCTGC TGCCCGAGGTGGACAAGGAGACAGTGGAGGCCGACATCGACCTGATCATGCAGGAGGCC GGCGCCGGCAGCGTGGAGACACCCAGAAGGCACATCAAGGTGACCACCTACCCCGGCGA GGAGATGATCGGCAGCTACGCCGTGCTGAGCCCCCAGGCCGTGCTGAACAGCGAGAAGC TGGCCTGCATCCACCCCCTGGCCGAGCAGGTGCTGGTGATGACCCACAAGGGCAGGGCC GGCAGATACAAGGTGGAGCCCTACCACGGCAGGGTGATCGTGCCCAGCGGCACCGCCAT CCCCATCCCCGACTTCCAGGCCCTGAGCGAGAGCGCCACCATCGTGTTCAACGAGAGGG AGTTCGTGAACAGGTACCTGCACCACATCGCCGTGAACGGCGGCGCCCTGAACACCGAC GAGGAGTACTACAAGGTGGTGAAGTCCACCGAGACAGACAGCGAGTACGTGTTCGACAT CGACGCCAAGAAGTGCGTGAAGAAGGGCGACGCCGGCCCCATGTGCCTGGTGGGCGAGC TGGTGGACCCCCCCTTCCACGAGTTCGCCTACGAGAGCCTGAAAACCAGGCCCGCCGCC CCCCACAAGGTGCCCACCATCGGCGTGTACGGCGTGCCCGGCAGCGGCAAGAGCGGCAT CATCAAGAGCGCCGTGACCAAGAGGGACCTGGTGGTGAGCGCCAAGAAGGAGAACTGCA TGGAGATCATCAAGGACGTGAAGAGGATGAGGGGCATGGACATCGCCGCCAGGACCGTG

[0273]

[0274] GACAGCGTGCTGCTGAACGGCGTGAAGCACAGCGTGGACACCCTGTACATCGACGAGGC

[0275] 12673398.1 CTTCGCCTGCCACGCCGGCACCCTGCTGGCCCTGATCGCCATCGTGAAGCCCAAGAAGG TGGTGCTGTGCGGCGACCCCAAGCAGTGCGGCTTCTTCAACATGATGTGCCTGAAGGTG CACTTCAACCACGAGATCTGCACCGAGGTGTACCACAAGAGCATCAGCAGGAGGTGCAC CAAGACCGTGACCAGCATCGTGAGCACCCTGTTCTACGACAAGAGAATGAGGACCGTGA ACCCCTGCAACGACAAGATCATCATCGACACCACCAGCACCACCAAGCCCCTGAAGGAC GACATCATCCTGACCTGCTTCAGGGGCTGGGTGAAGCAGCTGCAGATCGACTACAAGAA CCACGAGATCATGACCGCCGCCGCCAGCCAGGGCCTGACCAGGAAGGGCGTGTACGCCG TGAGGTACAAGGTGAACGAGAACCCCCTGTACGCCCAGACCAGCGAGCACGTGAACGTG CTGCTGACCAGAACCGAGAAGAGAATCGTGTGGAAAACCCTGGCCGGCGACCCCTGGAT CAAGACCCTGACCGCCAGCTACCCCGGCAACTTCACCGCCACCCTGGAGGAGTGGCAGG CCGAGCACGACGCCATCATGGCCAAGATCCTGGAGACACCCGCCAGCAGCGACGTGTTC CAGAACAAGGTGAACGTGTGCTGGGCCAAGGCCCTGGAGCCCGTGCTGGCCACCGCCAA CATCACCCTGACCAGGAGCCAGTGGGAGACAATCCCCGCCTTCAAGGACGACAAGGCCT ACAGCCCCGAGATGGCCCTGAACTTCTTCTGCACCAGGTTCTTCGGCGTGGACATCGAC AGCGGCCTGTTCAGCGCCCCCACCGTGCCCCTGACCTACACCAACGAGCACTGGGACAA CAGCCCCGGCCCCAACATGTACGGCCTGTGCATGAGGACCGCCAAGGAGCTGGCCAGAA GGTACCCCTGCATCCTGAAGGCCGTGGACACCGGCAGGGTGGCCGACGTGAGGACCGAC ACCATCAAGGACTACAACCCCCTGATCAACGTGGTGCCCCTGAACAGGAGACTGCCCCA CAGCCTGGTGGTGACCCACAGGTACACCGGCAACGGCGACTACAGCCAGCTGGTGACCA AGATGACCGGCAAGACCGTGCTGGTGGTGGGCACCCCCATGAACATCCCCGGCAAGAGG GTGGAGACACTGGGCCCCAGCCCCCAGTGCACCTACAAGGCCGAGCTGGACCTGGGCAT CCCCGCCGCCCTGGGCAAGTACGACATCATCTTCATCAACGTGAGGACCCCCTACAGAC ACCACCACTACCAGCAGTGCGAGGACCACGCCATCCACCACAGCATGCTGACCAGGAAG GCCGTGGACCACCTGAACAAGGGCGGCACCTGCATCGCCCTGGGCTACGGCACCGCCGA CAGAGCCACCGAGAACATCATCAGCGCCGTGGCCAGGAGCTTCAGGTTCAGCAGGGTGT GCCAGCCCAAGTGCGCCTGGGAGAACACCGAGGTGGCCTTCGTGTTCTTCGGCAAGGAC AACGGCAACCACCTGCAGGACCAGGACAGGCTGAGCGTGGTGCTGAACAACATCTACCA GGGCAGCACCCAGCACGAGGCCGGCAGAGCCCCCGCCTACAGGGTGGTGAGGGGCGACA TCACCAAGAGCAACGACGAGGTGATCGTGAACGCCGCCAACAACAAGGGCCAGCCCGGC GGCGGCGTGTGCGGCGCCCTGTACAGGAAGTGGCCCGGCGCCTTCGACAAGCAGCCCGT GGCCACCGGCAAGGCCCACCTGGTGAAGCACAGCCCCAACGTGATCCACGCCGTGGGCC CCAACTTCAGCAGACTGAGCGAGAACGAGGGCGACCAGAAGCTGAGCGAGGTGTACATG GACATCGCCAGGATCATCAACAACGAGAGGTTCACCAAGGTGAGCATCCCCCTGCTGAG CACCGGCATCTACGCCGGCGGCAAGGACAGGGTGATGCAGAGCCTGAACCACCTGTTCA CCGCCATGGACACCACCGACGCCGACATCACCATCTACTGCCTGGACAAGCAGTGGGAG AGCAGGATCAAGGAGGCCATCACCAGGAAGGAGAGCGTGGAGGAGCTGACCGAGGACGA CAGGCCCGTGGACATCGAGCTGGTGAGAGTGCACCCCCTGAGCAGCCTGGCCGGCAGGC CCGGCTACAGCACCACCGAGGGCAAGGTGTACAGCTACCTGGAGGGCACCAGATTCCAC CAGACCGCCAAGGACATCGCCGAGATCTACGCCATGTGGCCCAACAAGCAGGAGGCCAA CGAGCAGATCTGCCTGTACGTGCTGGGCGAGAGCATGAACAGCATCAGAAGCAAGTGCC CCGTGGAGGAGAGCGAGGCCAGCAGCCCCCCCCACACCATCCCCTGCCTGTGCAACTAC GCCATGACCGCCGAGAGGGTGTACAGACTGAGGATGGCCAAGAACGAGCAGTTCGCCGT GTGCAGCAGCTTCCAGCTGCCCAAGTACAGAATCACCGGCGTGCAGAAGATCCAGTGCA GCAAGCCCGTGATCTTCAGCGGCACCGTGCCCCCCGCCATCCACCCCAGAAAGTTCGCC AGCGTGACCGTGGAGGACACCCCCGTGGTGCAGCCCGAGAGGCTGGTGCCCAGGAGGCC CGCCCCCCCCGTGCCCGTGCCCGCCAGAATCCCCAGCCCCCCCTGCACCCCCACCAACG GCAGCACCACCAGCATCCAGAGCCTGGGCGAGGACCAGAGCGCCAGCGCCAGCGGCGGC GCCGAGATCAGCGTGGACCAGGTGAGCCTGTGGAGCATCCCCAGCGCCACCGGCTTCGA CGTGAGGACCAGCAGCAGCCTGAGCCTGGAGCAGCCCACCTTCCCCACCATGGTGGTGG AGGCCGAGATCCACGCCAGCCAGGGCAGCCTGTGGAGCATCCCCAGCATCACCGGCAGC GAAACCAGGGCCCCCAGCCCCCCCAGCCAGGACAGCAGACCCAGCACCCCCAGCGCCAG CGGCAGCCACACCAGCGTGGACCTGATCACCTTCGACAGCGTGGCCGAGATCCTGGAGG ACTTCAGCAGGAGCCCCTTCCAGTTCCTGAGCGAGATCAAGCCCATCCCCGCCCCCAGG ACCAGGGTGACCAACATGAGCAGGAGCGCCGACACCATCAAGCCCATCCCCAAGCCCAG AAAGTGCCAGGTGAAGTACACCCAGCCCCCCGGCGTGGCCAGGGCCATCAGCGCCGCCG AGTTCGACGAGTTCGTGCGGAGGCACTCGAATTGACGGTACGAAGCGGGTGCGTACATT TTCTCATCCGAGACGGGACAAGGGCACCTGCAACAAAAATCTACGCGGCAATGCAAACT CCAGTATCCAATCCTGGAGCGTTCCGTCCATGAGAAATTTTACGCCCCGCGCCTCGATC TCGAGAGGGAGAAGCTGCTGCAGAAGAAGCTGCAGCTGTGCGCCAGCGAGGGCAACAGG

[0276]

[0277] AGCAGATACCAGAGCAGGAAGGTGGAGAACATGAAGGCCATCACCGTGGAGAGGCTGCT

[0278] 12673398.1 GCAGGGCATCGGCAGCTACCTGAGCGCCGAGCCCCAGCCCGTGGAGTGCTACAAGGTGA CCTACCCCGCCCCCATGTACAGCAGCACCGCCAGCAACAGCTTCAGCAGCGCCGAGGTG GCCGTGAAGGTGTGCAACCTGGTGCTGCAGGAGAACTTCCCCACCGTGGCCAGCTACAA CATCACCGACGAGTACGACGCCTACCTGGACATGGTGGACGGCGCCAGCTGCTGCCTGG ACACCGCCACCTTCTGCCCCGCCAAGCTGAGGAGCTTCCCCAAGAAGCACAGCTACCTG AGGCCCGAGATCAGGAGCGCCGTGCCCAGCCCCATCCAGAACACCCTGCAGAACGTGCT GGCCGCCGCCACCAAGAGGAACTGCAACGTGACCCAGATGAGGGAGCTGCCCGTGCTGG ACAGCGCCGCCTTCAACGTGGAGTGCTTCAAGAAGTACGCCTGCAACGACGAGTACTGG GACTTCTACAAGACCAACCCCATCAGGCTGACCGCCGAGAACGTGACCCAGTACGTGAC CAAGCTGAAGGGCCCCAAGGCCGCCGCCCTGTTCGCCAAGACCCACAACCTGCAGCCCC TGCACGAGATCCCCATGGACAGGTTCGTGATGGACCTGAAGAGAGATGTGAAGGTGACC CCCGGCACCAAGCACACCGAGGAGAGGCCCAAGGTGCAGGTGATCCAGGCCGCCGACCC CCTGGCCACCGCCTACCTGTGCGGCATCCACAGGGAGCTGGTGAGGAGGCTGAACGCCG TGCTGCTGCCCAACATCCACACCCTGTTCGACATGAGCGCCGAGGACTTCGACGCCATC ATCGCCGAGCACTTCCAGTTCGGCGACAGCGTGCTGGAGACAGACATCGCCAGCTTCGA CAAGAGCGAGGACGACGCCATCGCCATGAGCGCCCTGATGATCCTGGAGGACCTGGGCG TGGACCAGGCCCTGCTGAACCTGATCGAGGCCGCCTTCGGCAACATCACCAGCGTGCAC CTGCCCACCGGCACCAGGTTCAAGTTCGGCGCCATGATGAAGTCCGGCATGTTCCTGAC CCTGTTCATCAACACCGTGGTGAACATCATGATCGCCAGCAGGGTGCTGAGGGAGAGGC TGACCACCAGCCCCTGCGCCGCCTTCATCGGCGACGACAACATCGTGAAGGGCGTGACC AGCGACGCCCTGATGGCCGAGAGGTGCGCCACCTGGCTGAACATGGAGGTGAAGATCAT CGACGCCGTGGTGGGCGTGAAGGCCCCCTACTTCTGCGGCGGCTTCATCGTGGTGGACC AGATCACCGGCACCGCCTGCAGGGTGGCCGACCCCCTGAAGAGACTGTTCAAGCTGGGC AAGCCCCTGCCCCTGGACGACGACCAGGACGTGGACAGGAGGAGAGCCCTGCACGACGA GGCCGCCAGGTGGAACAGAATCGGCATCACCGAGGAGCTGGTGAAGGCCGTGGAGAGCA GATACGAGGTGAACTACGTGAGCCTGATCATCACCGCCCTGACCACCCTGGCCAGCAGC GTGAGCAACTTCAAGCACATCAGGGGCCACCCCATCACCCTGTACGGC _

[0279] RNA AUGGAGAAAGUACACGUAGACUUGGACGCCGACAGCCCCUUCGUGAAGUCCCUGCAGAG 49 Sequence GUGCUUCCCCCACUUCGAGAUCGAGGCCACCCAGGUGACCGACAACGACCACGCCAACG CCAGGGCCUUCAGCCACCUGGCCACCAAGCUGAUCGAGGGCGAGGUGGACACCGACCAG GUGAUCCUGGACAUCGGCAGCGCCCCCGUGAGGCACACCCACAGCAAGCACAAGUACCA CUGCAUCUGCCCCAUGAAGUCCGCCGAGGACCCCGACAGGCUGUACAGGUACGCCGACA AGCUGAGAAAGAGCGACGUGACCGACAAGUGCAUCGCCAGCAAGGCCGCCGACCUGCUG ACCGUGAUGAGCACCCCCGACGCCGAGACACCCAGCCUGUGCAUGCACACCGACAGCAC CUGCAGGUACCACGGCAGCGUGGCCGUGUACCAGGACGUGUACGCCGUGCACGCCCCCA CCAGCAUCUACUACCAGGCCCUGAAGGGCGUGAGAACCAUCUACUGGAUCGGCUUCGAC ACCACCCCCUUCAUGUACAAGAACAUGGCCGGCGCCUACCCCACCUACAACACCAACUG GGCCGACGAGAGCGUGCUGGAGGCCAGGAACAUCGGCCUGGGCAGCAGCGACCUGCACG AGAAGUCCUUCGGCAAGGUGAGCAUCAUGAGGAAGAAGAAGCUGCAGCCCACCAACAAG GUGAUCUUCAGCGUGGGCAGCACCAUCUACACCGAGGAGAGGACCCUGCUGAGGAGCUG GCACCUGCCCAACGUGUUCCACCUGAAGGGCAAGACCAGCUUCACCGGCAGGUGCAACA CCAUCGUGAGCUGCGAGGGCUACGUGGUGAAGAAGAUCACCCUGAGCCCCGGCAUCUAC GGCAAGGUGGACAACCUGGCCAGCACCAUGCACAGGGAGGGCUUCCUGAGCUGCAAGGU GACCGACACCCUGAGGGGCGAGAGAGUGAGCUUCCCCGUGUGCACCUACGUGCCCGCCA CCCUGUGCGACCAGAUGACCGGCAUCCUGGCCACCGACGUGAGCGUGGACGACGCCCAG AAGCUGCUGGUGGGCCUGAACCAGAGGAUCGUGGUGAACGGCAGGACCCAGAGAAACAC CAACACCAUGCAGAACUACCUGCUGCCCGUGGUGGCCCAGGCCUUCAGCAGGUGGGCCA GGGAGCACAGGGCCGACCUGGAGGACGAGAAGGGCCUGGGCGUGAGAGAGAGAAGCCUG GUGAUGGGCUGCUGCUGGGCCUUCAAGACCCACAAGAUCACCAGCAUCUACAAGAGGCC CGGCACCCAGACCAUCAAGAAGGUGCCCGCCGUGUUCAACAGCUUCGUGAUCCCCCAGC CCACCAGCUACGGCCUGGACAUCGGCCUGAGGAGGAGAAUCAAGAUGCUGUUCGACGCC AAGAAGGCCCCCGCCCCCAUCAUCACCGAGGCCGACGUGGCCCACCUGAAGGGCCUGCA GGACGAGGCCGAGGCCGUGGCCGAGGCCGAGGCCGUGAGGGCCGCCCUGCCCCCCCUGC UGCCCGAGGUGGACAAGGAGACAGUGGAGGCCGACAUCGACCUGAUCAUGCAGGAGGCC GGCGCCGGCAGCGUGGAGACACCCAGAAGGCACAUCAAGGUGACCACCUACCCCGGCGA GGAGAUGAUCGGCAGCUACGCCGUGCUGAGCCCCCAGGCCGUGCUGAACAGCGAGAAGC UGGCCUGCAUCCACCCCCUGGCCGAGCAGGUGCUGGUGAUGACCCACAAGGGCAGGGCC GGCAGAUACAAGGUGGAGCCCUACCACGGCAGGGUGAUCGUGCCCAGCGGCACCGCCAU CCCCAUCCCCGACUUCCAGGCCCUGAGCGAGAGCGCCACCAUCGUGUUCAACGAGAGGG

[0280]

[0281] AGUUCGUGAACAGGUACCUGCACCACAUCGCCGUGAACGGCGGCGCCCUGAACACCGAC

[0282] 12673398.1 GAG GAGU ACU AC AAG GU G GU GAAGU C C AC C GAGAC AGAC AG C GAGU AC GU GUU C GAC AU CGACGCCAAGAAGUGCGUGAAGAAGGGCGACGCCGGCCCCAUGUGCCUGGUGGGCGAGC UGGUGGACCCCCCCUUCCACGAGUUCGCCUACGAGAGCCUGAAAACCAGGCCCGCCGCC CCCCACAAGGUGCCCACCAUCGGCGUGUACGGCGUGCCCGGCAGCGGCAAGAGCGGCAU CAUCAAGAGCGCCGUGACCAAGAGGGACCUGGUGGUGAGCGCCAAGAAGGAGAACUGCA UGGAGAUCAUCAAGGACGUGAAGAGGAUGAGGGGCAUGGACAUCGCCGCCAGGACCGUG GACAGCGUGCUGCUGAACGGCGUGAAGCACAGCGUGGACACCCUGUACAUCGACGAGGC CUUCGCCUGCCACGCCGGCACCCUGCUGGCCCUGAUCGCCAUCGUGAAGCCCAAGAAGG UGGUGCUGUGCGGCGACCCCAAGCAGUGCGGCUUCUUCAACAUGAUGUGCCUGAAGGUG CACUUCAACCACGAGAUCUGCACCGAGGUGUACCACAAGAGCAUCAGCAGGAGGUGCAC C AAGAC C GU GAC C AG C AU CGUGAGCACCCUGUUCUAC GAC AAGAGAAU GAG GAC C GU GA AC C C CU G C AAC GAC AAGAU C AU C AU C GAC AC C AC C AG C AC C AC C AAG C C C CU GAAG GAC GACAUCAUCCUGACCUGCUUCAGGGGCUGGGUGAAGCAGCUGCAGAUCGACUACAAGAA CCACGAGAUCAUGACCGCCGCCGCCAGCCAGGGCCUGACCAGGAAGGGCGUGUACGCCG UGAGGUACAAGGUGAACGAGAACCCCCUGUACGCCCAGACCAGCGAGCACGUGAACGUG CUGCUGACCAGAACCGAGAAGAGAAUCGUGUGGAAAACCCUGGCCGGCGACCCCUGGAU CAAGACCCUGACCGCCAGCUACCCCGGCAACUUCACCGCCACCCUGGAGGAGUGGCAGG CCGAGCACGACGCCAUCAUGGCCAAGAUCCUGGAGACACCCGCCAGCAGCGACGUGUUC CAGAACAAGGUGAACGUGUGCUGGGCCAAGGCCCUGGAGCCCGUGCUGGCCACCGCCAA CAUCACCCUGACCAGGAGCCAGUGGGAGACAAUCCCCGCCUUCAAGGACGACAAGGCCU ACAGCCCCGAGAUGGCCCUGAACUUCUUCUGCACCAGGUUCUUCGGCGUGGACAUCGAC AGCGGCCUGUUCAGCGCCCCCACCGUGCCCCUGACCUACACCAACGAGCACUGGGACAA CAGCCCCGGCCCCAACAUGUACGGCCUGUGCAUGAGGACCGCCAAGGAGCUGGCCAGAA GGUACCCCUGCAUCCUGAAGGCCGUGGACACCGGCAGGGUGGCCGACGUGAGGACCGAC ACCAUCAAGGACUACAACCCCCUGAUCAACGUGGUGCCCCUGAACAGGAGACUGCCCCA CAGCCUGGUGGUGACCCACAGGUACACCGGCAACGGCGACUACAGCCAGCUGGUGACCA AGAUGACCGGCAAGACCGUGCUGGUGGUGGGCACCCCCAUGAACAUCCCCGGCAAGAGG GUGGAGACACUGGGCCCCAGCCCCCAGUGCACCUACAAGGCCGAGCUGGACCUGGGCAU CCCCGCCGCCCUGGGCAAGUACGACAUCAUCUUCAUCAACGUGAGGACCCCCUACAGAC ACCACCACUACCAGCAGUGCGAGGACCACGCCAUCCACCACAGCAUGCUGACCAGGAAG GCCGUGGACCACCUGAACAAGGGCGGCACCUGCAUCGCCCUGGGCUACGGCACCGCCGA CAGAGCCACCGAGAACAUCAUCAGCGCCGUGGCCAGGAGCUUCAGGUUCAGCAGGGUGU GCCAGCCCAAGUGCGCCUGGGAGAACACCGAGGUGGCCUUCGUGUUCUUCGGCAAGGAC AACGGCAACCACCUGCAGGACCAGGACAGGCUGAGCGUGGUGCUGAACAACAUCUACCA GGGCAGCACCCAGCACGAGGCCGGCAGAGCCCCCGCCUACAGGGUGGUGAGGGGCGACA UCACCAAGAGCAACGACGAGGUGAUCGUGAACGCCGCCAACAACAAGGGCCAGCCCGGC GGCGGCGUGUGCGGCGCCCUGUACAGGAAGUGGCCCGGCGCCUUCGACAAGCAGCCCGU GGCCACCGGCAAGGCCCACCUGGUGAAGCACAGCCCCAACGUGAUCCACGCCGUGGGCC CCAACUUCAGCAGACUGAGCGAGAACGAGGGCGACCAGAAGCUGAGCGAGGUGUACAUG GACAUCGCCAGGAUCAUCAACAACGAGAGGUUCACCAAGGUGAGCAUCCCCCUGCUGAG CACCGGCAUCUACGCCGGCGGCAAGGACAGGGUGAUGCAGAGCCUGAACCACCUGUUCA CCGCCAUGGACACCACCGACGCCGACAUCACCAUCUACUGCCUGGACAAGCAGUGGGAG AGCAGGAUCAAGGAGGCCAUCACCAGGAAGGAGAGCGUGGAGGAGCUGACCGAGGACGA CAGGCCCGUGGACAUCGAGCUGGUGAGAGUGCACCCCCUGAGCAGCCUGGCCGGCAGGC CCGGCUACAGCACCACCGAGGGCAAGGUGUACAGCUACCUGGAGGGCACCAGAUUCCAC CAGACCGCCAAGGACAUCGCCGAGAUCUACGCCAUGUGGCCCAACAAGCAGGAGGCCAA CGAGCAGAUCUGCCUGUACGUGCUGGGCGAGAGCAUGAACAGCAUCAGAAGCAAGUGCC CCGUGGAGGAGAGCGAGGCCAGCAGCCCCCCCCACACCAUCCCCUGCCUGUGCAACUAC GCCAUGACCGCCGAGAGGGUGUACAGACUGAGGAUGGCCAAGAACGAGCAGUUCGCCGU GUGCAGCAGCUUCCAGCUGCCCAAGUACAGAAUCACCGGCGUGCAGAAGAUCCAGUGCA GCAAGCCCGUGAUCUUCAGCGGCACCGUGCCCCCCGCCAUCCACCCCAGAAAGUUCGCC AGCGUGACCGUGGAGGACACCCCCGUGGUGCAGCCCGAGAGGCUGGUGCCCAGGAGGCC CGCCCCCCCCGUGCCCGUGCCCGCCAGAAUCCCCAGCCCCCCCUGCACCCCCACCAACG GCAGCACCACCAGCAUCCAGAGCCUGGGCGAGGACCAGAGCGCCAGCGCCAGCGGCGGC GCCGAGAUCAGCGUGGACCAGGUGAGCCUGUGGAGCAUCCCCAGCGCCACCGGCUUCGA CGUGAGGACCAGCAGCAGCCUGAGCCUGGAGCAGCCCACCUUCCCCACCAUGGUGGUGG AGGCCGAGAUCCACGCCAGCCAGGGCAGCCUGUGGAGCAUCCCCAGCAUCACCGGCAGC GAAACCAGGGCCCCCAGCCCCCCCAGCCAGGACAGCAGACCCAGCACCCCCAGCGCCAG CGGCAGCCACACCAGCGUGGACCUGAUCACCUUCGACAGCGUGGCCGAGAUCCUGGAGG

[0283]

[0284] ACUUCAGCAGGAGCCCCUUCCAGUUCCUGAGCGAGAUCAAGCCCAUCCCCGCCCCCAGG

[0285] 12673398.1 ACCAGGGUGACCAACAUGAGCAGGAGCGCCGACACCAUCAAGCCCAUCCCCAAGCCCAG AAAGUGCCAGGUGAAGUACACCCAGCCCCCCGGCGUGGCCAGGGCCAUCAGCGCCGCCG AGUUCGACGAGUUCGUGCGGAGGCACUCGAAUUGACGGUACGAAGCGGGUGCGUACAUU UUCUCAUCCGAGACGGGACAAGGGCACCUGCAACAAAAAUCUACGCGGCAAUGCAAACU CCAGUAUCCAAUCCUGGAGCGUUCCGUCCAUGAGAAAUUUUACGCCCCGCGCCUCGAUC UCGAGAGGGAGAAGCUGCUGCAGAAGAAGCUGCAGCUGUGCGCCAGCGAGGGCAACAGG AGCAGAUACCAGAGCAGGAAGGUGGAGAACAUGAAGGCCAUCACCGUGGAGAGGCUGCU GCAGGGCAUCGGCAGCUACCUGAGCGCCGAGCCCCAGCCCGUGGAGUGCUACAAGGUGA CCUACCCCGCCCCCAUGUACAGCAGCACCGCCAGCAACAGCUUCAGCAGCGCCGAGGUG GCCGUGAAGGUGUGCAACCUGGUGCUGCAGGAGAACUUCCCCACCGUGGCCAGCUACAA CAUCACCGACGAGUACGACGCCUACCUGGACAUGGUGGACGGCGCCAGCUGCUGCCUGG ACACCGCCACCUUCUGCCCCGCCAAGCUGAGGAGCUUCCCCAAGAAGCACAGCUACCUG AGGCCCGAGAUCAGGAGCGCCGUGCCCAGCCCCAUCCAGAACACCCUGCAGAACGUGCU GGCCGCCGCCACCAAGAGGAACUGCAACGUGACCCAGAUGAGGGAGCUGCCCGUGCUGG ACAGCGCCGCCUUCAACGUGGAGUGCUUCAAGAAGUACGCCUGCAACGACGAGUACUGG GACUU CUACAAGAC CAAC C C GAU CAGGCU GAG C GC C GAGAAC GU GAG C CAGUAC GU GAG CAAGCUGAAGGGCCCCAAGGCCGCCGCCCUGUUCGCCAAGACCCACAACCUGCAGCCCC UGCACGAGAUCCCCAUGGACAGGUUCGUGAUGGACCUGAAGAGAGAUGUGAAGGUGACC CCCGGCACCAAGCACACCGAGGAGAGGCCCAAGGUGCAGGUGAUCCAGGCCGCCGACCC CCUGGCCACCGCCUACCUGUGCGGCAUCCACAGGGAGCUGGUGAGGAGGCUGAACGCCG UGCUGCUGCCCAACAUCCACACCCUGUUCGACAUGAGCGCCGAGGACUUCGACGCCAUC AUCGCCGAGCACUUCCAGUUCGGCGACAGCGUGCUGGAGACAGACAUCGCCAGCUUCGA CAAGAGCGAGGACGACGCCAUCGCCAUGAGCGCCCUGAUGAUCCUGGAGGACCUGGGCG UGGACCAGGCCCUGCUGAACCUGAUCGAGGCCGCCUUCGGCAACAUCACCAGCGUGCAC CUGCCCACCGGCACCAGGUUCAAGUUCGGCGCCAUGAUGAAGUCCGGCAUGUUCCUGAC CCUGUUCAUCAACACCGUGGUGAACAUCAUGAUCGCCAGCAGGGUGCUGAGGGAGAGGC UGACCACCAGCCCCUGCGCCGCCUUCAUCGGCGACGACAACAUCGUGAAGGGCGUGACC AGCGACGCCCUGAUGGCCGAGAGGUGCGCCACCUGGCUGAACAUGGAGGUGAAGAUCAU CGACGCCGUGGUGGGCGUGAAGGCCCCCUACUUCUGCGGCGGCUUCAUCGUGGUGGACC AGAUCACCGGCACCGCCUGCAGGGUGGCCGACCCCCUGAAGAGACUGUUCAAGCUGGGC AAGCCCCUGCCCCUGGACGACGACCAGGACGUGGACAGGAGGAGAGCCCUGCACGACGA GGCCGCCAGGUGGAACAGAAUCGGCAUCACCGAGGAGCUGGUGAAGGCCGUGGAGAGCA GAUACGAGGUGAACUACGUGAGCCUGAUCAUCACCGCCCUGACCACCCUGGCCAGCAGC GUGAGCAACUUCAAGCACAUCAGGGGCCACCCCAUCACCCUGUACGGC

[0286] Amino MEKVHVDLDADSPFVKSLQRCFPHFEIEATQVTDNDHANARAFSHLATKLIEGEVDTDQ 83 Acid VILDIGSAPVRHTHSKHKYHCICPMKSAEDPDRL YR YADKLRKSDVTDKCIASKAADLL contains Sequence TVMS TPDAE TPSL CMHTDS TCR YHGS VA VYQDVYA VHAPTS I YYQALKGVR TI YWIGFD SEQ ID NO:

[0287] TTPFMYKNMAGAYPTYNTNWADESVLEARNIGLGSSDLHEKSFGKVSIMRKKKLQPTNK 118 VIFSVGSTIYTEERTLLRSWHLPNVFHLKGKTSFTGRCNTIVSCEGYWKKITLSPGIY (italicized) GKVDNLASTMHREGFLSCKVTDTLRGERVSFPVCTYVPATLCDQMTGILATDVSVDDAQ and SEQ ID KLLVGLNQRIWNGRTQRNTNTMQNYLLPWAQAFSRWAREHRADLEDEKGLGVRERSL NO: 119 VMGCCWAFKTHKITSIYKRPGTQTIKKVPAVFNSFVIPQPTSYGLDIGLRRRIKMLFDA (underlined) KKAPAPIITEADVAHLKGLQDEAEAVAEAEAVRAALPPLLPEVDKETVEADIDLIMQEA GAGSVETPRRHIKVTTYPGEEMIGSYAVLSPQAVLNSEKLACIHPLAEQVLVMTHKGRA GRYKVEPYHGRVIVPSGTAIPIPDFQALSESATIVFNEREFVNRYLHHIAVNGGALNTD EEYYKWKSTETDSEYVFDIDAKKCVKKGDAGPMCLVGELVDPPFHEFAYESLKTRPAA PHKVPTIGVYGVPGSGKSGIIKSAVTKRDLWSAKKENCMEIIKDVKRMRGMDIAARTV DSVLLNGVKHSVDTLYIDEAFACHAGTLLALIAIVKPKKWLCGDPKQCGFFNMMCLKV HFNHEICTEVYHKSISRRCTKTVTSIVSTLFYDKRMRTVNPCNDKIIIDTTSTTKPLKD DIILTCFRGWVKQLQIDYKNHEIMTAAASQGLTRKGVYAVRYKVNENPLYAQTSEHVNV LLTRTEKRIVWKTLAGDPWIKTLTASYPGNFTATLEEWQAEHDAIMAKILETPASSDVF QNKVNVCWAKALEPVLATANITLTRSQWETIPAFKDDKAYSPEMALNFFCTRFFGVDID SGLFSAPTVPLTYTNEHWDNSPGPNMYGLCMRTAKELARRYPCILKAVDTGRVADVRTD TIKD YNPL INWPLNRRL PHSL WTHR YTGNGD YSQL VTKMTGKTVL WGTPMNIPGKR VETLGPSPQCTYKAELDLGIPAALGKYDIIFINVRTPYRHHHYQQCEDHAIHHSMLTRK AVDHLNKGGTCIALGYGTADRATENIISAVARSFRFSRVCQPKCAWENTEVAFVFFGKD NGNHLQDQDRLSWLNNIYQGSTQHEAGRAPAYRWRGDITKSNDEVIVNAANNKGQPG GGVCGALYREWPGAFDKQPVATGKAHLVKHSPNVIHAVGPNFSRLSENEGDQKLSEVYM DIARIINNERFTKVSIPLLSTGIYAGGKDRVMQSLNHLFTAMDTTDADITIYCLDKQWE

[0288]

[0289] SRIKEAITRKESVEELTEDDRPVDIELVRVHPLSSLAGRPGYSTTEGKVYSYLEGTRFH

[0290] 12673398.1 QTAKDIAEIYAMWPNKQEANEQICLYVLGESMNSIRSKCPVEESEASSPPHTIPCLCNY AMTAER VYRLRMAKNEQFA VCSSFQL PKYR I TGVQKIQ CSKPVIFSGTVPPA IHPRKFA SVTVEDTPWQPERLVPRRPAPPVPVPARIPSPPCTPTNGSTTSIQSLGEDQSASASGG AEISVDQVSLWSIPSATGFDVRTSSSLSLEQPTFPTMWEAEIHASQGSLWSIPSITGS ETRAPSPPSQDSRPSTPSASGSHTSVDLITFDSVAEILEDFSRSPFQFLSEIKPIPAPR TR VTNMSRSAD TIKPIPKPRKCQ VKYTQPPGVARA ISAAEFDEFVRRHSN*RYEAGAYI FSSETGQGHLQQKSTRQCKLQYPILERSVHEKFYAPRLDLEREKLLQKKLQLCASEGNR SRYQSRKVENMKAITVERLLQGIGSYLSAEPQPVECYKVTYPAPMYSSTASNSFSSAEV AVKVCNLVLQENFPTVASYNITDEYDAYLDMVDGASCCLDTATFCPAKLRSFPKKHSYL RPEIRSAVPSPIQNTLQNVLAAATKRNCNVTQMRELPVLDSAAFNVECFKKYACNDEYW DFYKTNPIRLTAENVTQYVTKLKGPKAAALFAKTHNLQPLHEIPMDRFVMDLKRDVKVT PGTKHTEERPKVQVIQAADPLATAYLCGIHRELVRRLNAVLLPNIHTLFDMSAEDFDAI IAEHFQFGDSVLETDIASFDKSEDDAIAMSALMILEDLGVDQALLNLIEAAFGNITSVH LPTGTRFKFGAMMKSGMFLTLFINTWNIMIASRVLRERLTTSPCAAFIGDDNIVKGVT SDALMAERCATWLNMEVKIIDAWGVKAPYFCGGFIWDQITGTACRVADPLKRLFKLG KPLPLDDDQDVDRRRALHDEAARWNRIGITEELVKAVESRYEVNYVSLIITALTTLASS VSNFKHIRGHPITLYG EEEV (Opal> Arg) Replicase

[0291] DNA ATGGAGAAAGTACACGTAGACTTGGACGCCGACAGCCCCTTCGTGAAGTCCCTGCAGAG 115 Sequence GTGCTTCCCCCACTTCGAGATCGAGGCCACCCAGGTGACCGACAACGACCACGCCAACG CCAGGGCCTTCAGCCACCTGGCCACCAAGCTGATCGAGGGCGAGGTGGACACCGACCAG GTGATCCTGGACATCGGCAGCGCCCCCGTGAGGCACACCCACAGCAAGCACAAGTACCA CTGCATCTGCCCCATGAAGTCCGCCGAGGACCCCGACAGGCTGTACAGGTACGCCGACA AGCTGAGAAAGAGCGACGTGACCGACAAGTGCATCGCCAGCAAGGCCGCCGACCTGCTG ACCGTGATGAGCACCCCCGACGCCGAGACACCCAGCCTGTGCATGCACACCGACAGCAC CTGCAGGTACCACGGCAGCGTGGCCGTGTACCAGGACGTGTACGCCGTGCACGCCCCCA CCAGCATCTACTACCAGGCCCTGAAGGGCGTGAGAACCATCTACTGGATCGGCTTCGAC ACCACCCCCTTCATGTACAAGAACATGGCCGGCGCCTACCCCACCTACAACACCAACTG GGCCGACGAGAGCGTGCTGGAGGCCAGGAACATCGGCCTGGGCAGCAGCGACCTGCACG AGAAGTCCTTCGGCAAGGTGAGCATCATGAGGAAGAAGAAGCTGCAGCCCACCAACAAG GTGATCTTCAGCGTGGGCAGCACCATCTACACCGAGGAGAGGACCCTGCTGAGGAGCTG GCACCTGCCCAACGTGTTCCACCTGAAGGGCAAGACCAGCTTCACCGGCAGGTGCAACA CCATCGTGAGCTGCGAGGGCTACGTGGTGAAGAAGATCACCCTGAGCCCCGGCATCTAC GGCAAGGTGGACAACCTGGCCAGCACCATGCACAGGGAGGGCTTCCTGAGCTGCAAGGT GACCGACACCCTGAGGGGCGAGAGAGTGAGCTTCCCCGTGTGCACCTACGTGCCCGCCA CCCTGTGCGACCAGATGACCGGCATCCTGGCCACCGACGTGAGCGTGGACGACGCCCAG AAGCTGCTGGTGGGCCTGAACCAGAGGATCGTGGTGAACGGCAGGACCCAGAGAAACAC CAACACCATGCAGAACTACCTGCTGCCCGTGGTGGCCCAGGCCTTCAGCAGGTGGGCCA GGGAGCACAGGGCCGACCTGGAGGACGAGAAGGGCCTGGGCGTGAGAGAGAGAAGCCTG GTGATGGGCTGCTGCTGGGCCTTCAAGACCCACAAGATCACCAGCATCTACAAGAGGCC CGGCACCCAGACCATCAAGAAGGTGCCCGCCGTGTTCAACAGCTTCGTGATCCCCCAGC CCACCAGCTACGGCCTGGACATCGGCCTGAGGAGGAGAATCAAGATGCTGTTCGACGCC AAGAAGGCCCCCGCCCCCATCATCACCGAGGCCGACGTGGCCCACCTGAAGGGCCTGCA GGACGAGGCCGAGGCCGTGGCCGAGGCCGAGGCCGTGAGGGCCGCCCTGCCCCCCCTGC TGCCCGAGGTGGACAAGGAGACAGTGGAGGCCGACATCGACCTGATCATGCAGGAGGCC GGCGCCGGCAGCGTGGAGACACCCAGAAGGCACATCAAGGTGACCACCTACCCCGGCGA GGAGATGATCGGCAGCTACGCCGTGCTGAGCCCCCAGGCCGTGCTGAACAGCGAGAAGC TGGCCTGCATCCACCCCCTGGCCGAGCAGGTGCTGGTGATGACCCACAAGGGCAGGGCC GGCAGATACAAGGTGGAGCCCTACCACGGCAGGGTGATCGTGCCCAGCGGCACCGCCAT CCCCATCCCCGACTTCCAGGCCCTGAGCGAGAGCGCCACCATCGTGTTCAACGAGAGGG AGTTCGTGAACAGGTACCTGCACCACATCGCCGTGAACGGCGGCGCCCTGAACACCGAC GAGGAGTACTACAAGGTGGTGAAGTCCACCGAGACAGACAGCGAGTACGTGTTCGACAT CGACGCCAAGAAGTGCGTGAAGAAGGGCGACGCCGGCCCCATGTGCCTGGTGGGCGAGC TGGTGGACCCCCCCTTCCACGAGTTCGCCTACGAGAGCCTGAAAACCAGGCCCGCCGCC CCCCACAAGGTGCCCACCATCGGCGTGTACGGCGTGCCCGGCAGCGGCAAGAGCGGCAT CATCAAGAGCGCCGTGACCAAGAGGGACCTGGTGGTGAGCGCCAAGAAGGAGAACTGCA TGGAGATCATCAAGGACGTGAAGAGGATGAGGGGCATGGACATCGCCGCCAGGACCGTG GACAGCGTGCTGCTGAACGGCGTGAAGCACAGCGTGGACACCCTGTACATCGACGAGGC CTTCGCCTGCCACGCCGGCACCCTGCTGGCCCTGATCGCCATCGTGAAGCCCAAGAAGG

[0292]

[0293] TGGTGCTGTGCGGCGACCCCAAGCAGTGCGGCTTCTTCAACATGATGTGCCTGAAGGTG 12673398.1 CACTTCAACCACGAGATCTGCACCGAGGTGTACCACAAGAGCATCAGCAGGAGGTGCAC CAAGACCGTGACCAGCATCGTGAGCACCCTGTTCTACGACAAGAGAATGAGGACCGTGA ACCCCTGCAACGACAAGATCATCATCGACACCACCAGCACCACCAAGCCCCTGAAGGAC GACATCATCCTGACCTGCTTCAGGGGCTGGGTGAAGCAGCTGCAGATCGACTACAAGAA CCACGAGATCATGACCGCCGCCGCCAGCCAGGGCCTGACCAGGAAGGGCGTGTACGCCG TGAGGTACAAGGTGAACGAGAACCCCCTGTACGCCCAGACCAGCGAGCACGTGAACGTG CTGCTGACCAGAACCGAGAAGAGAATCGTGTGGAAAACCCTGGCCGGCGACCCCTGGAT CAAGACCCTGACCGCCAGCTACCCCGGCAACTTCACCGCCACCCTGGAGGAGTGGCAGG CCGAGCACGACGCCATCATGGCCAAGATCCTGGAGACACCCGCCAGCAGCGACGTGTTC CAGAACAAGGTGAACGTGTGCTGGGCCAAGGCCCTGGAGCCCGTGCTGGCCACCGCCAA CATCACCCTGACCAGGAGCCAGTGGGAGACAATCCCCGCCTTCAAGGACGACAAGGCCT ACAGCCCCGAGATGGCCCTGAACTTCTTCTGCACCAGGTTCTTCGGCGTGGACATCGAC AGCGGCCTGTTCAGCGCCCCCACCGTGCCCCTGACCTACACCAACGAGCACTGGGACAA CAGCCCCGGCCCCAACATGTACGGCCTGTGCATGAGGACCGCCAAGGAGCTGGCCAGAA GGTACCCCTGCATCCTGAAGGCCGTGGACACCGGCAGGGTGGCCGACGTGAGGACCGAC ACCATCAAGGACTACAACCCCCTGATCAACGTGGTGCCCCTGAACAGGAGACTGCCCCA CAGCCTGGTGGTGACCCACAGGTACACCGGCAACGGCGACTACAGCCAGCTGGTGACCA AGATGACCGGCAAGACCGTGCTGGTGGTGGGCACCCCCATGAACATCCCCGGCAAGAGG GTGGAGACACTGGGCCCCAGCCCCCAGTGCACCTACAAGGCCGAGCTGGACCTGGGCAT CCCCGCCGCCCTGGGCAAGTACGACATCATCTTCATCAACGTGAGGACCCCCTACAGAC ACCACCACTACCAGCAGTGCGAGGACCACGCCATCCACCACAGCATGCTGACCAGGAAG GCCGTGGACCACCTGAACAAGGGCGGCACCTGCATCGCCCTGGGCTACGGCACCGCCGA CAGAGCCACCGAGAACATCATCAGCGCCGTGGCCAGGAGCTTCAGGTTCAGCAGGGTGT GCCAGCCCAAGTGCGCCTGGGAGAACACCGAGGTGGCCTTCGTGTTCTTCGGCAAGGAC AACGGCAACCACCTGCAGGACCAGGACAGGCTGAGCGTGGTGCTGAACAACATCTACCA GGGCAGCACCCAGCACGAGGCCGGCAGAGCCCCCGCCTACAGGGTGGTGAGGGGCGACA TCACCAAGAGCAACGACGAGGTGATCGTGAACGCCGCCAACAACAAGGGCCAGCCCGGC GGCGGCGTGTGCGGCGCCCTGTACAGGAAGTGGCCCGGCGCCTTCGACAAGCAGCCCGT GGCCACCGGCAAGGCCCACCTGGTGAAGCACAGCCCCAACGTGATCCACGCCGTGGGCC CCAACTTCAGCAGACTGAGCGAGAACGAGGGCGACCAGAAGCTGAGCGAGGTGTACATG GACATCGCCAGGATCATCAACAACGAGAGGTTCACCAAGGTGAGCATCCCCCTGCTGAG CACCGGCATCTACGCCGGCGGCAAGGACAGGGTGATGCAGAGCCTGAACCACCTGTTCA CCGCCATGGACACCACCGACGCCGACATCACCATCTACTGCCTGGACAAGCAGTGGGAG AGCAGGATCAAGGAGGCCATCACCAGGAAGGAGAGCGTGGAGGAGCTGACCGAGGACGA CAGGCCCGTGGACATCGAGCTGGTGAGAGTGCACCCCCTGAGCAGCCTGGCCGGCAGGC CCGGCTACAGCACCACCGAGGGCAAGGTGTACAGCTACCTGGAGGGCACCAGATTCCAC CAGACCGCCAAGGACATCGCCGAGATCTACGCCATGTGGCCCAACAAGCAGGAGGCCAA CGAGCAGATCTGCCTGTACGTGCTGGGCGAGAGCATGAACAGCATCAGAAGCAAGTGCC CCGTGGAGGAGAGCGAGGCCAGCAGCCCCCCCCACACCATCCCCTGCCTGTGCAACTAC GCCATGACCGCCGAGAGGGTGTACAGACTGAGGATGGCCAAGAACGAGCAGTTCGCCGT GTGCAGCAGCTTCCAGCTGCCCAAGTACAGAATCACCGGCGTGCAGAAGATCCAGTGCA GCAAGCCCGTGATCTTCAGCGGCACCGTGCCCCCCGCCATCCACCCCAGAAAGTTCGCC AGCGTGACCGTGGAGGACACCCCCGTGGTGCAGCCCGAGAGGCTGGTGCCCAGGAGGCC CGCCCCCCCCGTGCCCGTGCCCGCCAGAATCCCCAGCCCCCCCTGCACCCCCACCAACG GCAGCACCACCAGCATCCAGAGCCTGGGCGAGGACCAGAGCGCCAGCGCCAGCGGCGGC GCCGAGATCAGCGTGGACCAGGTGAGCCTGTGGAGCATCCCCAGCGCCACCGGCTTCGA CGTGAGGACCAGCAGCAGCCTGAGCCTGGAGCAGCCCACCTTCCCCACCATGGTGGTGG AGGCCGAGATCCACGCCAGCCAGGGCAGCCTGTGGAGCATCCCCAGCATCACCGGCAGC GAAACCAGGGCCCCCAGCCCCCCCAGCCAGGACAGCAGACCCAGCACCCCCAGCGCCAG CGGCAGCCACACCAGCGTGGACCTGATCACCTTCGACAGCGTGGCCGAGATCCTGGAGG ACTTCAGCAGGAGCCCCTTCCAGTTCCTGAGCGAGATCAAGCCCATCCCCGCCCCCAGG ACCAGGGTGACCAACATGAGCAGGAGCGCCGACACCATCAAGCCCATCCCCAAGCCCAG AAAGTGCCAGGTGAAGTACACCCAGCCCCCCGGCGTGGCCAGGGCCATCAGCGCCGCCG AGTTCGACGAGTTCGTGCGGAGGCACTCGAATCGGTACGAAGCGGGTGCGTACATTTTC TCATCCGAGACGGGACAAGGGCACCTGCAACAAAAATCTACGCGGCAATGCAAACTCCA GTATCCAATCCTGGAGCGTTCCGTCCATGAGAAATTTTACGCCCCGCGCCTCGATCTCG AGAGGGAGAAGCTGCTGCAGAAGAAGCTGCAGCTGTGCGCCAGCGAGGGCAACAGGAGC AGATACCAGAGCAGGAAGGT GGAGAACAT GAAGGCCAT CACCGT GGAGAGGCT GCT GCA GGGCATCGGCAGCTACCTGAGCGCCGAGCCCCAGCCCGTGGAGTGCTACAAGGTGACCT

[0294]

[0295] ACCCCGCCCCCATGTACAGCAGCACCGCCAGCAACAGCTTCAGCAGCGCCGAGGTGGCC 12673398.1 GTGAAGGTGTGCAACCTGGTGCTGCAGGAGAACTTCCCCACCGTGGCCAGCTACAACAT CACCGACGAGTACGACGCCTACCTGGACATGGTGGACGGCGCCAGCTGCTGCCTGGACA CCGCCACCTTCTGCCCCGCCAAGCTGAGGAGCTTCCCCAAGAAGCACAGCTACCTGAGG CCCGAGATCAGGAGCGCCGTGCCCAGCCCCATCCAGAACACCCTGCAGAACGTGCTGGC CGCCGCCACCAAGAGGAACTGCAACGTGACCCAGATGAGGGAGCTGCCCGTGCTGGACA GCGCCGCCTTCAACGTGGAGTGCTTCAAGAAGTACGCCTGCAACGACGAGTACTGGGAC TTCTACAAGACCAACCCCATCAGGCTGACCGCCGAGAACGTGACCCAGTACGTGACCAA GCTGAAGGGCCCCAAGGCCGCCGCCCTGTTCGCCAAGACCCACAACCTGCAGCCCCTGC ACGAGATCCCCATGGACAGGTTCGTGATGGACCTGAAGAGAGATGTGAAGGTGACCCCC GGCACCAAGCACACCGAGGAGAGGCCCAAGGTGCAGGTGATCCAGGCCGCCGACCCCCT GGCCACCGCCTACCTGTGCGGCATCCACAGGGAGCTGGTGAGGAGGCTGAACGCCGTGC TGCTGCCCAACATCCACACCCTGTTCGACATGAGCGCCGAGGACTTCGACGCCATCATC GCCGAGCACTTCCAGTTCGGCGACAGCGTGCTGGAGACAGACATCGCCAGCTTCGACAA GAGCGAGGACGACGCCATCGCCATGAGCGCCCTGATGATCCTGGAGGACCTGGGCGTGG ACCAGGCCCTGCTGAACCTGATCGAGGCCGCCTTCGGCAACATCACCAGCGTGCACCTG CCCACCGGCACCAGGTTCAAGTTCGGCGCCATGATGAAGTCCGGCATGTTCCTGACCCT GTTCATCAACACCGTGGTGAACATCATGATCGCCAGCAGGGTGCTGAGGGAGAGGCTGA CCACCAGCCCCTGCGCCGCCTTCATCGGCGACGACAACATCGTGAAGGGCGTGACCAGC GACGCCCTGATGGCCGAGAGGTGCGCCACCTGGCTGAACATGGAGGTGAAGATCATCGA CGCCGTGGTGGGCGTGAAGGCCCCCTACTTCTGCGGCGGCTTCATCGTGGTGGACCAGA TCACCGGCACCGCCTGCAGGGTGGCCGACCCCCTGAAGAGACTGTTCAAGCTGGGCAAG CCCCTGCCCCTGGACGACGACCAGGACGTGGACAGGAGGAGAGCCCTGCACGACGAGGC CGCCAGGTGGAACAGAATCGGCATCACCGAGGAGCTGGTGAAGGCCGTGGAGAGCAGAT ACGAGGTGAACTACGTGAGCCTGATCATCACCGCCCTGACCACCCTGGCCAGCAGCGTG AGCAACTTCAAGCACATCAGGGGCCACCCCATCACCCTGTACGGC RNA AUGGAGAAAGUACACGUAGACUUGGACGCCGACAGCCCCUUCGUGAAGUCCCUGCAGAG 116 Sequence GUGCUUCCCCCACUUCGAGAUCGAGGCCACCCAGGUGACCGACAACGACCACGCCAACG CCAGGGCCUUCAGCCACCUGGCCACCAAGCUGAUCGAGGGCGAGGUGGACACCGACCAG GUGAUCCUGGACAUCGGCAGCGCCCCCGUGAGGCACACCCACAGCAAGCACAAGUACCA CUGCAUCUGCCCCAUGAAGUCCGCCGAGGACCCCGACAGGCUGUACAGGUACGCCGACA AGCUGAGAAAGAGCGACGUGACCGACAAGUGCAUCGCCAGCAAGGCCGCCGACCUGCUG ACCGUGAUGAGCACCCCCGACGCCGAGACACCCAGCCUGUGCAUGCACACCGACAGCAC CUGCAGGUACCACGGCAGCGUGGCCGUGUACCAGGACGUGUACGCCGUGCACGCCCCCA CCAGCAUCUACUACCAGGCCCUGAAGGGCGUGAGAACCAUCUACUGGAUCGGCUUCGAC ACCACCCCCUUCAUGUACAAGAACAUGGCCGGCGCCUACCCCACCUACAACACCAACUG GGCCGACGAGAGCGUGCUGGAGGCCAGGAACAUCGGCCUGGGCAGCAGCGACCUGCACG AGAAGUCCUUCGGCAAGGUGAGCAUCAUGAGGAAGAAGAAGCUGCAGCCCACCAACAAG GUGAUCUUCAGCGUGGGCAGCACCAUCUACACCGAGGAGAGGACCCUGCUGAGGAGCUG GCACCUGCCCAACGUGUUCCACCUGAAGGGCAAGACCAGCUUCACCGGCAGGUGCAACA CCAUCGUGAGCUGCGAGGGCUACGUGGUGAAGAAGAUCACCCUGAGCCCCGGCAUCUAC GGCAAGGUGGACAACCUGGCCAGCACCAUGCACAGGGAGGGCUUCCUGAGCUGCAAGGU GACCGACACCCUGAGGGGCGAGAGAGUGAGCUUCCCCGUGUGCACCUACGUGCCCGCCA CCCUGUGCGACCAGAUGACCGGCAUCCUGGCCACCGACGUGAGCGUGGACGACGCCCAG AAGCUGCUGGUGGGCCUGAACCAGAGGAUCGUGGUGAACGGCAGGACCCAGAGAAACAC CAACACCAUGCAGAACUACCUGCUGCCCGUGGUGGCCCAGGCCUUCAGCAGGUGGGCCA GGGAGCACAGGGCCGACCUGGAGGACGAGAAGGGCCUGGGCGUGAGAGAGAGAAGCCUG GUGAUGGGCUGCUGCUGGGCCUUCAAGACCCACAAGAUCACCAGCAUCUACAAGAGGCC CGGCACCCAGACCAUCAAGAAGGUGCCCGCCGUGUUCAACAGCUUCGUGAUCCCCCAGC CCACCAGCUACGGCCUGGACAUCGGCCUGAGGAGGAGAAUCAAGAUGCUGUUCGACGCC AAGAAGGCCCCCGCCCCCAUCAUCACCGAGGCCGACGUGGCCCACCUGAAGGGCCUGCA GGACGAGGCCGAGGCCGUGGCCGAGGCCGAGGCCGUGAGGGCCGCCCUGCCCCCCCUGC UGCCCGAGGUGGACAAGGAGACAGUGGAGGCCGACAUCGACCUGAUCAUGCAGGAGGCC GGCGCCGGCAGCGUGGAGACACCCAGAAGGCACAUCAAGGUGACCACCUACCCCGGCGA GGAGAUGAUCGGCAGCUACGCCGUGCUGAGCCCCCAGGCCGUGCUGAACAGCGAGAAGC UGGCCUGCAUCCACCCCCUGGCCGAGCAGGUGCUGGUGAUGACCCACAAGGGCAGGGCC GGCAGAUACAAGGUGGAGCCCUACCACGGCAGGGUGAUCGUGCCCAGCGGCACCGCCAU CCCCAUCCCCGACUUCCAGGCCCUGAGCGAGAGCGCCACCAUCGUGUUCAACGAGAGGG AGUUCGUGAACAGGUACCUGCACCACAUCGCCGUGAACGGCGGCGCCCUGAACACCGAC GAG GAGU ACU AC AAG GU G GU GAAGU C C AC C GAGAC AGAC AG C GAGU AC GU GUU C GAC AU

[0296]

[0297] CGACGCCAAGAAGUGCGUGAAGAAGGGCGACGCCGGCCCCAUGUGCCUGGUGGGCGAGC 12673398.1 UGGUGGACCCCCCCUUCCACGAGUUCGCCUACGAGAGCCUGAAAACCAGGCCCGCCGCC CCCCACAAGGUGCCCACCAUCGGCGUGUACGGCGUGCCCGGCAGCGGCAAGAGCGGCAU CAUCAAGAGCGCCGUGACCAAGAGGGACCUGGUGGUGAGCGCCAAGAAGGAGAACUGCA UGGAGAUCAUCAAGGACGUGAAGAGGAUGAGGGGCAUGGACAUCGCCGCCAGGACCGUG GACAGCGUGCUGCUGAACGGCGUGAAGCACAGCGUGGACACCCUGUACAUCGACGAGGC CUUCGCCUGCCACGCCGGCACCCUGCUGGCCCUGAUCGCCAUCGUGAAGCCCAAGAAGG UGGUGCUGUGCGGCGACCCCAAGCAGUGCGGCUUCUUCAACAUGAUGUGCCUGAAGGUG CACUUCAACCACGAGAUCUGCACCGAGGUGUACCACAAGAGCAUCAGCAGGAGGUGCAC C AAGAC C GU GAG C AG GAU CGUGAGCACCCUGUUCUAC GAG AAGAGAAU GAG GAG C GU GA AC C C CU G C AAC GAC AAGAU C AU C AU C GAC AC C AC C AG C AC C AC C AAG C C C CU GAAG GAC GACAUCAUCCUGACCUGCUUCAGGGGCUGGGUGAAGCAGCUGCAGAUCGACUACAAGAA CCACGAGAUCAUGACCGCCGCCGCCAGCCAGGGCCUGACCAGGAAGGGCGUGUACGCCG UGAGGUACAAGGUGAACGAGAACCCCCUGUACGCCCAGACCAGCGAGCACGUGAACGUG CUGCUGACCAGAACCGAGAAGAGAAUCGUGUGGAAAACCCUGGCCGGCGACCCCUGGAU CAAGACCCUGACCGCCAGCUACCCCGGCAACUUCACCGCCACCCUGGAGGAGUGGCAGG CCGAGCACGACGCCAUCAUGGCCAAGAUCCUGGAGACACCCGCCAGCAGCGACGUGUUC CAGAACAAGGUGAACGUGUGCUGGGCCAAGGCCCUGGAGCCCGUGCUGGCCACCGCCAA CAUCACCCUGACCAGGAGCCAGUGGGAGACAAUCCCCGCCUUCAAGGACGACAAGGCCU ACAGCCCCGAGAUGGCCCUGAACUUCUUCUGCACCAGGUUCUUCGGCGUGGACAUCGAC AGCGGCCUGUUCAGCGCCCCCACCGUGCCCCUGACCUACACCAACGAGCACUGGGACAA CAGCCCCGGCCCCAACAUGUACGGCCUGUGCAUGAGGACCGCCAAGGAGCUGGCCAGAA GGUACCCCUGCAUCCUGAAGGCCGUGGACACCGGCAGGGUGGCCGACGUGAGGACCGAC ACCAUCAAGGACUACAACCCCCUGAUCAACGUGGUGCCCCUGAACAGGAGACUGCCCCA CAGCCUGGUGGUGACCCACAGGUACACCGGCAACGGCGACUACAGCCAGCUGGUGACCA AGAUGACCGGCAAGACCGUGCUGGUGGUGGGCACCCCCAUGAACAUCCCCGGCAAGAGG GUGGAGACACUGGGCCCCAGCCCCCAGUGCACCUACAAGGCCGAGCUGGACCUGGGCAU CCCCGCCGCCCUGGGCAAGUACGACAUCAUCUUCAUCAACGUGAGGACCCCCUACAGAC ACCACCACUACCAGCAGUGCGAGGACCACGCCAUCCACCACAGCAUGCUGACCAGGAAG GCCGUGGACCACCUGAACAAGGGCGGCACCUGCAUCGCCCUGGGCUACGGCACCGCCGA CAGAGCCACCGAGAACAUCAUCAGCGCCGUGGCCAGGAGCUUCAGGUUCAGCAGGGUGU GCCAGCCCAAGUGCGCCUGGGAGAACACCGAGGUGGCCUUCGUGUUCUUCGGCAAGGAC AACGGCAACCACCUGCAGGACCAGGACAGGCUGAGCGUGGUGCUGAACAACAUCUACCA GGGCAGCACCCAGCACGAGGCCGGCAGAGCCCCCGCCUACAGGGUGGUGAGGGGCGACA UCACCAAGAGCAACGACGAGGUGAUCGUGAACGCCGCCAACAACAAGGGCCAGCCCGGC GGCGGCGUGUGCGGCGCCCUGUACAGGAAGUGGCCCGGCGCCUUCGACAAGCAGCCCGU GGCCACCGGCAAGGCCCACCUGGUGAAGCACAGCCCCAACGUGAUCCACGCCGUGGGCC CCAACUUCAGCAGACUGAGCGAGAACGAGGGCGACCAGAAGCUGAGCGAGGUGUACAUG GACAUCGCCAGGAUCAUCAACAACGAGAGGUUCACCAAGGUGAGCAUCCCCCUGCUGAG CACCGGCAUCUACGCCGGCGGCAAGGACAGGGUGAUGCAGAGCCUGAACCACCUGUUCA CCGCCAUGGACACCACCGACGCCGACAUCACCAUCUACUGCCUGGACAAGCAGUGGGAG AGCAGGAUCAAGGAGGCCAUCACCAGGAAGGAGAGCGUGGAGGAGCUGACCGAGGACGA CAGGCCCGUGGACAUCGAGCUGGUGAGAGUGCACCCCCUGAGCAGCCUGGCCGGCAGGC CCGGCUACAGCACCACCGAGGGCAAGGUGUACAGCUACCUGGAGGGCACCAGAUUCCAC CAGACCGCCAAGGACAUCGCCGAGAUCUACGCCAUGUGGCCCAACAAGCAGGAGGCCAA CGAGCAGAUCUGCCUGUACGUGCUGGGCGAGAGCAUGAACAGCAUCAGAAGCAAGUGCC CCGUGGAGGAGAGCGAGGCCAGCAGCCCCCCCCACACCAUCCCCUGCCUGUGCAACUAC GCCAUGACCGCCGAGAGGGUGUACAGACUGAGGAUGGCCAAGAACGAGCAGUUCGCCGU GUGCAGCAGCUUCCAGCUGCCCAAGUACAGAAUCACCGGCGUGCAGAAGAUCCAGUGCA GCAAGCCCGUGAUCUUCAGCGGCACCGUGCCCCCCGCCAUCCACCCCAGAAAGUUCGCC AGCGUGACCGUGGAGGACACCCCCGUGGUGCAGCCCGAGAGGCUGGUGCCCAGGAGGCC CGCCCCCCCCGUGCCCGUGCCCGCCAGAAUCCCCAGCCCCCCCUGCACCCCCACCAACG GCAGCACCACCAGCAUCCAGAGCCUGGGCGAGGACCAGAGCGCCAGCGCCAGCGGCGGC GCCGAGAUCAGCGUGGACCAGGUGAGCCUGUGGAGCAUCCCCAGCGCCACCGGCUUCGA CGUGAGGACCAGCAGCAGCCUGAGCCUGGAGCAGCCCACCUUCCCCACCAUGGUGGUGG AGGCCGAGAUCCACGCCAGCCAGGGCAGCCUGUGGAGCAUCCCCAGCAUCACCGGCAGC GAAACCAGGGCCCCCAGCCCCCCCAGCCAGGACAGCAGACCCAGCACCCCCAGCGCCAG CGGCAGCCACACCAGCGUGGACCUGAUCACCUUCGACAGCGUGGCCGAGAUCCUGGAGG ACUUCAGCAGGAGCCCCUUCCAGUUCCUGAGCGAGAUCAAGCCCAUCCCCGCCCCCAGG ACCAGGGUGACCAACAUGAGCAGGAGCGCCGACACCAUCAAGCCCAUCCCCAAGCCCAG

[0298]

[0299] AAAGUGCCAGGUGAAGUACACCCAGCCCCCCGGCGUGGCCAGGGCCAUCAGCGCCGCCG

[0300] 12673398.1 AGUUCGACGAGUUCGUGCGGAGGCACUCGAAUcggCGGUACGAAGCGGGUGCGUACAUU UUCUCAUCCGAGACGGGACAAGGGCACCUGCAACAAAAAUCUACGCGGCAAUGCAAACU CCAGUAUCCAAUCCUGGAGCGUUCCGUCCAUGAGAAAUUUUACGCCCCGCGCCUCGAUC UCGAGAGGGAGAAGCUGCUGCAGAAGAAGCUGCAGCUGUGCGCCAGCGAGGGCAACAGG AGCAGAUACCAGAGCAGGAAGGUGGAGAACAUGAAGGCCAUCACCGUGGAGAGGCUGCU GCAGGGCAUCGGCAGCUACCUGAGCGCCGAGCCCCAGCCCGUGGAGUGCUACAAGGUGA CCUACCCCGCCCCCAUGUACAGCAGCACCGCCAGCAACAGCUUCAGCAGCGCCGAGGUG GCCGUGAAGGUGUGCAACCUGGUGCUGCAGGAGAACUUCCCCACCGUGGCCAGCUACAA CAUCACCGACGAGUACGACGCCUACCUGGACAUGGUGGACGGCGCCAGCUGCUGCCUGG ACACCGCCACCUUCUGCCCCGCCAAGCUGAGGAGCUUCCCCAAGAAGCACAGCUACCUG AGGCCCGAGAUCAGGAGCGCCGUGCCCAGCCCCAUCCAGAACACCCUGCAGAACGUGCU GGCCGCCGCCACCAAGAGGAACUGCAACGUGACCCAGAUGAGGGAGCUGCCCGUGCUGG ACAGCGCCGCCUUCAACGUGGAGUGCUUCAAGAAGUACGCCUGCAACGACGAGUACUGG GACUU CUACAAGAC CAAC C C GAU CAGGCU GAG C GC C GAGAAC GU GAG C CAGUAC GU GAG CAAGCUGAAGGGCCCCAAGGCCGCCGCCCUGUUCGCCAAGACCCACAACCUGCAGCCCC UGCACGAGAUCCCCAUGGACAGGUUCGUGAUGGACCUGAAGAGAGAUGUGAAGGUGACC CCCGGCACCAAGCACACCGAGGAGAGGCCCAAGGUGCAGGUGAUCCAGGCCGCCGACCC CCUGGCCACCGCCUACCUGUGCGGCAUCCACAGGGAGCUGGUGAGGAGGCUGAACGCCG UGCUGCUGCCCAACAUCCACACCCUGUUCGACAUGAGCGCCGAGGACUUCGACGCCAUC AUCGCCGAGCACUUCCAGUUCGGCGACAGCGUGCUGGAGACAGACAUCGCCAGCUUCGA CAAGAGCGAGGACGACGCCAUCGCCAUGAGCGCCCUGAUGAUCCUGGAGGACCUGGGCG UGGACCAGGCCCUGCUGAACCUGAUCGAGGCCGCCUUCGGCAACAUCACCAGCGUGCAC CUGCCCACCGGCACCAGGUUCAAGUUCGGCGCCAUGAUGAAGUCCGGCAUGUUCCUGAC CCUGUUCAUCAACACCGUGGUGAACAUCAUGAUCGCCAGCAGGGUGCUGAGGGAGAGGC UGACCACCAGCCCCUGCGCCGCCUUCAUCGGCGACGACAACAUCGUGAAGGGCGUGACC AGCGACGCCCUGAUGGCCGAGAGGUGCGCCACCUGGCUGAACAUGGAGGUGAAGAUCAU CGACGCCGUGGUGGGCGUGAAGGCCCCCUACUUCUGCGGCGGCUUCAUCGUGGUGGACC AGAUCACCGGCACCGCCUGCAGGGUGGCCGACCCCCUGAAGAGACUGUUCAAGCUGGGC AAGCCCCUGCCCCUGGACGACGACCAGGACGUGGACAGGAGGAGAGCCCUGCACGACGA GGCCGCCAGGUGGAACAGAAUCGGCAUCACCGAGGAGCUGGUGAAGGCCGUGGAGAGCA GAUACGAGGUGAACUACGUGAGCCUGAUCAUCACCGCCCUGACCACCCUGGCCAGCAGC GUGAGCAACUUCAAGCACAUCAGGGGCCACCCCAUCACCCUGUACGGC

[0301] Amino MEKVHVDLDADSPFVKSLQRCFPHFEIEATQVTDNDHANARAFSHLATKLIEGEVDTDQ 117 Acid VILDIGSAPVRHTHSKHKYHCICPMKSAEDPDRLYRYADKLRKSDVTDKCIASKAADLL Sequence TVMSTPDAETPSLCMHTDSTCRYHGSVAVYQDVYAVHAPTSIYYQALKGVRTIYWIGFD TTPFMYKNMAGAYPTYNTNWADESVLEARNIGLGSSDLHEKSFGKVSIMRKKKLQPTNK VIFSVGSTIYTEERTLLRSWHLPNVFHLKGKTSFTGRCNTIVSCEGYWKKITLSPGIY GKVDNLASTMHREGFLSCKVTDTLRGERVSFPVCTYVPATLCDQMTGILATDVSVDDAQ KLLVGLNQRIWNGRTQRNTNTMQNYLLPWAQAFSRWAREHRADLEDEKGLGVRERSL VMGCCWAFKTHKITSIYKRPGTQTIKKVPAVFNSFVIPQPTSYGLDIGLRRRIKMLFDA KKAPAPIITEADVAHLKGLQDEAEAVAEAEAVRAALPPLLPEVDKETVEADIDLIMQEA GAGSVETPRRHIKVTTYPGEEMIGSYAVLSPQAVLNSEKLACIHPLAEQVLVMTHKGRA GRYKVEPYHGRVIVPSGTAIPIPDFQALSESATIVFNEREFVNRYLHHIAVNGGALNTD EEYYKWKSTETDSEYVFDIDAKKCVKKGDAGPMCLVGELVDPPFHEFAYESLKTRPAA PHKVPTIGVYGVPGSGKSGIIKSAVTKRDLWSAKKENCMEIIKDVKRMRGMDIAARTV DSVLLNGVKHSVDTLYIDEAFACHAGTLLALIAIVKPKKWLCGDPKQCGFFNMMCLKV HFNHEICTEVYHKSISRRCTKTVTSIVSTLFYDKRMRTVNPCNDKIIIDTTSTTKPLKD DIILTCFRGWVKQLQIDYKNHEIMTAAASQGLTRKGVYAVRYKVNENPLYAQTSEHVNV LLTRTEKRIVWKTLAGDPWIKTLTASYPGNFTATLEEWQAEHDAIMAKILETPASSDVF QNKVNVCWAKALEPVLATANITLTRSQWETIPAFKDDKAYSPEMALNFFCTRFFGVDID SGLFSAPTVPLTYTNEHWDNSPGPNMYGLCMRTAKELARRYPCILKAVDTGRVADVRTD TIKDYNPLINWPLNRRLPHSLWTHRYTGNGDYSQLVTKMTGKTVLWGTPMNIPGKR VETLGPSPQCTYKAELDLGIPAALGKYDIIFINVRTPYRHHHYQQCEDHAIHHSMLTRK AVDHLNKGGTCIALGYGTADRATENIISAVARSFRFSRVCQPKCAWENTEVAFVFFGKD NGNHLQDQDRLSWLNNIYQGSTQHEAGRAPAYRWRGDITKSNDEVIVNAANNKGQPG GGVCGALYRKWPGAFDKQPVATGKAHLVKHSPNVIHAVGPNFSRLSENEGDQKLSEVYM DIARI INNERFTKVSIPLLSTGIYAGGKDRVMQSLNHLFTAMDTTDADITIYCLDKQWE SRIKEAITRKESVEELTEDDRPVDIELVRVHPLSSLAGRPGYSTTEGKVYSYLEGTRFH QTAKDIAEIYAMWPNKQEANEQICLYVLGESMNSIRSKCPVEESEASSPPHTIPCLCNY

[0302]

[0303] AMTAERVYRLRMAKNEQFAVCSSFQLPKYRITGVQKIQCSKPVIFSGTVPPAIHPRKFA

[0304] 12673398.1 SVTVEDTPWQPERLVPRRPAPPVPVPARIPSPPCTPTNGSTTSIQSLGEDQSASASGG AEISVDQVSLWSIPSATGFDVRTSSSLSLEQPTFPTMWEAEIHASQGSLWSIPSITGS ETRAPSPPSQDSRPSTPSASGSHTSVDLITFDSVAEILEDFSRSPFQFLSEIKPIPAPR TRVTNMSRSADTIKPIPKPRKCQVKYTQPPGVARAI SAAEFDEFVRRHSNRRYEAGAYI FSSETGQGHLQQKSTRQCKLQYPILERSVHEKFYAPRLDLEREKLLQKKLQLCASEGNR SRYQSRKVENMKAITVERLLQGIGSYLSAEPQPVECYKVTYPAPMYSSTASNSFSSAEV AVKVCNLVLQENFPTVASYNITDEYDAYLDMVDGASCCLDTATFCPAKLRSFPKKHSYL RPEIRSAVPSPIQNTLQNVLAAATKRNCNVTQMRELPVLDSAAFNVECFKKYACNDEYW DFYKTNPIRLTAENVTQYVTKLKGPKAAALFAKTHNLQPLHEIPMDRFVMDLKRDVKVT PGTKHTEERPKVQVIQAADPLATAYLCGIHRELVRRLNAVLLPNIHTLFDMSAEDFDAI IAEHFQFGDSVLETDIASFDKSEDDAIAMSALMILEDLGVDQALLNLIEAAFGNITSVH LPTGTRFKFGAMMKSGMFLTLFINTWNIMIASRVLRERLTTSPCAAFIGDDNIVKGVT SDALMAERCATWLNMEVKIIDAWGVKAPYFCGGFIWDQITGTACRVADPLKRLFKLG KPLPLDDDQDVDRRRALHDEAARWNRIGITEELVKAVESRYEVNYVSLIITALTTLASS VSNFKHIRGHPITLYG

[0305] Wildtype Semliki Forest Virus (SFV) Replicase

[0306] DNA ATGGCCGCCAAAGTACACGTTGACATCGAGGCCGACAGCCCCTTCATCAAGAGCCTGCA 9 Sequence GAAGGCCTTCCCCAGCTTCGAGGTGGAGAGCCTGCAGGTGACCCCCAACGACCACGCCA ACGCCAGGGCCTTCAGCCACCTGGCCACCAAGCTGATCGAGCAGGAGACAGACAAGGAC ACCCTGATCCTGGACATCGGCAGCGCCCCCAGCAGGAGGATGATGAGCACCCACAAGTA CCACTGCGTGTGCCCCATGAGGAGCGCCGAGGACCCCGAGAGGCTGGTGTGCTACGCCA AGAAGCTGGCCGCCGCCAGCGGCAAGGTGCTGGACAGGGAGATCGCCGGCAAGATCACC GACCTGCAGACCGTGATGGCCACCCCCGACGCCGAGAGCCCCACCTTCTGCCTGCACAC CGACGTGACCTGCAGAACCGCCGCCGAGGTGGCCGTGTACCAGGACGTGTACGCCGTGC ACGCCCCCACCAGCCTGTACCACCAGGCCATGAAGGGCGTGAGAACCGCCTACTGGATC GGCTTCGACACCACCCCCTTCATGTTCGACGCCCTGGCCGGCGCCTACCCCACCTACGC CACCAACTGGGCCGACGAGCAGGTGCTGCAGGCCAGAAACATCGGCCTGTGCGCCGCCA GCCTGACCGAGGGCAGGCTGGGCAAGCTGAGCATCCTGAGGAAGAAGCAGCTGAAGCCC TGCGACACCGTGATGTTCAGCGTGGGCAGCACCCTGTACACCGAGAGCAGAAAGCTGCT GAGAAGCTGGCACCTGCCCAGCGTGTTCCACCTGAAGGGCAAGCAGAGCTTCACCTGCA GGTGCGACACCATCGTGAGCTGCGAGGGCTACGTGGTGAAGAAGATCACCATGTGCCCC GGCCTGTACGGCAAGACCGTGGGCTACGCCGTGACCTACCACGCCGAGGGCTTCCTGGT GTGCAAGACCACCGACACCGTGAAGGGCGAGAGGGTGAGCTTCCCCGTGTGCACCTACG TGCCCAGCACCATCTGCGACCAGATGACCGGCATCCTGGCCACCGACGTGACCCCCGAG GACGCCCAGAAGCTGCTGGTGGGCCTGAACCAGAGGATCGTGGTGAACGGCAGGACCCA GAGAAACACCAACACCATGAAGAACTACCTGCTGCCCATCGTGGCCGTGGCCTTCAGCA AGTGGGCCAGAGAGTACAAGGCCGACCTGGACGACGAGAAGCCCCTGGGCGTGAGAGAG AGGAGCCTGACCTGCTGCTGCCTGTGGGCCTTCAAGACCAGGAAGATGCACACCATGTA CAAGAAGCCCGACACCCAGACCATCGTGAAGGTGCCCAGCGAGTTCAACAGCTTCGTGA TCCCCAGCCTGTGGAGCACCGGCCTGGCCATCCCCGTGAGGAGCAGGATCAAGATGCTG CTGGCCAAGAAAACCAAGAGGGAGCTGATCCCCGTGCTGGACGCCAGCAGCGCCAGGGA CGCCGAGCAGGAGGAGAAGGAGAGGCTGGAGGCCGAGCTGACCAGAGAGGCCCTGCCCC CCCTGGTGCCCATCGCCCCCGCCGAGACAGGCGTGGTGGACGTGGACGTGGAGGAGCTG GAGTACCACGCCGGCGCCGGCGTGGTGGAAACCCCCAGGAGCGCCCTGAAGGTGACCGC CCAGCCCAACGACGTGCTGCTGGGCAACTACGTGGTGCTGAGCCCCCAGACCGTGCTGA AAAGCAGCAAGCTGGCCCCCGTGCACCCCCTGGCCGAGCAGGTGAAGATCATCACCCAC AACGGCAGGGCCGGCAGGTACCAGGTGGACGGCTACGACGGCAGGGTGCTGCTGCCCTG CGGCAGCGCCATCCCCGTGCCCGAGTTCCAGGCCCTGAGCGAGAGCGCCACCATGGTGT ACAACGAGAGGGAGTTCGTGAACAGAAAGCTGTACCACATCGCCGTGCACGGCCCCAGC CTGAACACCGACGAGGAGAACTACGAGAAGGTGAGGGCCGAGAGAACCGACGCCGAGTA CGTGTTCGACGTGGACAAGAAGTGCTGCGTGAAGAGGGAGGAGGCCAGCGGCCTGGTGC TGGTGGGCGAGCTGACCAACCCCCCCTTCCACGAGTTCGCCTACGAGGGCCTGAAGATC AGGCCCAGCGCCCCCTACAAGACCACCGTGGTGGGCGTGTTCGGCGTGCCCGGCAGCGG CAAGAGCGCCATCATCAAGAGCCTGGTGACCAAGCACGACCTGGTGACCAGCGGCAAGA AGGAGAACTGCCAGGAGATCGTGAACGACGTGAAGAAGCACAGAGGCCTGGACATCCAG GCCAAGACCGTGGACAGCATCCTGCTGAACGGCTGCAGGAGGGCCGTGGACATCCTGTA CGTGGACGAGGCCTTCGCCTGCCACAGCGGCACCCTGCTGGCCCTGATCGCCCTGGTGA AGCCCAGAAGCAAGGTGGTGCTGTGCGGCGACCCCAAGCAGTGCGGCTTCTTCAACATG ATGCAGCTGAAGGTGAACTTCAACCACAACATCTGCACCGAGGTGTGCCACAAGAGCAT

[0307]

[0308] CAGCAGGAGGTGCACCAGGCCCGTGACCGCCATCGTGAGCACCCTGCACTACGGCGGCA 12673398.1 AGATGAGGACCACCAACCCCTGCAACAAGCCCATCATCATCGACACCACCGGCCAGACC AAGCCCAAGCCCGGCGACATCGTGCTGACCTGCTTCAGGGGCTGGGTGAAGCAGCTGCA GCTGGACTACAGGGGCCACGAGGTGATGACCGCCGCCGCCAGCCAGGGCCTGACCAGGA AGGGCGTGTACGCCGTGAGGCAGAAGGTGAACGAGAACCCCCTGTACGCCCCCGCCAGC GAGCACGTGAACGTGCTGCTGACCAGGACCGAGGACAGGCTGGTGTGGAAAACCCTGGC CGGCGACCCCTGGATCAAGGTGCTGAGCAACATCCCCCAGGGCAACTTCACCGCCACCC TGGAGGAGTGGCAGGAGGAGCACGACAAGATCATGAAGGTGATCGAGGGCCCCGCCGCC CCCGTGGACGCCTTCCAGAACAAGGCCAACGTGTGCTGGGCCAAGAGCCTGGTGCCCGT GCTGGACACCGCCGGCATCAGGCTGACCGCCGAGGAGTGGAGCACCATCATCACCGCCT TCAAGGAGGACAGGGCCTACAGCCCCGTGGTGGCCCTGAACGAGATCTGCACCAAGTAC TACGGCGTGGACCTGGACAGCGGCCTGTTCAGCGCCCCCAAGGTGAGCCTGTACTACGA GAACAACCACTGGGACAACAGGCCCGGCGGCAGAATGTACGGCTTCAACGCCGCCACCG CCGCCAGGCTGGAGGCCAGGCACACCTTCCTGAAGGGCCAGTGGCACACCGGCAAGCAG GCCGTGATCGCCGAGAGGAAGATCCAGCCCCTGAGCGTGCTGGACAACGTGATCCCCAT CAACAGAAGGCTGCCCCACGCCCTGGTGGCCGAGTACAAGACCGTGAAGGGCAGCAGGG TGGAGTGGCTGGTGAACAAGGTGAGGGGCTACCACGTGCTGCTGGTGAGCGAGTACAAC CTGGCCCTGCCCAGGAGAAGGGTGACCTGGCTGAGCCCCCTGAACGTGACCGGCGCCGA CAGGTGCTACGACCTGAGCCTGGGCCTGCCCGCCGACGCCGGCAGGTTCGACCTGGTGT TCGTGAACATCCACACCGAGTTCAGGATCCACCACTACCAGCAGTGCGTGGACCACGCC ATGAAGCTGCAGATGCTGGGCGGCGACGCCCTGAGGCTGCTGAAGCCCGGCGGCAGCCT GCTGATGAGGGCCTACGGCTACGCCGACAAGATCAGCGAGGCCGTGGTGAGCAGCCTGA GCAGGAAGTTCAGCAGCGCCAGAGTGCTGAGGCCCGACTGCGTGACCAGCAACACCGAG GTGTTCCTGCTGTTCAGCAACTTCGACAACGGCAAGAGGCCCAGCACCCTGCACCAGAT GAACACCAAGCTGAGCGCCGTGTACGCCGGCGAGGCCATGCACACCGCCGGCTGCGCCC CCAGCTACAGGGTGAAGAGGGCCGACATCGCCACCTGCACCGAGGCCGCCGTGGTGAAC GCCGCCAACGCCAGGGGCACCGTGGGCGACGGCGTGTGCAGGGCCGTGGCCAAGAAGTG GCCCAGCGCCTTCAAGGGCGAGGCCACCCCCGTGGGCACCATCAAGACCGTGATGTGCG GCAGCTACCCCGTGATCCACGCCGTGGCCCCCAACTTCAGCGCCACCACCGAGGCCGAG GGCGACAGGGAGCTGGCCGCCGTGTACAGGGCCGTGGCCGCCGAGGTGAACAGACTGAG CCTGAGCAGCGTGGCCATCCCCCTGCTGAGCACCGGCGTGTTCAGCGGCGGCAGAGATA GGCTGCAGCAGAGCCTGAACCACCTGTTCACCGCCATGGACGCCACCGACGCCGACGTG ACCAT CTACT GCAGGGACAAGAGCT GGGAGAAGAAGAT CCAGGAGGCCAT CGACAT GAG AACCGCCGTGGAGCTGCTGAACGACGACGTGGAGCTGACCACCGACCTGGTGAGGGTGC ACCCCGACAGCAGCCTGGTGGGCAGGAAGGGCTACAGCACCACCGACGGCAGCCTGTAC AGCTACTTCGAGGGCACCAAGTTCAACCAGGCCGCCATCGACATGGCCGAGATCCTGAC CCTGTGGCCCAGGCTGCAGGAGGCCAACGAGCAGATCTGCCTGTACGCCCTGGGCGAGA CAATGGACAACATCAGAAGCAAGTGCCCCGTGAACGACAGCGACAGCAGCACCCCCCCC AGGACCGTGCCCTGCCTGTGCAGATACGCCATGACCGCCGAGAGGATCGCCAGGCTGAG GAGCCACCAGGTGAAGTCCATGGTGGTGTGCAGCAGCTTCCCCCTGCCCAAGTACCACG TGGACGGCGTGCAGAAGGTGAAGTGCGAGAAGGTGCTGCTGTTCGACCCCACCGTGCCC AGCGTGGTGAGCCCCAGAAAGTACGCCGCCAGCACCACCGACCACAGCGACAGAAGCCT GAGGGGCTTCGACCTGGACTGGACCACCGACAGCAGCAGCACCGCCAGCGACACCATGA GCCTGCCCAGCCTGCAGAGCTGCGACATCGACAGCATCTACGAGCCCATGGCCCCCATC GTGGTGACCGCCGACGTGCACCCCGAGCCCGCCGGCATCGCCGACCTGGCCGCCGACGT GCACCCCGAGCCCGCCGACCACGTGGACCTGGAGAACCCCATCCCCCCCCCCAGACCCA AGAGGGCCGCCTACCTGGCCAGCAGAGCCGCCGAGAGGCCCGTGCCCGCCCCCAGGAAG CCCACCCCCGCCCCCAGGACCGCCTTCAGGAACAAGCTGCCCCTGACCTTCGGCGACTT CGACGAGCACGAGGTGGACGCCCTGGCCAGCGGCATCACCTTCGGCGACTTCGACGACG TGCTGAGGCTGGGCAGGGCCGGCGCCTACATCTTCAGCAGCGACACCGGCAGCGGCCAC CTGCAGCAGAAGTCCGTGAGACAGCACAACCTGCAGTGCGCCCAGCTGGACGCCGTGGA GGAGGAGAAGATGTACCCCCCCAAGCTGGACACCGAGAGGGAGAAGCTGCTGCTGCTGA AGATGCAGATGCACCCCAGCGAGGCCAACAAGAGCAGATACCAGAGCAGGAAGGTGGAG AACATGAAGGCCACCGTGGTGGACAGACTGACCAGCGGCGCCAGACTGTACACCGGCGC CGACGTGGGCAGGATCCCCACCTACGCCGTGAGATACCCCAGACCCGTGTACAGCCCCA CCGTGATCGAGAGATTCAGCAGCCCCGACGTGGCCATCGCCGCCTGCAACGAGTACCTG AGCAGGAACTACCCCACCGTGGCCAGCTACCAGATCACCGACGAGTACGACGCCTACCT GGACATGGTGGACGGCAGCGACAGCTGCCTGGACAGGGCCACCTTCTGCCCCGCCAAGC TGAGGTGCTACCCCAAGCACCACGCCTACCACCAGCCCACCGTGAGAAGCGCCGTGCCC AGCCCCTTCCAGAACACCCTGCAGAACGTGCTGGCCGCCGCCACCAAGAGGAACTGCAA

[0309]

[0310] CGTGACCCAGATGAGGGAGCTGCCCACCATGGACAGCGCCGTGTTCAACGTGGAGTGCT

[0311] 12673398.1 TCAAGAGGTACGCCTGCAGCGGCGAGTACTGGGAGGAGTACGCCAAGCAGCCCATCAGG ATCACCACCGAGAACATCACCACCTACGTGACCAAGCTGAAGGGCCCCAAGGCCGCCGC CCTGTTCGCCAAGACCCACAACCTGGTGCCCCTGCAGGAGGTGCCCATGGACAGATTCA CCGTGGACATGAAGAGGGACGTGAAGGTGACCCCCGGCACCAAGCACACCGAGGAGAGG CCCAAGGTGCAGGTGATCCAGGCCGCCGAGCCCCTGGCCACCGCCTACCTGTGCGGCAT CCACAGGGAGCTGGTGAGAAGGCTGAACGCCGTGCTGAGGCCCAACGTGCACACCCTGT TCGACATGAGCGCCGAGGACTTCGACGCCATCATCGCCAGCCACTTCCACCCCGGCGAC CCCGTGCTGGAGACAGACATCGCCAGCTTCGACAAGAGCCAGGACGACAGCCTGGCCCT GACCGGCCTGATGATCCTGGAGGACCTGGGCGTGGACCAGTACCTGCTGGACCTGATCG AGGCCGCCTTCGGCGAGATCAGCAGCTGCCACCTGCCCACCGGCACCAGATTCAAGTTC GGCGCCATGATGAAGTCCGGCATGTTCCTGACCCTGTTCATCAACACCGTGCTGAACAT CACCATCGCCAGCAGAGTGCTGGAGCAGAGGCTGACCGACAGCGCCTGCGCCGCCTTCA TCGGCGACGACAACATCGTGCACGGCGTGATCAGCGACAAGCTGATGGCCGAGAGGTGC GCCAGCTGGGTGAACATGGAGGTGAAGATCATCGACGCCGTGATGGGCGAGAAGCCCCC CTACTTCTGCGGCGGCTTCATCGTGTTCGACAGCGTGACCCAGACCGCCTGCAGGGTGA GCGACCCCCTGAAGAGGCTGTTCAAGCTGGGCAAGCCCCTGACCGCCGAGGACAAGCAG GACGAGGACAGAAGGAGGGCCCTGAGCGACGAGGTGAGCAAGTGGTTCAGGACCGGCCT GGGCGCCGAGCTGGAGGTGGCCCTGACCAGCAGGTACGAGGTGGAGGGCTGCAAGAGCA TCCTGATCGCCATGGCCACCCTGGCCAGAGACATCAAGGCCTTCAAGAAGCTGAGAGGC CCCGTGATCCACCTGTACGGCGGCCCCAGACTGGTGAGG _

[0312] RNA AUGGCCGCCAAAGUACACGUUGACAUCGAGGCCGACAGCCCCUUCAUCAAGAGCCUGCA 50 Sequence GAAGGCCUUCCCCAGCUUCGAGGUGGAGAGCCUGCAGGUGACCCCCAACGACCACGCCA ACGCCAGGGCCUUCAGCCACCUGGCCACCAAGCUGAUCGAGCAGGAGACAGACAAGGAC ACCCUGAUCCUGGACAUCGGCAGCGCCCCCAGCAGGAGGAUGAUGAGCACCCACAAGUA CCACUGCGUGUGCCCCAUGAGGAGCGCCGAGGACCCCGAGAGGCUGGUGUGCUACGCCA AGAAGCUGGCCGCCGCCAGCGGCAAGGUGCUGGACAGGGAGAUCGCCGGCAAGAUCACC GACCUGCAGACCGUGAUGGCCACCCCCGACGCCGAGAGCCCCACCUUCUGCCUGCACAC CGACGUGACCUGCAGAACCGCCGCCGAGGUGGCCGUGUACCAGGACGUGUACGCCGUGC ACGCCCCCACCAGCCUGUACCACCAGGCCAUGAAGGGCGUGAGAACCGCCUACUGGAUC GGCUUCGACACCACCCCCUUCAUGUUCGACGCCCUGGCCGGCGCCUACCCCACCUACGC CACCAACUGGGCCGACGAGCAGGUGCUGCAGGCCAGAAACAUCGGCCUGUGCGCCGCCA GCCUGACCGAGGGCAGGCUGGGCAAGCUGAGCAUCCUGAGGAAGAAGCAGCUGAAGCCC UGCGACACCGUGAUGUUCAGCGUGGGCAGCACCCUGUACACCGAGAGCAGAAAGCUGCU GAGAAGCUGGCACCUGCCCAGCGUGUUCCACCUGAAGGGCAAGCAGAGCUUCACCUGCA GGUGCGACACCAUCGUGAGCUGCGAGGGCUACGUGGUGAAGAAGAUCACCAUGUGCCCC GGCCUGUACGGCAAGACCGUGGGCUACGCCGUGACCUACCACGCCGAGGGCUUCCUGGU GUGCAAGACCACCGACACCGUGAAGGGCGAGAGGGUGAGCUUCCCCGUGUGCACCUACG UGCCCAGCACCAUCUGCGACCAGAUGACCGGCAUCCUGGCCACCGACGUGACCCCCGAG GACGCCCAGAAGCUGCUGGUGGGCCUGAACCAGAGGAUCGUGGUGAACGGCAGGACCCA GAGAAACACCAACACCAUGAAGAACUACCUGCUGCCCAUCGUGGCCGUGGCCUUCAGCA AGUGGGCCAGAGAGUACAAGGCCGACCUGGACGACGAGAAGCCCCUGGGCGUGAGAGAG AGGAGCCUGACCUGCUGCUGCCUGUGGGCCUUCAAGACCAGGAAGAUGCACACCAUGUA CAAGAAGCCCGACACCCAGACCAUCGUGAAGGUGCCCAGCGAGUUCAACAGCUUCGUGA UCCCCAGCCUGUGGAGCACCGGCCUGGCCAUCCCCGUGAGGAGCAGGAUCAAGAUGCUG CUGGCCAAGAAAACCAAGAGGGAGCUGAUCCCCGUGCUGGACGCCAGCAGCGCCAGGGA CGCCGAGCAGGAGGAGAAGGAGAGGCUGGAGGCCGAGCUGACCAGAGAGGCCCUGCCCC CCCUGGUGCCCAUCGCCCCCGCCGAGACAGGCGUGGUGGACGUGGACGUGGAGGAGCUG GAGUACCACGCCGGCGCCGGCGUGGUGGAAACCCCCAGGAGCGCCCUGAAGGUGACCGC CCAGCCCAACGACGUGCUGCUGGGCAACUACGUGGUGCUGAGCCCCCAGACCGUGCUGA AAAGCAGCAAGCUGGCCCCCGUGCACCCCCUGGCCGAGCAGGUGAAGAUCAUCACCCAC AACGGCAGGGCCGGCAGGUACCAGGUGGACGGCUACGACGGCAGGGUGCUGCUGCCCUG CGGCAGCGCCAUCCCCGUGCCCGAGUUCCAGGCCCUGAGCGAGAGCGCCACCAUGGUGU ACAACGAGAGGGAGUUCGUGAACAGAAAGCUGUACCACAUCGCCGUGCACGGCCCCAGC CUGAACACCGACGAGGAGAACUACGAGAAGGUGAGGGCCGAGAGAACCGACGCCGAGUA CGUGUUCGACGUGGACAAGAAGUGCUGCGUGAAGAGGGAGGAGGCCAGCGGCCUGGUGC UGGUGGGCGAGCUGACCAACCCCCCCUUCCACGAGUUCGCCUACGAGGGCCUGAAGAUC AGGCCCAGCGCCCCCUACAAGACCACCGUGGUGGGCGUGUUCGGCGUGCCCGGCAGCGG CAAGAGCGCCAUCAUCAAGAGCCUGGUGACCAAGCACGACCUGGUGACCAGCGGCAAGA AGGAGAACUGCCAGGAGAUCGUGAACGACGUGAAGAAGCACAGAGGCCUGGACAUCCAG

[0313]

[0314] GCCAAGACCGUGGACAGCAUCCUGCUGAACGGCUGCAGGAGGGCCGUGGACAUCCUGUA

[0315] 12673398.1 CGUGGACGAGGCCUUCGCCUGCCACAGCGGCACCCUGCUGGCCCUGAUCGCCCUGGUGA AGCCCAGAAGCAAGGUGGUGCUGUGCGGCGACCCCAAGCAGUGCGGCUUCUUCAACAUG AUGCAGCUGAAGGUGAACUUCAACCACAACAUCUGCACCGAGGUGUGCCACAAGAGCAU CAGCAGGAGGUGCACCAGGCCCGUGACCGCCAUCGUGAGCACCCUGCACUACGGCGGCA AGAUGAGGACCACCAACCCCUGCAACAAGCCCAUCAUCAUCGACACCACCGGCCAGACC AAGCCCAAGCCCGGCGACAUCGUGCUGACCUGCUUCAGGGGCUGGGUGAAGCAGCUGCA GCUGGACUACAGGGGCCACGAGGUGAUGACCGCCGCCGCCAGCCAGGGCCUGACCAGGA AGGGCGUGUACGCCGUGAGGCAGAAGGUGAACGAGAACCCCCUGUACGCCCCCGCCAGC GAGCACGUGAACGUGCUGCUGACCAGGACCGAGGACAGGCUGGUGUGGAAAACCCUGGC CGGCGACCCCUGGAUCAAGGUGCUGAGCAACAUCCCCCAGGGCAACUUCACCGCCACCC UGGAGGAGUGGCAGGAGGAGCACGACAAGAUCAUGAAGGUGAUCGAGGGCCCCGCCGCC CCCGUGGACGCCUUCCAGAACAAGGCCAACGUGUGCUGGGCCAAGAGCCUGGUGCCCGU GCUGGACACCGCCGGCAUCAGGCUGACCGCCGAGGAGUGGAGCACCAUCAUCACCGCCU UCAAGGAGGACAGGGCCUACAGCCCCGUGGUGGCCCUGAACGAGAUCUGCACCAAGUAC UACGGCGUGGACCUGGACAGCGGCCUGUUCAGCGCCCCCAAGGUGAGCCUGUACUACGA GAACAACCACUGGGACAACAGGCCCGGCGGCAGAAUGUACGGCUUCAACGCCGCCACCG CCGCCAGGCUGGAGGCCAGGCACACCUUCCUGAAGGGCCAGUGGCACACCGGCAAGCAG GCCGUGAUCGCCGAGAGGAAGAUCCAGCCCCUGAGCGUGCUGGACAACGUGAUCCCCAU CAACAGAAGGCUGCCCCACGCCCUGGUGGCCGAGUACAAGACCGUGAAGGGCAGCAGGG UGGAGUGGCUGGUGAACAAGGUGAGGGGCUACCACGUGCUGCUGGUGAGCGAGUACAAC CUGGCCCUGCCCAGGAGAAGGGUGACCUGGCUGAGCCCCCUGAACGUGACCGGCGCCGA CAGGUGCUACGACCUGAGCCUGGGCCUGCCCGCCGACGCCGGCAGGUUCGACCUGGUGU UCGUGAACAUCCACACCGAGUUCAGGAUCCACCACUACCAGCAGUGCGUGGACCACGCC AUGAAGCUGCAGAUGCUGGGCGGCGACGCCCUGAGGCUGCUGAAGCCCGGCGGCAGCCU GCUGAUGAGGGCCUACGGCUACGCCGACAAGAUCAGCGAGGCCGUGGUGAGCAGCCUGA GCAGGAAGUUCAGCAGCGCCAGAGUGCUGAGGCCCGACUGCGUGACCAGCAACACCGAG GUGUUCCUGCUGUUCAGCAACUUCGACAACGGCAAGAGGCCCAGCACCCUGCACCAGAU GAACACCAAGCUGAGCGCCGUGUACGCCGGCGAGGCCAUGCACACCGCCGGCUGCGCCC CCAGCUACAGGGUGAAGAGGGCCGACAUCGCCACCUGCACCGAGGCCGCCGUGGUGAAC GCCGCCAACGCCAGGGGCACCGUGGGCGACGGCGUGUGCAGGGCCGUGGCCAAGAAGUG GCCCAGCGCCUUCAAGGGCGAGGCCACCCCCGUGGGCACCAUCAAGACCGUGAUGUGCG GCAGCUACCCCGUGAUCCACGCCGUGGCCCCCAACUUCAGCGCCACCACCGAGGCCGAG GGCGACAGGGAGCUGGCCGCCGUGUACAGGGCCGUGGCCGCCGAGGUGAACAGACUGAG CCUGAGCAGCGUGGCCAUCCCCCUGCUGAGCACCGGCGUGUUCAGCGGCGGCAGAGAUA GGCUGCAGCAGAGCCUGAACCACCUGUUCACCGCCAUGGACGCCACCGACGCCGACGUG ACCAUCUACUGCAGGGACAAGAGCUGGGAGAAGAAGAUCCAGGAGGCCAUCGACAUGAG AACCGCCGUGGAGCUGCUGAACGACGACGUGGAGCUGACCACCGACCUGGUGAGGGUGC ACCCCGACAGCAGCCUGGUGGGCAGGAAGGGCUACAGCACCACCGACGGCAGCCUGUAC AGCUACUUCGAGGGCACCAAGUUCAACCAGGCCGCCAUCGACAUGGCCGAGAUCCUGAC CCUGUGGCCCAGGCUGCAGGAGGCCAACGAGCAGAUCUGCCUGUACGCCCUGGGCGAGA CAAUGGACAACAUCAGAAGCAAGUGCCCCGUGAACGACAGCGACAGCAGCACCCCCCCC AGGACCGUGCCCUGCCUGUGCAGAUACGCCAUGACCGCCGAGAGGAUCGCCAGGCUGAG GAGCCACCAGGUGAAGUCCAUGGUGGUGUGCAGCAGCUUCCCCCUGCCCAAGUACCACG UGGACGGCGUGCAGAAGGUGAAGUGCGAGAAGGUGCUGCUGUUCGACCCCACCGUGCCC AGCGUGGUGAGCCCCAGAAAGUACGCCGCCAGCACCACCGACCACAGCGACAGAAGCCU GAGGGGCUUCGACCUGGACUGGACCACCGACAGCAGCAGCACCGCCAGCGACACCAUGA GCCUGCCCAGCCUGCAGAGCUGCGACAUCGACAGCAUCUACGAGCCCAUGGCCCCCAUC GUGGUGACCGCCGACGUGCACCCCGAGCCCGCCGGCAUCGCCGACCUGGCCGCCGACGU GCACCCCGAGCCCGCCGACCACGUGGACCUGGAGAACCCCAUCCCCCCCCCCAGACCCA AGAGGGCCGCCUACCUGGCCAGCAGAGCCGCCGAGAGGCCCGUGCCCGCCCCCAGGAAG CCCACCCCCGCCCCCAGGACCGCCUUCAGGAACAAGCUGCCCCUGACCUUCGGCGACUU CGACGAGCACGAGGUGGACGCCCUGGCCAGCGGCAUCACCUUCGGCGACUUCGACGACG UGCUGAGGCUGGGCAGGGCCGGCGCCUACAUCUUCAGCAGCGACACCGGCAGCGGCCAC CUGCAGCAGAAGUCCGUGAGACAGCACAACCUGCAGUGCGCCCAGCUGGACGCCGUGGA GGAGGAGAAGAUGUACCCCCCCAAGCUGGACACCGAGAGGGAGAAGCUGCUGCUGCUGA AGAUGCAGAUGCACCCCAGCGAGGCCAACAAGAGCAGAUACCAGAGCAGGAAGGUGGAG AACAUGAAGGCCACCGUGGUGGACAGACUGACCAGCGGCGCCAGACUGUACACCGGCGC CGACGUGGGCAGGAUCCCCACCUACGCCGUGAGAUACCCCAGACCCGUGUACAGCCCCA CCGUGAUCGAGAGAUUCAGCAGCCCCGACGUGGCCAUCGCCGCCUGCAACGAGUACCUG

[0316]

[0317] AGCAGGAACUACCCCACCGUGGCCAGCUACCAGAUCACCGACGAGUACGACGCCUACCU

[0318] 12673398.1 GGACAUGGUGGACGGCAGCGACAGCUGCCUGGACAGGGCCACCUUCUGCCCCGCCAAGC UGAGGUGCUACCCCAAGCACCACGCCUACCACCAGCCCACCGUGAGAAGCGCCGUGCCC AGCCCCUUCCAGAACACCCUGCAGAACGUGCUGGCCGCCGCCACCAAGAGGAACUGCAA CGUGACCCAGAUGAGGGAGCUGCCCACCAUGGACAGCGCCGUGUUCAACGUGGAGUGCU UCAAGAGGUACGCCUGCAGCGGCGAGUACUGGGAGGAGUACGCCAAGCAGCCCAUCAGG AUCACCACCGAGAACAUCACCACCUACGUGACCAAGCUGAAGGGCCCCAAGGCCGCCGC CCUGUUCGCCAAGACCCACAACCUGGUGCCCCUGCAGGAGGUGCCCAUGGACAGAUUCA CCGUGGACAUGAAGAGGGACGUGAAGGUGACCCCCGGCACCAAGCACACCGAGGAGAGG CCCAAGGUGCAGGUGAUCCAGGCCGCCGAGCCCCUGGCCACCGCCUACCUGUGCGGCAU CCACAGGGAGCUGGUGAGAAGGCUGAACGCCGUGCUGAGGCCCAACGUGCACACCCUGU UCGACAUGAGCGCCGAGGACUUCGACGCCAUCAUCGCCAGCCACUUCCACCCCGGCGAC CCCGUGCUGGAGACAGACAUCGCCAGCUUCGACAAGAGCCAGGACGACAGCCUGGCCCU GACCGGCCUGAUGAUCCUGGAGGACCUGGGCGUGGACCAGUACCUGCUGGACCUGAUCG AGGCCGCCUUCGGCGAGAUCAGCAGCUGCCACCUGCCCACCGGCACCAGAUUCAAGUUC GGCGCCAUGAUGAAGUCCGGCAUGUUCCUGACCCUGUUCAUCAACACCGUGCUGAACAU CACCAUCGCCAGCAGAGUGCUGGAGCAGAGGCUGACCGACAGCGCCUGCGCCGCCUUCA UCGGCGACGACAACAUCGUGCACGGCGUGAUCAGCGACAAGCUGAUGGCCGAGAGGUGC GCCAGCUGGGUGAACAUGGAGGUGAAGAUCAUCGACGCCGUGAUGGGCGAGAAGCCCCC CUACUUCUGCGGCGGCUUCAUCGUGUUCGACAGCGUGACCCAGACCGCCUGCAGGGUGA GCGACCCCCUGAAGAGGCUGUUCAAGCUGGGCAAGCCCCUGACCGCCGAGGACAAGCAG GACGAGGACAGAAGGAGGGCCCUGAGCGACGAGGUGAGCAAGUGGUUCAGGACCGGCCU GGGCGCCGAGCUGGAGGUGGCCCUGACCAGCAGGUACGAGGUGGAGGGCUGCAAGAGCA UCCUGAUCGCCAUGGCCACCCUGGCCAGAGACAUCAAGGCCUUCAAGAAGCUGAGAGGC CCCGUGAUCCACCUGUACGGCGGCCCCAGACUGGUGAGG

[0319] Amino MAAKVHVDIEADSPFIKSLQKAFPSFEVESLQVTPNDHANARAFSHLATKLIEQETDKD 84 Acid TLILDIGSAPSRRMMSTHKYHCVCPMRSAEDPERLVCYAKKLAAASGKVLDREIAGKIT Sequence DLQTVMATPDAESPTFCLHTDVTCRTAAEVAVYQDVYAVHAPTSLYHQAMKGVRTAYWI GFDTTPFMFDALAGAYPTYATNWADEQVLQARNIGLCAASLTEGRLGKLSILRKKQLKP CDTVMFSVGSTLYTESRKLLRSWHLPSVFHLKGKQSFTCRCDTIVSCEGYWKKITMCP GLYGKTVGYAVTYHAEGFLVCKTTDTVKGERVSFPVCTYVPSTICDQMTGILATDVTPE DAQKLLVGLNQRIWNGRTQRNTNTMKNYLLPIVAVAFSKWAREYKADLDDEKPLGVRE RSLTCCCLWAFKTRKMHTMYKKPDTQTIVKVPSEFNSFVIPSLWSTGLAI PVRSRIKML LAKKTKRELIPVLDASSARDAEQEEKERLEAELTREALPPLVPIAPAETGWDVDVEEL EYHAGAGWETPRSALKVTAQPNDVLLGNYWLSPQTVLKSSKLAPVHPLAEQVKIITH NGRAGRYQVDGYDGRVLLPCGSAI PVPEFQALSESATMVYNEREFVNRKLYHIAVHGPS LNTDEENYEKVRAERTDAEYVFDVDKKCCVKREEASGLVLVGELTNPPFHEFAYEGLKI RPSAPYKTTWGVFGVPGSGKSAIIKSLVTKHDLVTSGKKENCQEIVNDVKKHRGLDIQ AKTVDSILLNGCRRAVDILYVDEAFACHSGTLLALIALVKPRSKWLCGDPKQCGFFNM MQLKVNFNHNICTEVCHKSISRRCTRPVTAIVSTLHYGGKMRTTNPCNKPIIIDTTGQT KPKPGDIVLTCFRGWVKQLQLDYRGHEVMTAAASQGLTRKGVYAVRQKVNENPLYAPAS EHVNVLLTRTEDRLVWKTLAGDPWIKVLSNIPQGNFTATLEEWQEEHDKIMKVIEGPAA PVDAFQNKANVCWAKSLVPVLDTAGIRLTAEEWSTIITAFKEDRAYSPWALNEICTKY YGVDLDSGLFSAPKVSLYYENNHWDNRPGGRMYGFNAATAARLEARHTFLKGQWHTGKQ AVIAERKIQPLSVLDNVIPINRRLPHALVAEYKTVKGSRVEWLVNKVRGYHVLLVSEYN LALPRRRVTWLSPLNVTGADRCYDLSLGLPADAGRFDLVFVNIHTEFRIHHYQQCVDHA MKLQMLGGDALRLLKPGGSLLMRAYGYADKISEAWSSLSRKFSSARVLRPDCVTSNTE VFLLFSNFDNGKRPSTLHQMNTKLSAVYAGEAMHTAGCAPSYRVKRADIATCTEAAWN AANARGTVGDGVCRAVAKKWPSAFKGEATPVGTIKTVMCGSYPVIHAVAPNFSATTEAE GDRELAAVYRAVAAEVNRLSLSSVAIPLLSTGVFSGGRDRLQQSLNHLFTAMDATDADV TIYCRDKSWEKKIQEAIDMRTAVELLNDDVELTTDLVRVHPDSSLVGRKGYSTTDGSLY SYFEGTKFNQAAIDMAEILTLWPRLQEANEQICLYALGETMDNIRSKCPVNDSDSSTPP RTVPCLCRYAMTAERIARLRSHQVKSMWCSSFPLPKYHVDGVQKVKCEKVLLFDPTVP SWSPRKYAASTTDHSDRSLRGFDLDWTTDSSSTASDTMSLPSLQSCDIDSIYEPMAPI WTADVHPEPAGIADLAADVHPEPADHVDLENPIPPPRPKRAAYLASRAAERPVPAPRK PTPAPRTAFRNKLPLTFGDFDEHEVDALASGITFGDFDDVLRLGRAGAYIFSSDTGSGH LQQKSVRQHNLQCAQLDAVEEEKMYPPKLDTEREKLLLLKMQMHPSEANKSRYQSRKVE NMKAT WDRLT S GARLYTGADVGRI PT YAVRYPRPVYS PTVI ERFS S PDVAI AACNEYL SRNYPTVASYQITDEYDAYLDMVDGSDSCLDRATFCPAKLRCYPKHHAYHQPTVRSAVP SPFQNTLQNVLAAATKRNCNVTQMRELPTMDSAVFNVECFKRYACSGEYWEEYAKQPIR

[0320]

[0321] ITTENITTYVTKLKGPKAAALFAKTHNLVPLQEVPMDRFTVDMKRDVKVTPGTKHTEER

[0322] 12673398.1 PKVQVIQAAEPLATAYLCGIHRELVRRLNAVLRPNVHTLFDMSAEDFDAIIASHFHPGD PVLETDIASFDKSQDDSLALTGLMILEDLGVDQYLLDLIEAAFGEISSCHLPTGTRFKF GAMMKSGMFLTLFINTVLNITIASRVLEQRLTDSACAAFIGDDNIVHGVISDKLMAERC ASWVNMEVKIIDAVMGEKPPYFCGGFIVFDSVTQTACRVSDPLKRLFKLGKPLTAEDKQ DEDRRRALSDEVSKWFRTGLGAELEVALTSRYEVEGCKSILIAMATLARDIKAFKKLRG PVIHLYGGPRLVR

[0323]

[0324] “RLE” Mutant SFV Replicase

[0325] DNA ATGGCCGCCAAAGTACACGTTGACATCGAGGCCGACAGCCCCTTCATCAAGAGCCTGCA 10 Sequence GAAGGCCTTCCCCAGCTTCGAGGTGGAGAGCCTGCAGGTGACCCCCAACGACCACGCCA ACGCCAGGGCCTTCAGCCACCTGGCCACCAAGCTGATCGAGCAGGAGACAGACAAGGAC ACCCTGATCCTGGACATCGGCAGCGCCCCCAGCAGGAGGATGATGAGCACCCACAAGTA CCACTGCGTGTGCCCCATGAGGAGCGCCGAGGACCCCGAGAGGCTGGTGTGCTACGCCA AGAAGCTGGCCGCCGCCAGCGGCAAGGTGCTGGACAGGGAGATCGCCGGCAAGATCACC GACCTGCAGACCGTGATGGCCACCCCCGACGCCGAGAGCCCCACCTTCTGCCTGCACAC CGACGTGACCTGCAGAACCGCCGCCGAGGTGGCCGTGTACCAGGACGTGTACGCCGTGC ACGCCCCCACCAGCCTGTACCACCAGGCCATGAAGGGCGTGAGAACCGCCTACTGGATC GGCTTCGACACCACCCCCTTCATGTTCGACGCCCTGGCCGGCGCCTACCCCACCTACGC CACCAACTGGGCCGACGAGCAGGTGCTGCAGGCCAGAAACATCGGCCTGTGCGCCGCCA GCCTGACCGAGGGCAGGCTGGGCAAGCTGAGCATCCTGAGGAAGAAGCAGCTGAAGCCC TGCGACACCGTGATGTTCAGCGTGGGCAGCACCCTGTACACCGAGAGCAGAAAGCTGCT GAGAAGCTGGCACCTGCCCAGCGTGTTCCACCTGAAGGGCAAGCAGAGCTTCACCTGCA GGTGCGACACCATCGTGAGCTGCGAGGGCTACGTGGTGAAGAAGATCACCATGTGCCCC GGCCTGTACGGCAAGACCGTGGGCTACGCCGTGACCTACCACGCCGAGGGCTTCCTGGT GTGCAAGACCACCGACACCGTGAAGGGCGAGAGGGTGAGCTTCCCCGTGTGCACCTACG TGCCCAGCACCATCTGCGACCAGATGACCGGCATCCTGGCCACCGACGTGACCCCCGAG GACGCCCAGAAGCTGCTGGTGGGCCTGAACCAGAGGATCGTGGTGAACGGCAGGACCCA GAGAAACACCAACACCATGAAGAACTACCTGCTGCCCATCGTGGCCGTGGCCTTCAGCA AGTGGGCCAGAGAGTACAAGGCCGACCTGGACGACGAGAAGCCCCTGGGCGTGAGAGAG AGGAGCCTGACCTGCTGCTGCCTGTGGGCCTTCAAGACCAGGAAGATGCACACCATGTA CAAGAAGCCCGACACCCAGACCATCGTGAAGGTGCCCAGCGAGTTCAACAGCTTCGTGA TCCCCAGCCTGTGGAGCACCGGCCTGGCCATCCCCGTGAGGAGCAGGATCAAGATGCTG CTGGCCAAGAAAACCAAGAGGGAGCTGATCCCCGTGCTGGACGCCAGCAGCGCCAGGGA CGCCGAGCAGGAGGAGAAGGAGAGGCTGGAGGCCGAGCTGACCAGAGAGGCCCTGCCCC CCCTGGTGCCCATCGCCCCCGCCGAGACAGGCGTGGTGGACGTGGACGTGGAGGAGCTG GAGTACCACGCCGGCGCCGGCGTGGTGGAAACCCCCAGGAGCGCCCTGAAGGTGACCGC CCAGCCCAACGACGTGCTGCTGGGCAACTACGTGGTGCTGAGCCCCCAGACCGTGCTGA AAAGCAGCAAGCTGGCCCCCGTGCACCCCCTGGCCGAGCAGGTGAAGATCATCACCCAC AACGGCAGGGCCGGCAGGTACCAGGTGGACGGCTACGACGGCAGGGTGCTGCTGCCCTG CGGCAGCGCCATCCCCGTGCCCGAGTTCCAGGCCCTGAGCGAGAGCGCCACCATGGTGT ACAACGAGAGGGAGTTCGTGAACAGAAAGCTGTACCACATCGCCGTGCACGGCCCCAGC CTGAACACCGACGAGGAGAACTACGAGAAGGTGAGGGCCGAGAGAACCGACGCCGAGTA CGTGTTCGACGTGGACAAGAAGTGCTGCGTGAAGAGGGAGGAGGCCAGCGGCCTGGTGC TGGTGGGCGAGCTGACCAACCCCCCCTTCCACGAGTTCGCCTACGAGGGCCTGAAGATC AGGCCCAGCGCCCCCTACAAGACCACCGTGGTGGGCGTGTTCGGCGTGCCCGGCAGCGG CAAGAGCGCCATCATCAAGAGCCTGGTGACCAAGCACGACCTGGTGACCAGCGGCAAGA AGGAGAACTGCCAGGAGATCGTGAACGACGTGAAGAAGCACAGAGGCCTGGACATCCAG GCCAAGACCGTGGACAGCATCCTGCTGAACGGCTGCAGGAGGGCCGTGGACATCCTGTA CGTGGACGAGGCCTTCGCCTGCCACAGCGGCACCCTGCTGGCCCTGATCGCCCTGGTGA AGCCCAGAAGCAAGGTGGTGCTGTGCGGCGACCCCAAGCAGTGCGGCTTCTTCAACATG ATGCAGCTGAAGGTGAACTTCAACCACAACATCTGCACCGAGGTGTGCCACAAGAGCAT CAGCAGGAGGTGCACCAGGCCCGTGACCGCCATCGTGAGCACCCTGCACTACGGCGGCA AGATGAGGACCACCAACCCCTGCAACAAGCCCATCATCATCGACACCACCGGCCAGACC AAGCCCAAGCCCGGCGACATCGTGCTGACCTGCTTCAGGGGCTGGGTGAAGCAGCTGCA GCTGGACTACAGGGGCCACGAGGTGATGACCGCCGCCGCCAGCCAGGGCCTGACCAGGA AGGGCGTGTACGCCGTGAGGCAGAAGGTGAACGAGAACCCCCTGTACGCCCCCGCCAGC GAGCACGTGAACGTGCTGCTGACCAGGACCGAGGACAGGCTGGTGTGGAAAACCCTGGC CGGCGACCCCTGGATCAAGGTGCTGAGCAACATCCCCCAGGGCAACTTCACCGCCACCC TGGAGGAGTGGCAGGAGGAGCACGACAAGATCATGAAGGTGATCGAGGGCCCCGCCGCC CCCGTGGACGCCTTCCAGAACAAGGCCAACGTGTGCTGGGCCAAGAGCCTGGTGCCCGT

[0326]

[0327] GCTGGACACCGCCGGCATCAGGCTGACCGCCGAGGAGTGGAGCACCATCATCACCGCCT

[0328] 12673398.1 TCAAGGAGGACAGGGCCTACAGCCCCGTGGTGGCCCTGAACGAGATCTGCACCAAGTAC TACGGCGTGGACCTGGACAGCGGCCTGTTCAGCGCCCCCAAGGTGAGCCTGTACTACGA GAACAACCACTGGGACAACAGGCCCGGCGGCAGAATGTACGGCTTCAACGCCGCCACCG CCGCCAGGCTGGAGGCCAGGCACACCTTCCTGAAGGGCCAGTGGCACACCGGCAAGCAG GCCGTGATCGCCGAGAGGAAGATCCAGCCCCTGAGCGTGCTGGACAACGTGATCCCCAT CAACAGAAGGCTGCCCCACGCCCTGGTGGCCGAGTACAAGACCGTGAAGGGCAGCAGGG TGGAGTGGCTGGTGAACAAGGTGAGGGGCTACCACGTGCTGCTGGTGAGCGAGTACAAC CTGGCCCTGCCCAGGAGAAGGGTGACCTGGCTGAGCCCCCTGAACGTGACCGGCGCCGA CAGGTGCTACGACCTGAGCCTGGGCCTGCCCGCCGACGCCGGCAGGTTCGACCTGGTGT TCGTGAACATCCACACCGAGTTCAGGATCCACCACTACCAGCAGTGCGTGGACCACGCC ATGAAGCTGCAGATGCTGGGCGGCGACGCCCTGAGGCTGCTGAAGCCCGGCGGCAGCCT GCTGATGAGGGCCTACGGCTACGCCGACAAGATCAGCGAGGCCGTGGTGAGCAGCCTGA GCAGGAAGTTCAGCAGCGCCAGAGTGCTGAGGCCCGACTGCGTGACCAGCAACACCGAG GTGTTCCTGCTGTTCAGCAACTTCGACAACGGCAAGAGGCCCAGCACCCTGCACCAGAT GAACACCAAGCTGAGCGCCGTGTACGCCGGCGAGGCCATGCACACCGCCGGCTGCGCCC CCAGCTACAGGGTGAAGAGGGCCGACATCGCCACCTGCACCGAGGCCGCCGTGGTGAAC GCCGCCAACGCCAGGGGCACCGTGGGCGACGGCGTGTGCAGGGCCGTGGCCAAGAAGTG GCCCAGCGCCTTCAAGGGCGAGGCCACCCCCGTGGGCACCATCAAGACCGTGATGTGCG GCAGCTACCCCGTGATCCACGCCGTGGCCCCCAACTTCAGCGCCACCACCGAGGCCGAG GGCGACAGGGAGCTGGCCGCCGTGTACAGGGCCGTGGCCGCCGAGGTGAACAGACTGAG CCTGAGCAGCGTGGCCATCCCCCTGCTGAGCACCGGCGTGTTCAGCGGCGGCAGAGATA GGCTGCAGCAGAGCCTGAACCACCTGTTCACCGCCATGGACGCCACCGACGCCGACGTG ACCAT CTACT GCAGGGACAAGAGCT GGGAGAAGAAGAT CCAGGAGGCCAT CGACAT GAG AACCGCCGTGGAGCTGCTGAACGACGACGTGGAGCTGACCACCGACCTGGTGAGGGTGC ACCCCGACAGCAGCCTGGTGGGCAGGAAGGGCTACAGCACCACCGACGGCAGCCTGTAC AGCTACTTCGAGGGCACCAAGTTCAACCAGGCCGCCATCGACATGGCCGAGATCCTGAC CCTGTGGCCCAGGCTGCAGGAGGCCAACGAGCAGATCTGCCTGTACGCCCTGGGCGAGA CAATGGACAACATCAGAAGCAAGTGCCCCGTGAACGACAGCGACAGCAGCACCCCCCCC AGGACCGTGCCCTGCCTGTGCAGATACGCCATGACCGCCGAGAGGATCGCCAGGCTGAG GAGCCACCAGGTGAAGTCCATGGTGGTGTGCAGCAGCTTCCCCCTGCCCAAGTACCACG TGGACGGCGTGCAGAAGGTGAAGTGCGAGAAGGTGCTGCTGTTCGACCCCACCGTGCCC AGCGTGGTGAGCCCCAGAAAGTACGCCGCCAGCACCACCGACCACAGCGACAGAAGCCT GAGGGGCTTCGACCTGGACTGGACCACCGACAGCAGCAGCACCGCCAGCGACACCATGA GCCTGCCCAGCCTGCAGAGCTGCGACATCGACAGCATCTACGAGCCCATGGCCCCCATC GTGGTGACCGCCGACGTGCACCCCGAGCCCGCCGGCATCGCCGACCTGGCCGCCGACGT GCACCCCGAGCCCGCCGACCACGTGGACCTGGAGAACCCCATCCCCCCCCCCAGACCCA AGAGGGCCGCCTACCTGGCCAGCAGAGCCGCCGAGAGGCCCGTGCCCGCCCCCAGGAAG CCCACCCCCGCCCCCAGGACCGCCTTCAGGAACAAGCTGCCCCTGACCTTCGGCGACTT CGACGAGCACGAGGTGGACGCCCTGGCCAGCGGCATCACCTTCGGCGACTTCGACGACG TGCTGAGGCTGGGCAGGGCCGGCGCCTACATCTTCAGCAGCGACACCGGCAGCGGCCAC CTGCAGCAGAAGTCCGTGAGACAGCACAACCTGCAGTGCGCCCAGCTGGACGCCGTGGA GGAGGAGAAGATGTACCCCCCCAAGCTGGACACCGAGAGGGAGAAGCTGCTGCTGCTGA AGATGCAGATGCACCCCAGCGAGGCCAACAAGAGCAGATACCAGAGCAGGAAGGTGGAG AACATGAAGGCCACCGTGGTGGACAGACTGACCAGCGGCGCCAGACTGTACACCGGCGC CGACGTGGGCAGGATCCCCACCTACGCCGTGAGATACCCCAGACCCGTGTACAGCCCCA CCGTGATCGAGAGATTCAGCAGCCCCGACGTGGCCATCGCCGCCTGCAACGAGTACCTG AGCAGGAACTACCCCACCGTGGCCAGCTACCAGATCACCGACGAGTACGACGCCTACCT GGACATGGTGGACGGCAGCGACAGCTGCCTGGACAGGGCCACCTTCTGCCCCGCCAAGC TGAGGTGCTACCCCAAGCACCACGCCTACCACCAGCCCACCGTGAGAAGCGCCGTGCCC AGCCCCTTCCAGAACACCCTGCAGAACGTGCTGGCCGCCGCCACCAAGAGGAACTGCAA CGTGACCCAGATGAGGGAGCTGCCCACCATGGACAGCGCCGTGTTCAACGTGGAGTGCT TCAAGAGGTACGCCTGCAGCGGCGAGTACTGGGAGGAGTACGCCAAGCAGCCCATCAGG ATCACCACCGAGAACATCACCACCTACGTGACCAAGCTGAAGGGCCCCAAGGCCGCCGC CCTGTTCGCCAAGACCCACAACCTGGTGCCCCTGCAGGAGGTGCCCATGGACAGATTCA CCGTGGACATGAAGAGGGACGTGAAGGTGACCCCCGGCACCAAGCACACCGAGGAGAGG CCCAAGGTGCAGGTGATCCAGGCCGCCGAGCCCCTGGCCACCGCCTACCTGTGCGGCAT CCACAGGGAGCTGGTGAGAAGGCTGAACGCCGTGCTGAGGCCCAACGTGCACACCCTGT TCGACATGAGCGCCGAGGACTTCGACGCCATCATCGCCAGCCACTTCCACCCCGGCGAC CCCGTGCTGGAGACAGACATCGCCAGCTTCGACAAGAGCCAGGACGACAGCCTGGCCCT

[0329]

[0330] GACCGGCCTGATGATCCTGGAGGACCTGGGCGTGGACCAGTACCTGCTGGACCTGATCG

[0331] 12673398.1 AGGCCGCCTTCGGCGAGATCAGCAGCTGCCACCTGCCCACCGGCACCAGATTCAAGTTC GGCGCCATGATGAAGTCCGGCATGTTCCTGACCCTGTTCATCAACACCGTGCTGAACAT CACCATCGCCAGCAGAGTGCTGGAGCAGAGGCTGACCGACAGCGCCTGCGCCGCCTTCA TCGGCGACGACAACATCGTGCACGGCGTGATCAGCGACAAGCTGATGGCCGAGAGGTGC GCCAGCTGGGTGAACATGGAGGTGAAGATCATCGACGCCGTGATGGGCGAGAAGCCCCC CTACTTCTGCGGCGGCTTCATCGTGTTCGACAGCGTGACCCAGACCGCCTGCAGGGTGA GCGACCCCCTGAAGAGGCTGTTCAAGCTGGGCAAGCCCCTGACCGCCGAGGACAAGCAG GACGAGGACAGAAGGAGGGCCCTGAGCGACGAGGTGAGCAAGTGGTTCAGGACCGGCCT GGGCGCCGAGCTGGAGGTGGCCCTGACCAGCAGGTACGAGGTGGAGGGCTGCAAGAGCA TCCTGATCGCCATGGCCACCCTGGCCAGAGACATCAAGGCCTTCAAGAAGCTGAGAGGC CCCGTGATCCACCTGTACGGCGGCCCCAGACTGGTGAGG RNA AUGGCCGCCAAAGUACACGUUGACAUCGAGGCCGACAGCCCCUUCAUCAAGAGCCUGCA

[0332] Sequence GAAGGCCUUCCCCAGCUUCGAGGUGGAGAGCCUGCAGGUGACCCCCAACGACCACGCCA ACGCCAGGGCCUUCAGCCACCUGGCCACCAAGCUGAUCGAGCAGGAGACAGACAAGGAC ACCCUGAUCCUGGACAUCGGCAGCGCCCCCAGCAGGAGGAUGAUGAGCACCCACAAGUA CCACUGCGUGUGCCCCAUGAGGAGCGCCGAGGACCCCGAGAGGCUGGUGUGCUACGCCA AGAAGCUGGCCGCCGCCAGCGGCAAGGUGCUGGACAGGGAGAUCGCCGGCAAGAUCACC GACCUGCAGACCGUGAUGGCCACCCCCGACGCCGAGAGCCCCACCUUCUGCCUGCACAC CGACGUGACCUGCAGAACCGCCGCCGAGGUGGCCGUGUACCAGGACGUGUACGCCGUGC ACGCCCCCACCAGCCUGUACCACCAGGCCAUGAAGGGCGUGAGAACCGCCUACUGGAUC GGCUUCGACACCACCCCCUUCAUGUUCGACGCCCUGGCCGGCGCCUACCCCACCUACGC CACCAACUGGGCCGACGAGCAGGUGCUGCAGGCCAGAAACAUCGGCCUGUGCGCCGCCA GCCUGACCGAGGGCAGGCUGGGCAAGCUGAGCAUCCUGAGGAAGAAGCAGCUGAAGCCC UGCGACACCGUGAUGUUCAGCGUGGGCAGCACCCUGUACACCGAGAGCAGAAAGCUGCU GAGAAGCUGGCACCUGCCCAGCGUGUUCCACCUGAAGGGCAAGCAGAGCUUCACCUGCA GGUGCGACACCAUCGUGAGCUGCGAGGGCUACGUGGUGAAGAAGAUCACCAUGUGCCCC GGCCUGUACGGCAAGACCGUGGGCUACGCCGUGACCUACCACGCCGAGGGCUUCCUGGU GUGCAAGACCACCGACACCGUGAAGGGCGAGAGGGUGAGCUUCCCCGUGUGCACCUACG UGCCCAGCACCAUCUGCGACCAGAUGACCGGCAUCCUGGCCACCGACGUGACCCCCGAG GACGCCCAGAAGCUGCUGGUGGGCCUGAACCAGAGGAUCGUGGUGAACGGCAGGACCCA GAGAAACACCAACACCAUGAAGAACUACCUGCUGCCCAUCGUGGCCGUGGCCUUCAGCA AGUGGGCCAGAGAGUACAAGGCCGACCUGGACGACGAGAAGCCCCUGGGCGUGAGAGAG AGGAGCCUGACCUGCUGCUGCCUGUGGGCCUUCAAGACCAGGAAGAUGCACACCAUGUA CAAGAAGCCCGACACCCAGACCAUCGUGAAGGUGCCCAGCGAGUUCAACAGCUUCGUGA UCCCCAGCCUGUGGAGCACCGGCCUGGCCAUCCCCGUGAGGAGCAGGAUCAAGAUGCUG CUGGCCAAGAAAACCAAGAGGGAGCUGAUCCCCGUGCUGGACGCCAGCAGCGCCAGGGA CGCCGAGCAGGAGGAGAAGGAGAGGCUGGAGGCCGAGCUGACCAGAGAGGCCCUGCCCC CCCUGGUGCCCAUCGCCCCCGCCGAGACAGGCGUGGUGGACGUGGACGUGGAGGAGCUG GAGUACCACGCCGGCGCCGGCGUGGUGGAAACCCCCAGGAGCGCCCUGAAGGUGACCGC CCAGCCCAACGACGUGCUGCUGGGCAACUACGUGGUGCUGAGCCCCCAGACCGUGCUGA AAAGCAGCAAGCUGGCCCCCGUGCACCCCCUGGCCGAGCAGGUGAAGAUCAUCACCCAC AACGGCAGGGCCGGCAGGUACCAGGUGGACGGCUACGACGGCAGGGUGCUGCUGCCCUG CGGCAGCGCCAUCCCCGUGCCCGAGUUCCAGGCCCUGAGCGAGAGCGCCACCAUGGUGU ACAACGAGAGGGAGUUCGUGAACAGAAAGCUGUACCACAUCGCCGUGCACGGCCCCAGC CUGAACACCGACGAGGAGAACUACGAGAAGGUGAGGGCCGAGAGAACCGACGCCGAGUA CGUGUUCGACGUGGACAAGAAGUGCUGCGUGAAGAGGGAGGAGGCCAGCGGCCUGGUGC UGGUGGGCGAGCUGACCAACCCCCCCUUCCACGAGUUCGCCUACGAGGGCCUGAAGAUC AGGCCCAGCGCCCCCUACAAGACCACCGUGGUGGGCGUGUUCGGCGUGCCCGGCAGCGG CAAGAGCGCCAUCAUCAAGAGCCUGGUGACCAAGCACGACCUGGUGACCAGCGGCAAGA AGGAGAACUGCCAGGAGAUCGUGAACGACGUGAAGAAGCACAGAGGCCUGGACAUCCAG GCCAAGACCGUGGACAGCAUCCUGCUGAACGGCUGCAGGAGGGCCGUGGACAUCCUGUA CGUGGACGAGGCCUUCGCCUGCCACAGCGGCACCCUGCUGGCCCUGAUCGCCCUGGUGA AGCCCAGAAGCAAGGUGGUGCUGUGCGGCGACCCCAAGCAGUGCGGCUUCUUCAACAUG AUGCAGCUGAAGGUGAACUUCAACCACAACAUCUGCACCGAGGUGUGCCACAAGAGCAU CAGCAGGAGGUGCACCAGGCCCGUGACCGCCAUCGUGAGCACCCUGCACUACGGCGGCA AGAUGAGGACCACCAACCCCUGCAACAAGCCCAUCAUCAUCGACACCACCGGCCAGACC AAGCCCAAGCCCGGCGACAUCGUGCUGACCUGCUUCAGGGGCUGGGUGAAGCAGCUGCA GCUGGACUACAGGGGCCACGAGGUGAUGACCGCCGCCGCCAGCCAGGGCCUGACCAGGA AGGGCGUGUACGCCGUGAGGCAGAAGGUGAACGAGAACCCCCUGUACGCCCCCGCCAGC

[0333]

[0334] GAGCACGUGAACGUGCUGCUGACCAGGACCGAGGACAGGCUGGUGUGGAAAACCCUGGC

[0335] 12673398.1 CGGCGACCCCUGGAUCAAGGUGCUGAGCAACAUCCCCCAGGGCAACUUCACCGCCACCC UGGAGGAGUGGCAGGAGGAGCACGACAAGAUCAUGAAGGUGAUCGAGGGCCCCGCCGCC CCCGUGGACGCCUUCCAGAACAAGGCCAACGUGUGCUGGGCCAAGAGCCUGGUGCCCGU GCUGGACACCGCCGGCAUCAGGCUGACCGCCGAGGAGUGGAGCACCAUCAUCACCGCCU UCAAGGAGGACAGGGCCUACAGCCCCGUGGUGGCCCUGAACGAGAUCUGCACCAAGUAC UACGGCGUGGACCUGGACAGCGGCCUGUUCAGCGCCCCCAAGGUGAGCCUGUACUACGA GAACAACCACUGGGACAACAGGCCCGGCGGCAGAAUGUACGGCUUCAACGCCGCCACCG CCGCCAGGCUGGAGGCCAGGCACACCUUCCUGAAGGGCCAGUGGCACACCGGCAAGCAG GCCGUGAUCGCCGAGAGGAAGAUCCAGCCCCUGAGCGUGCUGGACAACGUGAUCCCCAU CAACAGAAGGCUGCCCCACGCCCUGGUGGCCGAGUACAAGACCGUGAAGGGCAGCAGGG UGGAGUGGCUGGUGAACAAGGUGAGGGGCUACCACGUGCUGCUGGUGAGCGAGUACAAC CUGGCCCUGCCCAGGAGAAGGGUGACCUGGCUGAGCCCCCUGAACGUGACCGGCGCCGA CAGGUGCUACGACCUGAGCCUGGGCCUGCCCGCCGACGCCGGCAGGUUCGACCUGGUGU UCGUGAACAUCCACACCGAGUUCAGGAUCCACCACUACCAGCAGUGCGUGGACCACGCC AUGAAGCUGCAGAUGCUGGGCGGCGACGCCCUGAGGCUGCUGAAGCCCGGCGGCAGCCU GCUGAUGAGGGCCUACGGCUACGCCGACAAGAUCAGCGAGGCCGUGGUGAGCAGCCUGA GCAGGAAGUUCAGCAGCGCCAGAGUGCUGAGGCCCGACUGCGUGACCAGCAACACCGAG GUGUUCCUGCUGUUCAGCAACUUCGACAACGGCAAGAGGCCCAGCACCCUGCACCAGAU GAACACCAAGCUGAGCGCCGUGUACGCCGGCGAGGCCAUGCACACCGCCGGCUGCGCCC CCAGCUACAGGGUGAAGAGGGCCGACAUCGCCACCUGCACCGAGGCCGCCGUGGUGAAC GCCGCCAACGCCAGGGGCACCGUGGGCGACGGCGUGUGCAGGGCCGUGGCCAAGAAGUG GCCCAGCGCCUUCAAGGGCGAGGCCACCCCCGUGGGCACCAUCAAGACCGUGAUGUGCG GCAGCUACCCCGUGAUCCACGCCGUGGCCCCCAACUUCAGCGCCACCACCGAGGCCGAG GGCGACAGGGAGCUGGCCGCCGUGUACAGGGCCGUGGCCGCCGAGGUGAACAGACUGAG CCUGAGCAGCGUGGCCAUCCCCCUGCUGAGCACCGGCGUGUUCAGCGGCGGCAGAGAUA GGCUGCAGCAGAGCCUGAACCACCUGUUCACCGCCAUGGACGCCACCGACGCCGACGUG ACCAUCUACUGCAGGGACAAGAGCUGGGAGAAGAAGAUCCAGGAGGCCAUCGACAUGAG AACCGCCGUGGAGCUGCUGAACGACGACGUGGAGCUGACCACCGACCUGGUGAGGGUGC ACCCCGACAGCAGCCUGGUGGGCAGGAAGGGCUACAGCACCACCGACGGCAGCCUGUAC AGCUACUUCGAGGGCACCAAGUUCAACCAGGCCGCCAUCGACAUGGCCGAGAUCCUGAC CCUGUGGCCCAGGCUGCAGGAGGCCAACGAGCAGAUCUGCCUGUACGCCCUGGGCGAGA CAAUGGACAACAUCAGAAGCAAGUGCCCCGUGAACGACAGCGACAGCAGCACCCCCCCC AGGACCGUGCCCUGCCUGUGCAGAUACGCCAUGACCGCCGAGAGGAUCGCCAGGCUGAG GAGCCACCAGGUGAAGUCCAUGGUGGUGUGCAGCAGCUUCCCCCUGCCCAAGUACCACG UGGACGGCGUGCAGAAGGUGAAGUGCGAGAAGGUGCUGCUGUUCGACCCCACCGUGCCC AGCGUGGUGAGCCCCAGAAAGUACGCCGCCAGCACCACCGACCACAGCGACAGAAGCCU GAGGGGCUUCGACCUGGACUGGACCACCGACAGCAGCAGCACCGCCAGCGACACCAUGA GCCUGCCCAGCCUGCAGAGCUGCGACAUCGACAGCAUCUACGAGCCCAUGGCCCCCAUC GUGGUGACCGCCGACGUGCACCCCGAGCCCGCCGGCAUCGCCGACCUGGCCGCCGACGU GCACCCCGAGCCCGCCGACCACGUGGACCUGGAGAACCCCAUCCCCCCCCCCAGACCCA AGAGGGCCGCCUACCUGGCCAGCAGAGCCGCCGAGAGGCCCGUGCCCGCCCCCAGGAAG CCCACCCCCGCCCCCAGGACCGCCUUCAGGAACAAGCUGCCCCUGACCUUCGGCGACUU CGACGAGCACGAGGUGGACGCCCUGGCCAGCGGCAUCACCUUCGGCGACUUCGACGACG UGCUGAGGCUGGGCAGGGCCGGCGCCUACAUCUUCAGCAGCGACACCGGCAGCGGCCAC CUGCAGCAGAAGUCCGUGAGACAGCACAACCUGCAGUGCGCCCAGCUGGACGCCGUGGA GGAGGAGAAGAUGUACCCCCCCAAGCUGGACACCGAGAGGGAGAAGCUGCUGCUGCUGA AGAUGCAGAUGCACCCCAGCGAGGCCAACAAGAGCAGAUACCAGAGCAGGAAGGUGGAG AACAUGAAGGCCACCGUGGUGGACAGACUGACCAGCGGCGCCAGACUGUACACCGGCGC CGACGUGGGCAGGAUCCCCACCUACGCCGUGAGAUACCCCAGACCCGUGUACAGCCCCA CCGUGAUCGAGAGAUUCAGCAGCCCCGACGUGGCCAUCGCCGCCUGCAACGAGUACCUG AGCAGGAACUACCCCACCGUGGCCAGCUACCAGAUCACCGACGAGUACGACGCCUACCU GGACAUGGUGGACGGCAGCGACAGCUGCCUGGACAGGGCCACCUUCUGCCCCGCCAAGC UGAGGUGCUACCCCAAGCACCACGCCUACCACCAGCCCACCGUGAGAAGCGCCGUGCCC AGCCCCUUCCAGAACACCCUGCAGAACGUGCUGGCCGCCGCCACCAAGAGGAACUGCAA CGUGACCCAGAUGAGGGAGCUGCCCACCAUGGACAGCGCCGUGUUCAACGUGGAGUGCU UCAAGAGGUACGCCUGCAGCGGCGAGUACUGGGAGGAGUACGCCAAGCAGCCCAUCAGG AUCACCACCGAGAACAUCACCACCUACGUGACCAAGCUGAAGGGCCCCAAGGCCGCCGC CCUGUUCGCCAAGACCCACAACCUGGUGCCCCUGCAGGAGGUGCCCAUGGACAGAUUCA CCGUGGACAUGAAGAGGGACGUGAAGGUGACCCCCGGCACCAAGCACACCGAGGAGAGG

[0336]

[0337] CCCAAGGUGCAGGUGAUCCAGGCCGCCGAGCCCCUGGCCACCGCCUACCUGUGCGGCAU

[0338] 12673398.1 CCACAGGGAGCUGGUGAGAAGGCUGAACGCCGUGCUGAGGCCCAACGUGCACACCCUGU UCGACAUGAGCGCCGAGGACUUCGACGCCAUCAUCGCCAGCCACUUCCACCCCGGCGAC CCCGUGCUGGAGACAGACAUCGCCAGCUUCGACAAGAGCCAGGACGACAGCCUGGCCCU GACCGGCCUGAUGAUCCUGGAGGACCUGGGCGUGGACCAGUACCUGCUGGACCUGAUCG AGGCCGCCUUCGGCGAGAUCAGCAGCUGCCACCUGCCCACCGGCACCAGAUUCAAGUUC GGCGCCAUGAUGAAGUCCGGCAUGUUCCUGACCCUGUUCAUCAACACCGUGCUGAACAU CACCAUCGCCAGCAGAGUGCUGGAGCAGAGGCUGACCGACAGCGCCUGCGCCGCCUUCA UCGGCGACGACAACAUCGUGCACGGCGUGAUCAGCGACAAGCUGAUGGCCGAGAGGUGC GCCAGCUGGGUGAACAUGGAGGUGAAGAUCAUCGACGCCGUGAUGGGCGAGAAGCCCCC CUACUUCUGCGGCGGCUUCAUCGUGUUCGACAGCGUGACCCAGACCGCCUGCAGGGUGA GCGACCCCCUGAAGAGGCUGUUCAAGCUGGGCAAGCCCCUGACCGCCGAGGACAAGCAG GACGAGGACAGAAGGAGGGCCCUGAGCGACGAGGUGAGCAAGUGGUUCAGGACCGGCCU GGGCGCCGAGCUGGAGGUGGCCCUGACCAGCAGGUACGAGGUGGAGGGCUGCAAGAGCA UCCUGAUCGCCAUGGCCACCCUGGCCAGAGACAUCAAGGCCUUCAAGAAGCUGAGAGGC CCCGUGAUCCACCUGUACGGCGGCCCCAGACUGGUGAGG

[0339] Amino MAAKVHVDIEADSPFIKSLQKAFPSFEVESLQVTPNDHANARAFSHLATKLIEQETDKD 85 Acid TLILDIGSAPSRRMMSTHKYHCVCPMRSAEDPERLVCYAKKLAAASGKVLDREIAGKIT Sequence DLQTVMATPDAESPTFCLHTDVTCRTAAEVAVYQDVYAVHAPTSLYHQAMKGVRTAYWI GFDTTPFMFDALAGAYPTYATNWADEQVLQARNIGLCAASLTEGRLGKLSILRKKQLKP CDTVMFSVGSTLYTESRKLLRSWHLPSVFHLKGKQSFTCRCDTIVSCEGYWKKITMCP GLYGKTVGYAVTYHAEGFLVCKTTDTVKGERVSFPVCTYVPSTICDQMTGILATDVTPE DAQKLLVGLNQRIWNGRTQRNTNTMKNYLLPIVAVAFSKWAREYKADLDDEKPLGVRE RSLTCCCLWAFKTRKMHTMYKKPDTQTIVKVPSEFNSFVIPSLWSTGLAI PVRSRIKML LAKKTKRELIPVLDASSARDAEQEEKERLEAELTREALPPLVPIAPAETGWDVDVEEL EYHAGAGWETPRSALKVTAQPNDVLLGNYWLSPQTVLKSSKLAPVHPLAEQVKIITH NGRAGRYQVDGYDGRVLLPCGSAI PVPEFQALSESATMVYNEREFVNRKLYHIAVHGPS LNTDEENYEKVRAERTDAEYVFDVDKKCCVKREEASGLVLVGELTNPPFHEFAYEGLKI RPSAPYKTTWGVFGVPGSGKSAIIKSLVTKHDLVTSGKKENCQEIVNDVKKHRGLDIQ AKTVDSILLNGCRRAVDILYVDEAFACHSGTLLALIALVKPRSKWLCGDPKQCGFFNM MQLKVNFNHNICTEVCHKSISRRCTRPVTAIVSTLHYGGKMRTTNPCNKPIIIDTTGQT KPKPGDIVLTCFRGWVKQLQLDYRGHEVMTAAASQGLTRKGVYAVRQKVNENPLYAPAS EHVNVLLTRTEDRLVWKTLAGDPWIKVLSNIPQGNFTATLEEWQEEHDKIMKVIEGPAA PVDAFQNKANVCWAKSLVPVLDTAGIRLTAEEWSTIITAFKEDRAYSPWALNEICTKY YGVDLDSGLFSAPKVSLYYENNHWDNRPGGRMYGFNAATAARLEARHTFLKGQWHTGKQ AVIAERKIQPLSVLDNVIPINRRLPHALVAEYKTVKGSRVEWLVNKVRGYHVLLVSEYN LALPRRRVTWLSPLNVTGADRCYDLSLGLPADAGRFDLVFVNIHTEFRIHHYQQCVDHA MKLQMLGGDALRLLKPGGSLLMRAYGYADKISEAWSSLSRKFSSARVLRPDCVTSNTE VFLLFSNFDNGKRPSTLHQMNTKLSAVYAGEAMHTAGCAPSYRVKRADIATCTEAAWN AANARGTVGDGVCRAVAKKWPSAFKGEATPVGTIKTVMCGSYPVIHAVAPNFSATTEAE GDRELAAVYRAVAAEVNRLSLSSVAIPLLSTGVFSGGRDRLQQSLNHLFTAMDATDADV TIYCRDKSWEKKIQEAIDMRTAVELLNDDVELTTDLVRVHPDSSLVGRKGYSTTDGSLY SYFEGTKFNQAAIDMAEILTLWPRLQEANEQICLYALGETMDNIRSKCPVNDSDSSTPP RTVPCLCRYAMTAERIARLRSHQVKSMWCSSFPLPKYHVDGVQKVKCEKVLLFDPTVP SWSPRKYAASTTDHSDRSLRGFDLDWTTDSSSTASDTMSLPSLQSCDIDSIYEPMAPI WTADVHPEPAGIADLAADVHPEPADHVDLENPIPPPRPKRAAYLASRAAERPVPAPRK PTPAPRTAFRNKLPLTFGDFDEHEVDALASGITFGDFDDVLRLGRAGAYIFSSDTGSGH LQQKSVRQHNLQCAQLDAVEEEKMYPPKLDTEREKLLLLKMQMHPSEANKSRYQSRKVE NMKAT WDRLT S GARLYTGADVGRI PT YAVRYPRPVYS PTVI ERFS S PDVAI AACNEYL SRNYPTVASYQITDEYDAYLDMVDGSDSCLDRATFCPAKLRCYPKHHAYHQPTVRSAVP SPFQNTLQNVLAAATKRNCNVTQMRELPTMDSAVFNVECFKRYACSGEYWEEYAKQPIR ITTENITTYVTKLKGPKAAALFAKTHNLVPLQEVPMDRFTVDMKRDVKVTPGTKHTEER PKVQVIQAAEPLATAYLCGIHRELVRRLNAVLRPNVHTLFDMSAEDFDAIIASHFHPGD PVLETDIASFDKSQDDSLALTGLMILEDLGVDQYLLDLIEAAFGEISSCHLPTGTRFKF GAMMKSGMFLTLFINTVLNITIASRVLEQRLTDSACAAFIGDDNIVHGVISDKLMAERC ASWVNMEVKIIDAVMGEKPPYFCGGFIVFDSVTQTACRVSDPLKRLFKLGKPLTAEDKQ DEDRRRALSDEVSKWFRTGLGAELEVALTSRYEVEGCKSILIAMATLARDIKAFKKLRG

[0340]

[0341] PVIHLYGGPRLVR

[0342] 12673398.1 Table 4, Illustrative EEEV Replicase non-structural protein (nsP) Sequences

[0343] EEEV nSP Sequences SEQID NO: nsPl / nsP2

[0344] Amino MEKVHVDLDADSPFVKSLQRCFPHFEIEATQVTDNDHANARAFSHLATKLIEGEVDT 86 Acid DQVILDIGSAPVRHTHSKHKYHCICPMKSAEDPDRLYRYADKLRKSDVTDKCIASKA Sequence ADLLTVMSTPDAETPSLCMHTDSTCRYHGSVAVYQDVYAVHAPTSIYYQALKGVRTI YWI GFDTT P FMYKNMAGAYPT YNTNWADES VLEARNI GLGS S DLHEKS FGKVS IMRK KKLQPTNKVIFSVGSTIYTEERTLLRSWHLPNVFHLKGKTSFTGRCNTIVSCEGYW KKITLSPGIYGKVDNLASTMHREGFLSCKVTDTLRGERVSFPVCTYVPATLCDQMTG ILATDVSVDDAQKLLVGLNQRIWNGRTQRNTNTMQNYLLPWAQAFSRWAREHRAD LEDEKGLGVRERSLVMGCCWAFKTHKITSIYKRPGTQTIKKVPAVFNSFVIPQPTSY GLDIGLRRRIKMLFDAKKAPAPIITEADVAHLKGLQDEAEAVAEAEAVRAALPPLLP EVDKETVEADIDLIMQEAGAGSVETPRRHIKVTTYPGEEMIGSYAVLSPQAVLNSEK LACIHPLAEQVLVMTHKGRAGRYKVEPYHGRVIVPSGTAIPIPDFQALSESATIVFN EREFVNRYLHHIAVNGGALNTDEEYYKWKSTETDSEYVFDIDAKKCVKKGDAGPMC LVGELVDPPFHEFAYESLKTRPAAPHKVPTIGVYGVPGSGKSGIIKSAVTKRDLWS AKKENCMEIIKDVKRMRGMDIAARTVDSVLLNGVKHSVDTLYIDEAFACHAGTLLAL IAIVKPKKWLCGDPKQCGFFNMMCLKVHFNHEICTEVYHKSISRRCTKTVTSIVST LFYDKRMRTVNPCNDKIIIDTTSTTKPLKDDIILTCFRGWVKQLQIDYKNHEIMTAA ASQGLTRKGVYAVRYKVNENPLYAQTSEHVNVLLTRTEKRIVWKTLAGDPWIKTLTA S Y P GN FT AT L E EWQAEH DAI MAK ILETPASSDVFQN KVN VCWAKAL E P VLAT AN I T L TRSQWETIPAFKDDKAYSPEMALNFFCTRFFGVDIDSGLFSAPTVPLTYTNEHWDNS PGPNMYGLCMRTAKELARRYPCILKAVDTGRVADVRTDTIKDYNPLINWPLNRRLP HSLWTHRYTGNGDYSQLVTKMTGKTVLWGTPMNIPGKRVETLGPSPQCTYKAELD LGIPAALGKYDIIFINVRTPYRHHHYQQCEDHAIHHSMLTRKAVDHLNKGGTCIALG YGTADRATENIISAVARSFRFSRVCQPKCAWENTEVAFVFFGKDNGNHLQDQDRLSV VLNNIYQGSTQHEAGR

[0345] nsP3

[0346] Amino APAYRWRGDITKSNDEVIVNAANNKGQPGGGVCGALYRKWPGAFDKQPVATGKAHL 87 Acid VKHSPNVIHAVGPNFSRLSENEGDQKLSEVYMDIARIINNERFTKVSIPLLSTGIYA Sequence GGKDRVMQSLNHLFTAMDTTDADITIYCLDKQWESRIKEAITRKESVEELTEDDRPV (version 1) DIELVRVHPLSSLAGRPGYSTTEGKVYSYLEGTRFHQTAKDIAEIYAMWPNKQEANE QICLYVLGESMNSIRSKCPVEESEASSPPHTIPCLCNYAMTAERVYRLRMAKNEQFA VCSSFQLPKYRITGVQKIQCSKPVIFSGTVPPAIHPRKFASVTVEDTPWQPERLVP RRPAPPVPVPARIPSPPCTPTNGSTTSIQSLGEDQSASASGGAEISVDQVSLWSIPS ATGFDVRTSSSLSLEQPTFPTMWEAEIHASQGSLWSIPSITGSETRAPSPPSQDSR PSTPSASGSHTSVDLITFDSVAEILEDFSRSPFQFLSEIKPIPAPRTRVTNMSRSAD TIKPIPKP RKCQ VK YT Q P P GVARAI S AAE FD E FVRRH S N

[0347] Amino APAYRWRGDITKSNDEVIVNAANNKGQPGGGVCGALYRKWPGAFDKQPVATGKAHL 88 Acid VKHSPNVIHAVGPNFSRLSENEGDQKLSEVYMDIARIINNERFTKVSIPLLSTGIYA Sequence GGKDRVMQSLNHLFTAMDTTDADITIYCLDKQWESRIKEAITRKESVEELTEDDRPV (version 2) DIELVRVHPLSSLAGRPGYSTTEGKVYSYLEGTRFHQTAKDIAEIYAMWPNKQEANE QICLYVLGESMNSIRSKCPVEESEASSPPHTIPCLCNYAMTAERVYRLRMAKNEQFA VCSSFQLPKYRITGVQKIQCSKPVIFSGTVPPAIHPRKFASVTVEDTPWQPERLVP RRPAPPVPVPARIPSPPCTPTNGSTTSIQSLGEDQSASASGGAEISVDQVSLWSIPS ATGFDVRTSSSLSLEQPTFPTMWEAEIHASQGSLWSIPSITGSETRAPSPPSQDSR PSTPSASGSHTSVDLITFDSVAEILEDFSRSPFQFLSEIKPIPAPRTRVTNMSRSAD TIKPIPKP RKCQ VK YT Q P P GVARAI S AAE FD E FVRRH S N RYEAGA

[0348] nsP4

[0349] Amino YIFSSETGQGHLQQKSTRQCKLQYPILERSVHEKFYAPRLDLEREKLLQKKLQLCAS 89 Acid EGNRSRYQSRKVENMKAITVERLLQGIGSYLSAEPQPVECYKVTYPAPMYSSTASNS Sequence FSSAEVAVKVCNLVLQENFPTVASYNITDEYDAYLDMVDGASCCLDTATFCPAKLRS FPKKHSYLRPEIRSAVPSPIQNTLQNVLAAATKRNCNVTQMRELPVLDSAAFNVECF KKYACNDEYWDFYKTNPIRLTAENVTQYVTKLKGPKAAALFAKTHNLQPLHEIPMDR

[0350]

[0351] FVMDLKRDVKVTPGTKHTEERPKVQVIQAADPLATAYLCGIHRELVRRLNAVLLPNI

[0352] 12673398.1 HTLFDMSAEDFDAIIAEHFQFGDSVLETDIASFDKSEDDAIAMSALMILEDLGVDQA LLNLIEAAFGNITSVHLPTGTRFKFGAMMKSGMFLTLFINTWNIMIASRVLRERLT TSPCAAFIGDDNIVKGVTSDALMAERCATWLNMEVKIIDAWGVKAPYFCGGFIWD QITGTACRVADPLKRLFKLGKPLPLDDDQDVDRRRALHDEAARWNRIGITEELVKAV

[0353]

[0354] ESRYEVNYVSLIITALTTLASSVSNFKHIRGHPITLYG

[0355] Table 5, Illustrative Immune Modulating Protein (IMP) Sequences

[0356] IMP Rational Name SEQID NO: dominant negative Protein Kinase R (dnPKR)

[0357] DNA ATGGCAGGCGACCTGTCTGCCGGGTTCTTCATGGAAGAGTTGAACACCTACCGGCAA 11 Sequence AAACAGGGCGTGGTTCTGAAATATCAGGAACTCCCTAACTCCGGCCCTCCCCATGAC CGGAGATTCACCTTCCAGGTGATCATTGACGGAAGAGAGTTTCCTGAGGGAGAGGGC CGAAGCAAGAAGGAAGCCAAGAATGCTGCCGCCAAGCTAGCCGTGGAGATACTGAAC AAGGAGAAGAAAGCTGTGAGCCCCCTGTTACTAACCACAACCAACTCAAGTGAAGGC CTGAGCATGGGCAACTACATAGGTTTAATTAACCGAATCGCCCAGAAAAAAAGACTC ACCGTGAATTATGAGCAGTGTGCCTCTGGCGTCCACGGCCCAGAAGGGTTCCATTAC AAAT GCAAGAT GGGGCAGAAGGAATACT CTATT GGCACAGGTAGCACCAAGCAGGAG GCGAAGCAACTGGCCGCCAAACTCGCGTACCTTCAGATTCTGTCCGAGGAGACGAGT GTGAAGAGTGACTACCTGTCCAGCGGCTCATTTGCCACCACCTGTGAGAGCCAGTCC AACTCTCTGGTGACTAGTACCCTTGCCTCTGAGTCCTCCTCGGAAGGGGACTTCTCT GCTGACACCAGCGAGATCAACTCGAACTCTGACTCCCTCAACAGCAGTAGCTTGCTC AT GAAT GGT CTAAGAAACAACCAGAGGAAGGCAAAGAGAT CACT GGCT CCCAGATT C GAT CT GCCT GACAT GAAGGAGACAAAGTACACAGT GGATAAACGCTT CGGGAT GGAT TTCAAAGAGATTGAACTGATCGGGAGCGGAGGATTCGGCCAGGTCTTTAAGGCCAAA CACCGGATCGACGGCAAAACCTATGTCATCAGGCGCGTTAAGTACAACAATGAGAAG GCTGAGCGGGAGGTAAAGGCCCTAGCCAAACTAGACCACGTGAATATCGTGCACTAC AATGGCTGCTGGGACGGCTTCGATTATGATCCTGAGACATCTGATGACAGTCTGGAA AGTAGCGACTATGACCCTGAAAACTCTAAGAACTCCTCCAGATCTAAAACCAAATGC CTCTTCATTCAGATGGAGTTCTGTGACAAGGGAACCTTGGAGCAGTGGATTGAAAAG AGAAGAGGAGAGAAGCTGGATAAGGTTCTGGCCCTGGAGCTGTTTGAACAAATCACA AAGGGAGTAGATTACATCCATTCCAAGAAGCTGATCCACCGGGACCTGAAGCCCAGC AACATCTTCCTGGTGGACACAAAGCAAGTGAAAATTGGTGACTTTGGACTCGTGACG AGCCTGAAGAATGACGGCAAACGGACAAGATCTAAGGGCACTCTGCGCTACATGTCT CCTGAACAGATCAGCAGCCAGGACTACGGAAAGGAAGTGGACCTCTACGCTCTGGGC CTCATACTGGCAGAGCTGTTACACGTTTGCGATACAGCCTTTGAGACAAGCAAGTTT TT CACAGAT CT GAGAGAT GGTAT CAT CT CAGATAT CTTT GACAAAAAGGAAAAGACC CT GCT GCAGAAGCT GCT GT CCAAGAAGCCAGAGGACAGACCTAACACAAGCGAAAT C CTGAGAACGCTAACTGTGTGGAAAAAAAGCCCGGAGAAAAATGAGCGGCACACGTGC RNA AUGGCAGGCGACCUGUCUGCCGGGUUCUUCAUGGAAGAGUUGAACACCUACCGGCAA 52 Sequence AAACAGGGCGUGGUUCUGAAAUAUCAGGAACUCCCUAACUCCGGCCCUCCCCAUGAC CGGAGAUUCACCUUCCAGGUGAUCAUUGACGGAAGAGAGUUUCCUGAGGGAGAGGGC CGAAGCAAGAAGGAAGCCAAGAAUGCUGCCGCCAAGCUAGCCGUGGAGAUACUGAAC AAGGAGAAGAAAGCUGUGAGCCCCCUGUUACUAACCACAACCAACUCAAGUGAAGGC CUGAGCAUGGGCAACUACAUAGGUUUAAUUAACCGAAUCGCCCAGAAAAAAAGACUC ACCGUGAAUUAUGAGCAGUGUGCCUCUGGCGUCCACGGCCCAGAAGGGUUCCAUUAC AAAUGCAAGAUGGGGCAGAAGGAAUACUCUAUUGGCACAGGUAGCACCAAGCAGGAG GCGAAGCAACUGGCCGCCAAACUCGCGUACCUUCAGAUUCUGUCCGAGGAGACGAGU GUGAAGAGUGACUACCUGUCCAGCGGCUCAUUUGCCACCACCUGUGAGAGCCAGUCC AACUCUCUGGUGACUAGUACCCUUGCCUCUGAGUCCUCCUCGGAAGGGGACUUCUCU GCUGACACCAGCGAGAUCAACUCGAACUCUGACUCCCUCAACAGCAGUAGCUUGCUC AUGAAUGGUCUAAGAAACAACCAGAGGAAGGCAAAGAGAUCACUGGCUCCCAGAUUC GAUCUGCCUGACAUGAAGGAGACAAAGUACACAGUGGAUAAACGCUUCGGGAUGGAU UUCAAAGAGAUUGAACUGAUCGGGAGCGGAGGAUUCGGCCAGGUCUUUAAGGCCAAA CACCGGAUCGACGGCAAAACCUAUGUCAUCAGGCGCGUUAAGUACAACAAUGAGAAG GCUGAGCGGGAGGUAAAGGCCCUAGCCAAACUAGACCACGUGAAUAUCGUGCACUAC

[0358]

[0359] AAUGGCUGCUGGGACGGCUUCGAUUAUGAUCCUGAGACAUCUGAUGACAGUCUGGAA

[0360] 12673398.1 AGUAGC GACUAU GAG C CU GAAAACU CUAAGAACU C CU C CAGAU CUAAAAC CAAAU GC CUCUUCAUUCAGAUGGAGUUCUGUGACAAGGGAACCUUGGAGCAGUGGAUUGAAAAG AGAAGAGGAGAGAAGCUGGAUAAGGUUCUGGCCCUGGAGCUGUUUGAACAAAUCACA AAGGGAGUAGAUUACAUCCAUUCCAAGAAGCUGAUCCACCGGGACCUGAAGCCCAGC AACAUCUUCCUGGUGGACACAAAGCAAGUGAAAAUUGGUGACUUUGGACUCGUGACG AGCCUGAAGAAUGACGGCAAACGGACAAGAUCUAAGGGCACUCUGCGCUACAUGUCU CCUGAACAGAUCAGCAGCCAGGACUACGGAAAGGAAGUGGACCUCUACGCUCUGGGC CUCAUACUGGCAGAGCUGUUACACGUUUGCGAUACAGCCUUUGAGACAAGCAAGUUU UU CACAGAU CU GAGAGAU GGUAU CAU CU CAGAU AU CUUU GACAAAAAGGAAAAGAC C CUGCUGCAGAAGCUGCUGUCCAAGAAGCCAGAGGACAGACCUAACACAAGCGAAAUC CUGAGAACGCUAACUGUGUGGAAAAAAAGCCCGGAGAAAAAUGAGCGGCACACGUGC

[0361] Amino MAGDLSAGFFMEELNTYRQKQGWLKYQELPNSGPPHDRRFTFQVIIDGREFPEGEG 90 Acid RSKKEAKNAAAKLAVEILNKEKKAVSPLLLTTTNSSEGLSMGNYIGLINRIAQKKRL Sequence TVNYEQCASGVHGPEGFHYKCKMGQKEYSIGTGSTKQEAKQLAAKLAYLQILSEETS VKSDYLSSGSFATTCESQSNSLVTSTLASESSSEGDFSADTSEINSNSDSLNSSSLL MNGLRNNQRKAKRSLAPRFDLPDMKETKYTVDKRFGMDFKEIELIGSGGFGQVFKAK HRIDGKTYVIRRVKYNNEKAEREVKALAKLDHVNIVHYNGCWDGFDYDPETSDDSLE SSDYDPENSKNSSRSKTKCLFIQMEFCDKGTLEQWIEKRRGEKLDKVLALELFEQIT KGVDYIHSKKLIHRDLKPSNIFLVDTKQVKIGDFGLVTSLKNDGKRTRSKGTLRYMS PEQISSQDYGKEVDLYALGLILAELLHVCDTAFETSKFFTDLRDGIISDIFDKKEKT LLQKLLSKKPEDRPNTSEILRTLTVWKKSPEKNERHTC

[0362] dominant negative Zinc Finger Antiviral Protein (ZAP) C88R (dbZAPC88R)

[0363] DNA ATGGCCGACCCTGGTGTTTGCTGCTTCATCACCAAAATCCTCTGTGCTCATGGCGGC 12 Sequence CGCATGACTCTTGAGGAGCTGCTGGGAGAGATCCGGCTGCCTGAGGCTCAGCTCTAT GAGCTGCTGGAAACCGCAGGCCCAGACCGATTTGTCCTGTTGGAGACTGGCGGTCAG GCTGGCATCACTCGGTCTGTTGTGGCTACCACAAGAGCCCGGGTGTGCAGGAGAAAA TACTGCCAAAGACCTTGCGATTCCCTGCATCTTAGAAAGCTCAACCTCCTGGGCCGT TGCCACTATGCCCAAAGCCAAAGAAACCTGTGCAAGTACTCTCATGACGTACTGAGT GAACAGAATTTCCAGATTCTGAAGAACCATGAACTCAGCGGCCTGAACCAGGAGGAG CTGGCTTGCCTGCTGGTACAGTCTGACCCGTTCTTTTTACCCGAAATCTGTAAGAGC TACAAAGGAGAAGGACGCAAGCAGACCTGCGGGCAGCCCCAGCCCTGTGAGAGGTTA CACATCTGTGAACATTTTACACGAGGGAACTGTTCTTATCTCAATTGCCTGAGAAGC CACAACCTCATGGACAGAAAGGTCCTGACCATCATGAGGGAACACGGCCTGAGCCCT GATGTGGTGCAGAACATCCAAGATATCTGCAACAACAAACACGCGAGAAGAAACCCA CCGGGCACCCGGGCCGCACACCCACACAGACGGGGAGGCGCCCACAGGGACAGATCC AAGTCTCGGGATCGCTTCCTCCACAACAGTCTGGAGTTTCTGAGCCCCGTCGTGAGC CCCCTCGGCTCAGGTCCACCTAGCCCAGATGTGACCTCTTGCAAAGACAGCCTGGAG GACGTGTCAGTGGATGTCACCCAGAAGTTTAAGTACCTGGGGACCCACGACAGGGCC CAGCTGTCGCCTGTGTCCAGCAAAGCAGCCGGCGTCCAAGGTCCTTCTCAAATGCGA GCCTCTCAGGAGTTCTCTGAGGACGGCAATCTGGATGACATTTTTAGCCGGAACAGG TCAGACTCAAGCTCCTCCCGGGCCTCAGCTGCCAAGGTGGCTCAAAGAAACGAGGCA GTGGCGATGAAAATGGGGATGGAGGTGAAGGGGAAAAAAGAAGCCCCTGACATTGAT AGAGTACCATTCTTGAACAGCTACATTGATGGGGTGACGATGGAAAAAGCCTCCGTG AGCGGCATCCCTGGAAAGAAGTTCACCGCCAACGACCTGGAGAACCTGTTGCTGCTG AATGACACATGGAAAAACGTGGCTAAGCCTCAGGATCTGCAAACCACAGGCCGGATA ACTGACTCAGGGCAGGATAAGGCCTTCCTGCAGAATAAGTATGGCGGCAACCCTGTG TGGGCATCAGCCAGCACTCACAATGCCCCCAATGGAAGTAGTCAGATTATGGACGAG ACACCTAATGTGTCAAAAAGTAGTACTAGTGGGTTTGCCATCAAACCTGCCATAGCC GGCGGCAAGGAGGCCGTTTACAGTGGTGTTCAGAGCCCACGAAGTCAAGTGTTAGCC GTGCCAGGCGAGGCGACCACACCTGTGCAGAGCAACCGTCTGCCACAGAGTCCTCTG AGCAGCAGCTCCCACCGGGCCGCCGCCTCTGGCAGCCCTGGAAAGAATAGCACCCAC ACCTCCGTGAGCCCCGCAATCGAGAGCAGCAGAATGACATCCGATCCCGATGAATAC CTGCTGAGGTACATTCTTAACCCCCTGTTCCGCATGGACAATCATGGACCCAAGGAA ATCTGTCAAGACCACCTCTACAAGGGCTGCCAGCAGTCACACTGTGACAGAAGTCAC TTCCACCTGCCCTACCGCTGGCAGATGTTTGTGTACACAACCTGGAGAGACTTTCAA GACATGGAGTCTATTGAGCAAGCCTACTGTGATCCTCACGTCGAGCTGATTCTGATT GAGAACCACCAGATAAACTTTCAGAAGATGACCTGTGACAGCTACCCCATCAGACGG CTGAGCACTCCTAGTTACGAGGAGAAGCCTCTGAGCGCTGTGTTTGCTACGAAGTGG

[0364]

[0365] ATCTGGTACTGGAAGAATGAATTCAACGAGTATATCCAATACGGAAACGAAAGTCCT

[0366] 12673398.1 GGCCACACAAGCTCTGACATTAATTCCGCCTATCTGGAGTCCTTCTTCCAGAGCTGC CCAAGAGGTGTGCTCCCTTTCCAGGCAGGCTCTCAGAAGTATGAATTGTCCTTCCAG GGGATGATCCAGACAAACATCGCCAGCAAGACACAGCGCCACGTCGTAAGACGGCCT GTGTTCGTGAGCTCCAATGATGTGGAGCAGAAGAGGAGAGGTCCAGAGtga RNA AUGGCCGACCCUGGUGUUUGCUGCUUCAUCACCAAAAUCCUCUGUGCUCAUGGCGGC 53 Sequence CGCAUGACUCUUGAGGAGCUGCUGGGAGAGAUCCGGCUGCCUGAGGCUCAGCUCUAU GAGCUGCUGGAAACCGCAGGCCCAGACCGAUUUGUCCUGUUGGAGACUGGCGGUCAG GCUGGCAUCACUCGGUCUGUUGUGGCUACCACAAGAGCCCGGGUGUGCAGGAGAAAA UACUGCCAAAGACCUUGCGAUUCCCUGCAUCUUAGAAAGCUCAACCUCCUGGGCCGU U GC CACUAU GC C CAAAGC CAAAGAAAC CU GU GCAAGUACU CU GAU GAG GUACU GAGU GAACAGAAUUUCCAGAUUCUGAAGAACCAUGAACUCAGCGGCCUGAACCAGGAGGAG CUGGCUUGCCUGCUGGUACAGUCUGACCCGUUCUUUUUACCCGAAAUCUGUAAGAGC UACAAAGGAGAAGGACGCAAGCAGACCUGCGGGCAGCCCCAGCCCUGUGAGAGGUUA CACAU CU GU GAACAUUUUACAC GAGGGAACU GUU CUUAU CU CAAUU GC CU GAGAAGC CACAACCUCAUGGACAGAAAGGUCCUGACCAUCAUGAGGGAACACGGCCUGAGCCCU GAU GU G GU G C AGAAC AU C C AAGAU AU CU G C AAC AAC AAAC AC G C GAGAAGAAAC C C A CCGGGCACCCGGGCCGCACACCCACACAGACGGGGAGGCGCCCACAGGGACAGAUCC AAGUCUCGGGAUCGCUUCCUCCACAACAGUCUGGAGUUUCUGAGCCCCGUCGUGAGC CCCCUCGGCUCAGGUCCACCUAGCCCAGAUGUGACCUCUUGCAAAGACAGCCUGGAG GACGUGUCAGUGGAUGUCACCCAGAAGUUUAAGUACCUGGGGACCCACGACAGGGCC CAGCUGUCGCCUGUGUCCAGCAAAGCAGCCGGCGUCCAAGGUCCUUCUCAAAUGCGA GCCUCUCAGGAGUUCUCUGAGGACGGCAAUCUGGAUGACAUUUUUAGCCGGAACAGG UCAGACUCAAGCUCCUCCCGGGCCUCAGCUGCCAAGGUGGCUCAAAGAAACGAGGCA GUGGCGAUGAAAAUGGGGAUGGAGGUGAAGGGGAAAAAAGAAGCCCCUGACAUUGAU AGAGUACCAUUCUUGAACAGCUACAUUGAUGGGGUGACGAUGGAAAAAGCCUCCGUG AGCGGCAUCCCUGGAAAGAAGUUCACCGCCAACGACCUGGAGAACCUGUUGCUGCUG AAUGACACAUGGAAAAACGUGGCUAAGCCUCAGGAUCUGCAAACCACAGGCCGGAUA ACUGACUCAGGGCAGGAUAAGGCCUUCCUGCAGAAUAAGUAUGGCGGCAACCCUGUG UGGGCAUCAGCCAGCACUCACAAUGCCCCCAAUGGAAGUAGUCAGAUUAUGGACGAG ACACCUAAUGUGUCAAAAAGUAGUACUAGUGGGUUUGCCAUCAAACCUGCCAUAGCC GGCGGCAAGGAGGCCGUUUACAGUGGUGUUCAGAGCCCACGAAGUCAAGUGUUAGCC GUGCCAGGCGAGGCGACCACACCUGUGCAGAGCAACCGUCUGCCACAGAGUCCUCUG AGCAGCAGCUCCCACCGGGCCGCCGCCUCUGGCAGCCCUGGAAAGAAUAGCACCCAC ACCUCCGUGAGCCCCG C AAU C GAGAG C AG C AGAAU GAC AU C C GAU C C C GAU GAAU AC CUGCUGAGGUACAUUCUUAACCCCCUGUUCCGCAUGGACAAUCAUGGACCCAAGGAA AUCUGUCAAGACCACCUCUACAAGGGCUGCCAGCAGUCACACUGUGACAGAAGUCAC UUCCACCUGCCCUACCGCUGGCAGAUGUUUGUGUACACAACCUGGAGAGACUUUCAA GACAU GGAGU CUAUU GAGCAAGC CUACU GU GAU C CU CAC GU C GAGCU GAUU CU GAUU GAGAAC CAC CAGAUAAACUUU CAGAAGAU GAC CU GU GACAGCUAC C C CAU CAGAC GG CUGAGCACUCCUAGUUACGAGGAGAAGCCUCUGAGCGCUGUGUUUGCUACGAAGUGG AU CU GGUACU GGAAGAAU GAAUU CAAC GAGU AU AU C CAAUAC GGAAAC GAAAGU C CU GGCCACACAAGCUCUGACAUUAAUUCCGCCUAUCUGGAGUCCUUCUUCCAGAGCUGC CCAAGAGGUGUGCUCCCUUUCCAGGCAGGCUCUCAGAAGUAUGAAUUGUCCUUCCAG GGGAUGAUCCAGACAAACAUCGCCAGCAAGACACAGCGCCACGUCGUAAGACGGCCU GUGUUCGUGAGCUCCAAUGAUGUGGAGCAGAAGAGGAGAGGUCCAGAGtga Amino MADPGVCCFITKILCAHGGRMTLEELLGEIRLPEAQLYELLETAGPDRFVLLETGGQ 91 Acid AGITRSWATTRARVCRRKYCQRPCDSLHLRKLNLLGRCHYAQSQRNLCKYSHDVLS Sequence EQNFQILKNHELSGLNQEELACLLVQSDPFFLPEICKSYKGEGRKQTCGQPQPCERL HICEHFTRGNCSYLNCLRSHNLMDRKVLTIMREHGLSPDWQNIQDICNNKHARRNP PGTRAAHPHRRGGAHRDRSKSRDRFLHNSLEFLSPWSPLGSGPPSPDVTSCKDSLE DVSVDVTQKFKYLGTHDRAQLSPVSSKAAGVQGPSQMRASQEFSEDGNLDDIFSRNR SDS S S SRASAAKVAQRNEAVAMKMGMEVKGKKEAPDI DRVPFLNS YI DGVTMEKASV SGIPGKKFTANDLENLLLLNDTWKNVAKPQDLQTTGRITDSGQDKAFLQNKYGGNPV WASASTHNAPNGSSQIMDETPNVSKSSTSGFAIKPAIAGGKEAVYSGVQSPRSQVLA VPGEATTPVQSNRLPQSPLSSSSHRAAASGSPGKNSTHTSVSPAIESSRMTSDPDEY LLRYILNPLFRMDNHGPKEICQDHLYKGCQQSHCDRSHFHLPYRWQMFVYTTWRDFQ DMESIEQAYCDPHVELILIENHQINFQKMTCDSYPIRRLSTPSYEEKPLSAVFATKW IWYWKNEFNEYIQYGNESPGHTSSDINSAYLESFFQSCPRGVLPFQAGSQKYELSFQ

[0367]

[0368] GMIQTNIASKTQRHWRRPVFVSSNDVEQKRRGPE*

[0369] 12673398.1 dominant negative Mitochondrial Viral Signaling Protein

[0370] DNA ATGCCTTTTGCAGAGGACAAGACCTACAAAGGCTGTGAGCTCGTGGACCTGGCAGAT 13 Sequence GAGGTGGCCTCAGTGTATCAGTCTTACCAGCCCCGGACATCTGATCGGCCCCCGGAC CCCCTGGAGCCACCTTCTCTGCCTGCGGAAAGGCCTGGTCCTCCAACACCTGCAGCA GCTCACAGCATCCCGTACAACAGCTGTCGAGAGAAGGAACCTTCCTACCCAATGCCC GTGCAGGAGACACAGGCCCCAGAGTCACCTGGGGAGAACTCTGAACAGGCCCTGCAG ACTCTGTCACCACGAGCCATCCCACGGAACCCAGATGGAGGCCCTCTGGAGAGCAGC AGTGACCTCGCAGCCCTGAGCCCTCTGACATCTAGTGGCCACCAGGAGCAAGACACA GAACTGGGCAGTACACACACAGCCGGCGCCACCAGCAGCCTGACACCTTCTAGAGGC CCAGTATCGCCTTCCGTGTCGTTCCAGCCACTGGCCCGCTCTACCCCCCGTGCCAGC AGACTTCCTGGTCCCACCGGCTCTGTGGTGAGCACAGGCACTAGCTTCTCTAGCAGC AGCCCAGGCCTGGCATCAGCAGGCGCCGCAGAGGGAAAGCAGGGCGCCGAGAGTGAC CAGGCTGAGCCCATCATCTGCTCTAGCGGAGCTGAGGCCCCTGCCAACTCTCTGCCA TCGAAGGTCCCTACCACCCTCATGCCTGTGAACACAGTCGCCCTTAAGGTGCCTGCT AACCCTGCCAGTGTGAGCACTGTGCCTTCAAAATTGCCAACGTCCAGCAAACCCCCT GGTGCGGTGCCCAGCAATGCTCTGACCAACCCTGCCCCCTCCAAGCTGCCCATCAAC TCTACCCGGGCCGGAATGGTTCCTAGCAAAGTTCCTACTTCCATGGTGTTAACCAAG GTGAGCGCCAGCACCGTGCCAACAGATGGCAGCTCAAGAAATGAGGAGACTCCTGCC GCCCCGACCCCTGCTGGCGCCACTGGCGGTAGTAGTGCCTGGCTGGACTCCTCTTCC GAAAACAGAGGCCTCGGCTCTGAGCTGTCTAAGCCAGGAGTCCTGGCCAGTCAGGTG GATAGCCCCTTCTCTGGCTGCTTTGAAGATTTAGCCATTTCTGCCTCCACCTCCCTG GGCATGGGACCGTGCCATGGTCCTGAAGAGAATGAATATAAAAGTGAAGGGACCTTC GGTATTCATGTAGCAGAAAATCCCAGTATCCAGCTGCTCGAGGGAAACCCTGGCCCT CCCGCAGACCCTGATGGGGGCCCCAGGCCCCAGGCTGACAGAAAGTTCCAAGAACGG GAAGTCCCCTGCCACCGGCCCTCGCCCGGGGCCCTTTGGCTTCAGGTGGCTGTAACA GGAGTGCTGGTTGTGACCCTCCTGGTGGTTCTGTACCGGAGAAGATTGCACTGA RNA AUGCCUUUUGCAGAGGACAAGACCUACAAAGGCUGUGAGCUCGUGGACCUGGCAGAU 54 Sequence GAGGUGGCCUCAGUGUAUCAGUCUUACCAGCCCCGGACAUCUGAUCGGCCCCCGGAC CCCCUGGAGCCACCUUCUCUGCCUGCGGAAAGGCCUGGUCCUCCAACACCUGCAGCA GCUCACAGCAUCCCGUACAACAGCUGUCGAGAGAAGGAACCUUCCUACCCAAUGCCC GUGCAGGAGACACAGGCCCCAGAGUCACCUGGGGAGAACUCUGAACAGGCCCUGCAG ACUCUGUCACCACGAGCCAUCCCACGGAACCCAGAUGGAGGCCCUCUGGAGAGCAGC AGUGACCUCGCAGCCCUGAGCCCUCUGACAUCUAGUGGCCACCAGGAGCAAGACACA GAACUGGGCAGUACACACACAGCCGGCGCCACCAGCAGCCUGACACCUUCUAGAGGC CCAGUAUCGCCUUCCGUGUCGUUCCAGCCACUGGCCCGCUCUACCCCCCGUGCCAGC AGACUUCCUGGUCCCACCGGCUCUGUGGUGAGCACAGGCACUAGCUUCUCUAGCAGC AGCCCAGGCCUGGCAUCAGCAGGCGCCGCAGAGGGAAAGCAGGGCGCCGAGAGUGAC CAGGCUGAGCCCAUCAUCUGCUCUAGCGGAGCUGAGGCCCCUGCCAACUCUCUGCCA UCGAAGGUCCCUACCACCCUCAUGCCUGUGAACACAGUCGCCCUUAAGGUGCCUGCU AACCCUGCCAGUGUGAGCACUGUGCCUUCAAAAUUGCCAACGUCCAGCAAACCCCCU GGUGCGGUGCCCAGCAAUGCUCUGACCAACCCUGCCCCCUCCAAGCUGCCCAUCAAC UCUACCCGGGCCGGAAUGGUUCCUAGCAAAGUUCCUACUUCCAUGGUGUUAACCAAG GUGAGCGCCAGCACCGUGCCAACAGAUGGCAGCUCAAGAAAUGAGGAGACUCCUGCC GCCCCGACCCCUGCUGGCGCCACUGGCGGUAGUAGUGCCUGGCUGGACUCCUCUUCC GAAAACAGAGGCCUCGGCUCUGAGCUGUCUAAGCCAGGAGUCCUGGCCAGUCAGGUG GAUAGCCCCUUCUCUGGCUGCUUUGAAGAUUUAGCCAUUUCUGCCUCCACCUCCCUG GGCAUGGGACCGUGCCAUGGUCCUGAAGAGAAUGAAUAUAAAAGUGAAGGGACCUUC GGUAUUCAUGUAGCAGAAAAUCCCAGUAUCCAGCUGCUCGAGGGAAACCCUGGCCCU CCCGCAGACCCUGAUGGGGGCCCCAGGCCCCAGGCUGACAGAAAGUUCCAAGAACGG GAAGUCCCCUGCCACCGGCCCUCGCCCGGGGCCCUUUGGCUUCAGGUGGCUGUAACA GGAGUGCUGGUUGUGACCCUCCUGGUGGUUCUGUACCGGAGAAGAUUGCACUGA

[0371] Amino MPFAEDKTYKGCELVDLADEVASVYQSYQPRTSDRPPDPLEPPSLPAERPGPPTPAA 92 Acid AHSIPYNSCREKEPSYPMPVQETQAPESPGENSEQALQTLSPRAIPRNPDGGPLESS Sequence SDLAALSPLTSSGHQEQDTELGSTHTAGATSSLTPSRGPVSPSVSFQPLARSTPRAS RLPGPTGSWSTGTSFSSSSPGLASAGAAEGKQGAESDQAEPIICSSGAEAPANSLP SKVPTTLMPVNTVALKVPANPASVSTVPSKLPTSSKPPGAVPSNALTNPAPSKLPIN STRAGMVPSKVPTSMVLTKVSASTVPTDGSSRNEETPAAPTPAGATGGSSAWLDSSS

[0372]

[0373] ENRGLGSELSKPGVLASQVDSPFSGCFEDLAI SASTSLGMGPCHGPEENEYKSEGTF

[0374] 12673398.1 GIHVAENPSIQLLEGNPGPPADPDGGPRPQADRKFQEREVPCHRPSPGALWLQVAVT GVLWTLLWLYRRRLH

[0375] Microprotein in Antiviral Immunity 1 (MA VII)

[0376] DNA ATGACCTCAGTTTCAACACAGTTGTCCTTAGTCCTCATGTCACTGCTTTTGGTGCTG 14 Sequence CCTGTTGTGGAAGCAGTAGAAGCCGGTGATGCAATCGCCCTTTTGTTAGGTGTGGTT CTCAGCATTACAGGCATTTGTGCCTGCTTGGGGGTATATGCACGAAAAAGAAATGGA CAGATG RNA AU GAG CU CAGUUU CAACACAGUU GU C CUUAGU C CU GAU GU CACU GCUUUU GGU GCU G 55 Sequence CCUGUUGUGGAAGCAGUAGAAGCCGGUGAUGCAAUCGCCCUUUUGUUAGGUGUGGUU CUCAGCAUUACAGGCAUUUGUGCCUGCUUGGGGGUAUAUGCACGAAAAAGAAAUGGA CAGAUG

[0377] Amino MTSVSTQLSLVLMSLLLVLPWEAVEAGDAIALLLGWLSITGICACLGVYARKRNG 93 Acid QM

[0378] Sequence

[0379] Suppressor of Cytokine Signaling 1 (SOCS1)

[0380] DNA ATGGTGGCCCACAACCAGGTGGCTGCAGACAATGCTGTGTCCACAGCAGCTGAACCT 15 Sequence AGAAGGCGGCCAGAGCCCTCCTCTTCCAGCTCAAGCTCCCCAGCAGCCCCTGCCAGA CCTCGGCCGTGCCCTGCAGTGCCAGCTCCAGCTCCTGGAGACACCCACTTCCGGACC TTCCGCTCTCATGCTGATTACAGGAGGATCACCAGAGCCTCCGCCCTGCTGGATGCC TGTGGCTTCTACTGGGGCCCTTTGAGCGTCCACGGTGCCCATGAGAGACTGCGCGCA GAGCCAGTGGGGACTTTTCTGGTGAGAGATAGCCGACAGAGAAACTGTTTCTTTGCT TTGTCTGTGAAGATGGCCAGTGGCCCCACCAGCATTAGAGTGCACTTTCAGGCAGGC CGTTTCCACCTGGATGGGAGCCGGGAAAGCTTCGACTGCCTGTTTGAACTCCTGGAG CACTATGTCGCAGCCCCTAGACGCATGCTGGGCGCCCCCCTCAGACAACGGCGTGTC AGACCCCTGCAGGAGCTGTGCAGGCAGAGAATCGTTGCCACAGTTGGAAGGGAAAAT CTGGCCCGCATCCCTCTGAACCCTGTGTTGAGAGACTACCTCAGCTCCTTCCCTTTC CAGATG RNA AUGGUGGCCCACAACCAGGUGGCUGCAGACAAUGCUGUGUCCACAGCAGCUGAACCU 56 Sequence AGAAGGCGGCCAGAGCCCUCCUCUUCCAGCUCAAGCUCCCCAGCAGCCCCUGCCAGA CCUCGGCCGUGCCCUGCAGUGCCAGCUCCAGCUCCUGGAGACACCCACUUCCGGACC UUCCGCUCUCAUGCUGAUUACAGGAGGAUCACCAGAGCCUCCGCCCUGCUGGAUGCC UGUGGCUUCUACUGGGGCCCUUUGAGCGUCCACGGUGCCCAUGAGAGACUGCGCGCA GAGCCAGUGGGGACUUUUCUGGUGAGAGAUAGCCGACAGAGAAACUGUUUCUUUGCU UUGUCUGUGAAGAUGGCCAGUGGCCCCACCAGCAUUAGAGUGCACUUUCAGGCAGGC CGUUUCCACCUGGAUGGGAGCCGGGAAAGCUUCGACUGCCUGUUUGAACUCCUGGAG CACUAUGUCGCAGCCCCUAGACGCAUGCUGGGCGCCCCCCUCAGACAACGGCGUGUC AGACCCCUGCAGGAGCUGUGCAGGCAGAGAAUCGUUGCCACAGUUGGAAGGGAAAAU CUGGCCCGCAUCCCUCUGAACCCUGUGUUGAGAGACUACCUCAGCUCCUUCCCUUUC CAGAUC

[0381] Amino MVAHNQVAADNAVS TAAE P RRRP E PSSSSSSS PAAPARP RP C PAVPAPAP GDTH FRT 94 Acid FRSHADYRRITRASALLDACGFYWGPLSVHGAHERLRAEPVGTFLVRDSRQRNCFFA Sequence LSVKMASGPTSIRVHFQAGRFHLDGSRESFDCLFELLEHYVAAPRRMLGAPLRQRRV RPLQELCRQRIVATVGRENLARIPLNPVLRDYLSSFPFQI

[0382] Suppressor of Cytokine Signaling 3 (SOCS3)

[0383] DNA ATGGTGACACACAGCAAGTTCCCTGCAGCCGGCATGTCGCGGCCTTTGGACACCTCC 16 Sequence CTGAGACTTAAGACCTTCTCCTCGAAGTCTGAATACCAGCTGGTGGTGAATGCTGTG CGGAAACTGCAGGAGTCAGGGTTCTACTGGAGTGCTGTCACCGGGGGAGAAGCCAAC CTTCTGCTCAGTGCTGAGCCTGCCGGCACCTTCCTGATTAGAGATTCCTCTGACCAG CGCCACTTCTTTACTCTGTCTGTGAAAACCCAGAGTGGTACAAAAAACCTGCGAATC CAGTGTGAGGGAGGCAGCTTCAGCCTGCAGTCAGACCCTAGAAGCACGCAGCCAGTG CCCCGCTTTGACTGCGTCCTGAAGCTGGTTCACCATTACATGCCACCCCCAGGGGCC CCTAGCTTCCCTAGTCCCCCTACAGAGCCCAGCTCTGAAGTGCCAGAGCAGCCTTCA GCCCAGCCCCTGCCAGGCAGCCCTCCCAGACGGGCCTACTACATCTACAGCGGCGGG GAGAAGATCCCACTGGTCCTGTCTCGGCCTCTGTCCAGCAACGTTGCCACCCTGCAG CACCTCTGCAGAAAGACCGTGAACGGCCACCTGGACTCCTATGAAAAAGTGACCCAG

[0384]

[0385] CTACCTGGACCCATCCGAGAGTTTCTGGATCAGTATGATGCCCCCCTG

[0386] 12673398.1 RNA AUGGUGACACACAGCAAGUUCCCUGCAGCCGGCAUGUCGCGGCCUUUGGACACCUCC 57 Sequence CU GAGACUUAAGAC CUU CU C CU C GAAGU CU GAAUAC CAGCU GGU GGU GAAU GCU GU G CGGAAACUGCAGGAGUCAGGGUUCUACUGGAGUGCUGUCACCGGGGGAGAAGCCAAC CUUCUGCUCAGUGCUGAGCCUGCCGGCACCUUCCUGAUUAGAGAUUCCUCUGACCAG C GC CACUU CUUUACU CU GU CU GU GAAAAC C CAGAGU GGUACAAAAAAC CU GC GAAU C CAGUGUGAGGGAGGCAGCUUCAGCCUGCAGUCAGACCCUAGAAGCACGCAGCCAGUG CCCCGCUUUGACUGCGUCCUGAAGCUGGUUCACCAUUACAUGCCACCCCCAGGGGCC CCUAGCUUCCCUAGUCCCCCUACAGAGCCCAGCUCUGAAGUGCCAGAGCAGCCUUCA GCCCAGCCCCUGCCAGGCAGCCCUCCCAGACGGGCCUACUACAUCUACAGCGGCGGG GAGAAGAUCCCACUGGUCCUGUCUCGGCCUCUGUCCAGCAACGUUGCCACCCUGCAG CAC CU CU GCAGAAAGAC C GU GAAC GGC CAC CU GGACU C CUAU GAAAAAGU GAC C CAG CUACCUGGACCCAUCCGAGAGUUUCUGGAUCAGUAUGAUGCCCCCCUG

[0387] Amino MVTHSKFPAAGMSRPLDTSLRLKTFSSKSEYQLWNAVRKLQESGFYWSAVTGGEAN 95 Acid LLLSAEPAGTFLIRDSSDQRHFFTLSVKTQSGTKNLRIQCEGGSFSLQSDPRSTQPV Sequence PRFDCVLKLVHHYMPPPGAPSFPSPPTEPSSEVPEQPSAQPLPGSPPRRAYYIYSGG EKIPLVLSRPLSSNVATLQHLCRKTVNGHLDSYEKVTQLPGPIREFLDQYDAPL

[0388] Vaccinia Virus (VACV) F1L

[0389] DNA ATGCTGTCCATGTTCATGTGCAACAACATTGTGGATTATGTGGACGACATCGATAAT 17 Sequence GGCATT GT CCAAGACATT GAAGAT GAGGCTAGCAACAAT GT GGACCAT GACTAT GT C TACCCATTACCTGAGAACATGGTGTATAGATTTGATAAAAGCACCAACATCCTGGAC TACCT GAGCACAGAACGGGACCACGT CAT GAT GGCT GT GAGATACTACAT GAGCAAG CAGAGGCTGGATGACCTGTACCGCCAGCTGCCCACCAAGACACGAAGCTACATTGAT AT CATAAACAT CTACT GT GACAAGGT GAGCAAT GACTACAACCGGGATAT GAATATT AT GT AT GACAT GGC CT C CAC GAAGT CT T T CACAGT GT AT GAT AT CAAT AAT GAAGT T AACACAATCCTCATGGATAACAAAGGCCTAGGAGTGAGGCTGGCCACCATCTCTTTC ATCACAGAGTTGGGCCGGCGCTGCATGAACCCTGTGGAGACAATCAAGATGTTCACC CT GCT GAGCCACACAAT CT GT GAT GATTACTT CGT GGACTACAT CACAGACAT CT CA CCCCCAGACAACACTATCCCTAACACTTCTACCCGGGAGTACCTGAAGCTCATTGGA ATCACCGCCATCATGTTTGCAACCTACAAGACTCTGAAATACATGATAGGCtga RNA AU G CU GU C C AU GUU C AU GU G C AAC AAC AUU GU G GAUU AU GU G GAC GAC AU C GAU AAU 58 Sequence GGCAUUGUCCAAGACAUUGAAGAUGAGGCUAGCAACAAUGUGGACCAUGACUAUGUC UAC C CAUUAC CU GAGAACAU GGU GUAUAGAUUU GAUAAAAGCAC CAACAU C CU GGAC UACCUGAGCACAGAACGGGACCACGUCAUGAUGGCUGUGAGAUACUACAUGAGCAAG CAGAGGCUGGAUGACCUGUACCGCCAGCUGCCCACCAAGACACGAAGCUACAUUGAU AU C AU AAAC AU CU ACU GU GAC AAG GU GAG C AAU GACU AC AAC C G G GAU AU GAAU AUU AU GUAU GACAU GGC CU C CAC GAAGU CUUU CACAGU GUAU GAU AU CAAUAAU GAAGUU AACACAAUCCUCAUGGAUAACAAAGGCCUAGGAGUGAGGCUGGCCACCAUCUCUUUC AUCACAGAGUUGGGCCGGCGCUGCAUGAACCCUGUGGAGACAAUCAAGAUGUUCACC CU GCU GAGC CACACAAU CU GU GAU GAUUACUU C GU GGACUACAU CACAGACAU CU CA C C C C CAGACAACACUAU C C CUAACACUU CUAC C C GGGAGUAC CU GAAGCU CAUU GGA AU CAC C GC CAU CAU GUUU GCAAC CUACAAGACU CU GAAAUACAU GAUAGGC t g a Amino MLSMFMCNNIVDYVDDIDNGIVQDIEDEASNNVDHDYVYPLPENMVYRFDKSTNILD 96 Acid YLSTERDHVMMAVRYYMSKQRLDDLYRQLPTKTRSYIDIINIYCDKVSNDYNRDMNI Sequence MYDMASTKSFTVYDINNEVNTILMDNKGLGVRLATISFITELGRRCMNPVETIKMFT LLSHTICDDYFVDYITDISPPDNTIPNTSTREYLKLIGITAIMFATYKTLKYMIG* Vaccinia Virus (VACV) RNA-binding protein E3 (E3L)

[0390] DNA ATGAGCAAGATCTACATCGACGAGCGGAGCAACGCCGAGATTGTGTGCGAGGCCATC 18 Sequence AAGACCATCGGAATCGAAGGCGCCACAGCCGCTCAGCTGACCAGACAGCTGAACATG GAAAAGCGGGAAGTGAACAAGGCCCTGTACGACCTGCAGAGAAGCGCCATGGTGTAC AGCAGCGACGACATCCCTCCTCGGTGGTTTATGACCACAGAGGCCGACAAGCCTGAC GCCGATGCTATGGCCGACGTGATCATCGACGACGTGTCCCGCGAGAAGTCCATGAGA GAGGACCACAAGAGCTTCGACGATGTGATCCCCGCCAAGAAGATCATCGATTGGAAG GGCGCCAATCCTGTGACCGTGATCAACGAGTACTGCCAGATCACCAGAAGAGACTGG TCCTTCCGGATCGAGAGCGTGGGCCCTAGCAATAGCCCTACCTTCTACGCCTGCGTG GACATCGACGGCAGAGTGTTCGATAAGGCCGACGGCAAGAGCAAGCGGGACGCCAAA AACAATGCCGCCAAGCTGGCCGTGGATAAGCTGCTGGGCTATGTGATCATCCGGTTC RNA AUGAGCAAGAUCUACAUCGACGAGCGGAGCAACGCCGAGAUUGUGUGCGAGGCCAUC 59

[0391]

[0392] Sequence AAGACCAUCGGAAUCGAAGGCGCCACAGCCGCUCAGCUGACCAGACAGCUGAACAUG

[0393] 12673398.1 GAAAAGCGGGAAGUGAACAAGGCCCUGUACGACCUGCAGAGAAGCGCCAUGGUGUAC AGCAGCGACGACAUCCCUCCUCGGUGGUUUAUGACCACAGAGGCCGACAAGCCUGAC GCCGAUGCUAUGGCCGACGUGAUCAUCGACGACGUGUCCCGCGAGAAGUCCAUGAGA GAG GAG C AC AAGAG CUU C GAC GAU GU GAU C C C C G C C AAGAAGAU C AU C GAUU G GAAG GGCGCCAAUCCUGUGACCGUGAUCAACGAGUACUGCCAGAUCACCAGAAGAGACUGG UCCUUCCGGAUCGAGAGCGUGGGCCCUAGCAAUAGCCCUACCUUCUACGCCUGCGUG GACAUCGACGGCAGAGUGUUCGAUAAGGCCGACGGCAAGAGCAAGCGGGACGCCAAA AACAAUGCCGCCAAGCUGGCCGUGGAUAAGCUGCUGGGCUAUGUGAUCAUCCGGUUC

[0394] Amino MS KI YI DERSNAEI VCEAI KT I GI EGATAAQLTRQLNMEKREVNKALYDLQRSAMVY 97 Acid SSDDIPPRWFMTTEADKPDADAMADVIIDDVSREKSMREDHKSFDDVI PAKKIIDWK Sequence GANPVTVINEYCQITRRDWSFRIESVGPSNSPTFYACVDIDGRVFDKADGKSKRDAK NNAAKLAVDKLLGYVI I RF

[0395] Vaccinia Vims (VACV) soluble IFN-a / 0 receptor B18 (B18R)

[0396] DNA ATGGGCACCATGAAGATGATGGTGCACATCTACTTCGTGTCCCTGCTGCTGCTCCTG 19 Sequence TTCCACAGCTACGC CAT C GAC AT C GAGAAC GAGAT CACCGAGTTCTT C AAC AAGAT G CGGGACACCCTGCCTGCCAAGGACAGCAAGTGGCTGAATCCCGCCTGTATGTTCGGC GGCACAATGAACGATATCGCCGCTCTGGGCGAGCCCTTCAGCGCTAAGTGTCCTCCT ATCGAGGACAGCCTGCTGAGCCACCGGTACAAGGACTACGTGGTCAAGTGGGAGAGA CTCGAGAAGAACCGGCGGAGACAGGTGTCCAACAAGAGAGTGAAGCACGGCGACCTG TGGATCGCCAACTACACCAGCAAGTTCAGCAATCGGAGATACCTGTGCACCGTGACC ACCAAGAACGGCGATTGTGTGCAGGGAATCGTGCGGAGCCACATCAGAAAGCCTCCA AGCTGCATCCCCAAGACCTACGAGCTGGGCACCCACGATAAGTACGGCATCGATCTG TACTGCGGCATCCTGTACGCCAAGCACTACAACAACATCACCTGGTATAAGGACAAC AAAGAGATCAACATCGACGACATCAAGTACAGCCAGACCGGCAAAGAGCTGATCATT CACAACCCCGAGCTGGAAGATAGCGGCAGATACGACTGCTACGTGCACTACGACGAC GTGCGGATCAAGAACGACATCGTGGTGTCCCGGTGCAAGATCCTGACAGTGATCCCC AGCCAGGACCACCGGTTCAAGCTGATTCTGGACCCCAAGATCAACGTGACCATCGGC GAGCCCGCCAACATCACATGTACCGCCGTGTCTACCTCTCTGCTGATCGACGATGTG CTGATTGAGTGGGAGAACCCCAGCGGCTGGCTGATCGGCTTCGACTTCGATGTGTAC AGCGTGCTGACAAGCAGAGGCGGCATCACAGAGGCCACACTGTACTTCGAGAACGTG ACCGAAGAGTACATCGGCAACACCTACAAGTGCAGGGGCCACAACTACTACTTCGAA AAGACCCTGACCACCACCGTGGTGCTGGAAG RNA AUGGGCACCAUGAAGAUGAUGGUGCACAUCUACUUCGUGUCCCUGCUGCUGCUCCUG 60 Sequence UU C CACAGCUAC GC CAU C GACAU C GAGAAC GAGAU CAC C GAGUU CUU CAACAAGAU G CGGGACACCCUGCCUGCCAAGGACAGCAAGUGGCUGAAUCCCGCCUGUAUGUUCGGC GGCACAAUGAACGAUAUCGCCGCUCUGGGCGAGCCCUUCAGCGCUAAGUGUCCUCCU AUCGAGGACAGCCUGCUGAGCCACCGGUACAAGGACUACGUGGUCAAGUGGGAGAGA CUCGAGAAGAACCGGCGGAGACAGGUGUCCAACAAGAGAGUGAAGCACGGCGACCUG UGGAUCGCCAACUACACCAGCAAGUUCAGCAAUCGGAGAUACCUGUGCACCGUGACC ACCAAGAACGGCGAUUGUGUGCAGGGAAUCGUGCGGAGCCACAUCAGAAAGCCUCCA AGCUGCAUCCCCAAGACCUACGAGCUGGGCACCCACGAUAAGUACGGCAUCGAUCUG UACUGCGGCAUCCUGUACGCCAAGCACUACAACAACAUCACCUGGUAUAAGGACAAC AAAGAGAU C AAC AU C GAC GACAU C AAGU AC AG C C AGAC C G G C AAAGAG CU GAU C AUU CACAACCCCGAGCUGGAAGAUAGCGGCAGAUACGACUGCUACGUGCACUACGACGAC GUGCGGAUCAAGAACGACAUCGUGGUGUCCCGGUGCAAGAUCCUGACAGUGAUCCCC AGCCAGGACCACCGGUUCAAGCUGAUUCUGGACCCCAAGAUCAACGUGACCAUCGGC GAGCCCGCCAACAUCACAUGUACCGCCGUGUCUACCUCUCUGCUGAUCGACGAUGUG CUGAUUGAGUGGGAGAACCCCAGCGGCUGGCUGAUCGGCUUCGACUUCGAUGUGUAC AGCGUGCUGACAAGCAGAGGCGGCAUCACAGAGGCCACACUGUACUUCGAGAACGUG ACCGAAGAGUACAUCGGCAACACCUACAAGUGCAGGGGCCACAACUACUACUUCGAA AAGAC C CU GAC CAC CAC C GU G GU G CU G GAAG

[0397] Amino MGTMKMMVHIYFVSLLLLLFHSYAIDIENEITEFFNKMRDTLPAKDSKWLNPACMFG 98 Acid GTMNDIAALGEPFSAKCPPIEDSLLSHRYKDYWKWERLEKNRRRQVSNKRVKHGDL Sequence WIANYTSKFSNRRYLCTVTTKNGDCVQGIVRSHIRKPPSCIPKTYELGTHDKYGIDL YCGILYAKHYNNITWYKDNKEINIDDIKYSQTGKELIIHNPELEDSGRYDCYVHYDD VRIKNDIWSRCKILTVIPSQDHRFKLILDPKINVTIGEPANITCTAVSTSLLIDDV LIEWENPSGWLIGFDFDVYSVLTSRGGITEATLYFENVTEEYIGNTYKCRGHNYYFE

[0398]

[0399] KTLTTTWLE

[0400] 12673398.1 Herpes Simplex Virus (HSV) US1

[0401] DNA ATGGCAGACATTCCTCCAGACCCCCCAGCCCTGAACACTACCCCAGCTAATCATGCC 20 Sequence CCTCCAAGCCCCCCCCCGGGCTCTAGAAAGAGGCGGCGGCCTGTTCTGCCCTCCAGC T CAGAGAGCGAAGGAAAGCCT GACACT GAGT CT GAAT CCAGCT CGACAGAGAGTAGC GAGGATGAGGCTGGCGACCTGCGCGGAGGCCGGCGGCGATCCCCCCGGGAGCTGGGT GGGAGATACTTCCTGGACCTGTCCGCTGAGAGCACCACAGGGACAGAATCAGAAGGC ACTGGTCCCAGTGATGACGACGATGATGATGCCAGTGATGGCTGGTTGGTGGACACT CCTCCCCGCAAATCCAAAAGACCCCGCATTAACCTCCGGCTTACCAGCAGCCCAGAT CGGCGGGCCGGAGTGGTGTTCCCTGAGGTCTGGAGATCGGACCGGCCGATCCGGGCT GCCCAGCCACAGGCCCCTGCCTCACTGCCTGGCATCGCCCATGCCCACAGACGGAGT GCGAGACAAGCGCAGATGAGATCAGGAGCAGCCTGGACCCTGGACCTGCACTATATC AGGCAATGTGTGAACCAGCTGTTCCGCATCTTACGCGCGGCCCCGAACCCCCCTGGC AGTGCAAACAGACTGCGTCACCTGGTGAGAGACTGCTACCTGATGGGCTACTGCCGC ACCAGGCTGGGGCCCCGCACCTGGGGCCGGCTGCTCCAGATCTCGGGGGGCACATGG GACGTGCGCCTGAGAAATGCAATCAGAGAGGTGGAGGCACACTTTGAACCTGCCGCT GAGCCTGTGTGTGAACTCCCCTGCCTCAATGCCCGACGGTATGGCCCTGAGTGTGAT GTAGGAAACCT GGAGACAAACGGAGGCAGCACCT CT GAT GAT GAAAT CT CT GAT GCC ACAGACAGTGACGACACTCTGGCCTCCCACAGTGACACCGAAGGGGGCCCTTCCCCC GCTGGCAGAGAAAACCCGGAGAGTGCCAGCGGTGGCGCCATTGCTGCAAGACTGGAA TGTGAGTTTGGAACTTTTGACTGGACCTCTGAGGAGGGCTCTCAGCCCTGGCTGTCA GCTGTGGTGGCAGATACAAGCTCTGCAGAGCGATCTGGCCTCCCTGCCCCGGGCGCC TGCCGAGCCACGGAAGCCCCAGAACGTGAGGACGGCTGCAGAAAGATGAGGTTCCCA GCAGCTTGCCCTTACCCATGTGGCCATACCTTCCTTCGGCCT RNA AUGGCAGACAUUCCUCCAGACCCCCCAGCCCUGAACACUACCCCAGCUAAUCAUGCC 61 Sequence CCUCCAAGCCCCCCCCCGGGCUCUAGAAAGAGGCGGCGGCCUGUUCUGCCCUCCAGC UCAGAGAGCGAAGGAAAGCCUGACACUGAGUCUGAAUCCAGCUCGACAGAGAGUAGC GAGGAUGAGGCUGGCGACCUGCGCGGAGGCCGGCGGCGAUCCCCCCGGGAGCUGGGU GGGAGAUACUUCCUGGACCUGUCCGCUGAGAGCACCACAGGGACAGAAUCAGAAGGC ACUGGUCCCAGUGAUGACGACGAUGAUGAUGCCAGUGAUGGCUGGUUGGUGGACACU CCUCCCCGCAAAUCCAAAAGACCCCGCAUUAACCUCCGGCUUACCAGCAGCCCAGAU CGGCGGGCCGGAGUGGUGUUCCCUGAGGUCUGGAGAUCGGACCGGCCGAUCCGGGCU GCCCAGCCACAGGCCCCUGCCUCACUGCCUGGCAUCGCCCAUGCCCACAGACGGAGU GCGAGACAAGCGCAGAUGAGAUCAGGAGCAGCCUGGACCCUGGACCUGCACUAUAUC AGGCAAUGUGUGAACCAGCUGUUCCGCAUCUUACGCGCGGCCCCGAACCCCCCUGGC AGUGCAAACAGACUGCGUCACCUGGUGAGAGACUGCUACCUGAUGGGCUACUGCCGC ACCAGGCUGGGGCCCCGCACCUGGGGCCGGCUGCUCCAGAUCUCGGGGGGCACAUGG GACGUGCGCCUGAGAAAUGCAAUCAGAGAGGUGGAGGCACACUUUGAACCUGCCGCU GAGCCUGUGUGUGAACUCCCCUGCCUCAAUGCCCGACGGUAUGGCCCUGAGUGUGAU GUAGGAAACCUGGAGACAAACGGAGGCAGCACCUCUGAUGAUGAAAUCUCUGAUGCC ACAGACAGUGACGACACUCUGGCCUCCCACAGUGACACCGAAGGGGGCCCUUCCCCC GCUGGCAGAGAAAACCCGGAGAGUGCCAGCGGUGGCGCCAUUGCUGCAAGACUGGAA UGUGAGUUUGGAACUUUUGACUGGACCUCUGAGGAGGGCUCUCAGCCCUGGCUGUCA GCUGUGGUGGCAGAUACAAGCUCUGCAGAGCGAUCUGGCCUCCCUGCCCCGGGCGCC UGCCGAGCCACGGAAGCCCCAGAACGUGAGGACGGCUGCAGAAAGAUGAGGUUCCCA GCAGCUUGCCCUUACCCAUGUGGCCAUACCUUCCUUCGGCCU

[0402] Amino MADIPPDPPALNTTPANHAPPSPPPGSRKRRRPVLPSSSESEGKPDTESESSSTESS 99 Acid EDEAGDLRGGRRRSPRELGGRYFLDLSAESTTGTESEGTGPSDDDDDDASDGWLVDT Sequence PPRKSKRPRINLRLTSSPDRRAGWFPEVWRSDRPIRAAQPQAPASLPGIAHAHRRS ARQAQMRSGAAWTLDLHYIRQCVNQLFRILRAAPNPPGSANRLRHLVRDCYLMGYCR TRLGPRTWGRLLQISGGTWDVRLRNAIREVEAHFEPAAEPVCELPCLNARRYGPECD VGNLETNGGSTSDDEISDATDSDDTLASHSDTEGGPSPAGRENPESASGGAIAARLE CEFGTFDWTSEEGSQPWLSAWADTSSAERSGLPAPGACRATEAPEREDGCRKMRFP AACPYPCGHTFLRP

[0403] Herpes Simplex Virus (HSV) US 11

[0404] DNA ATGGCCAGCGGAGTGAGCCCAGCCCACCCTCAGACACCTGTGGGCGCAGGCAGCCGC 21 Sequence GACCTGTCGCTGAAAGGCACCCCATCTGATGGCATGCAGCCTCGAGGAGCTGACACC

[0405] CTGGAGGGCCACAGCCTGCCCACAGATGGACCTCCCCACAGGGGTGGCGACCACGAC

[0406]

[0407] CCTGCTGCTGGGAAGAGAGGCGACTCCGGGCTGCTCCGGGTCTGCGCTGCCCTGAGC

[0408] 12673398.1 ATCCCTAAGCCATCTGAAGCCGTGAGGCCCAGCCGGATTCCTAGAGCCCCTAGAGTT CCCCGGGAGCCCCGGGTGCCCAGAGAACCAAGAGAACCTCGCGTGCCACGGTCCCCT CGAGAGCCACGGGTTCCTCGCATCCCCCGGGATCCCCGGCCTCCACGTCCACCCCGG GTGCCCAGGGAGCCACGGCCTCCAAGGGAGCCTCGGGCCACCAGAGGCCTGGCC RNA AUGGCCAGCGGAGUGAGCCCAGCCCACCCUCAGACACCUGUGGGCGCAGGCAGCCGC 62 Sequence GACCUGUCGCUGAAAGGCACCCCAUCUGAUGGCAUGCAGCCUCGAGGAGCUGACACC CUGGAGGGCCACAGCCUGCCCACAGAUGGACCUCCCCACAGGGGUGGCGACCACGAC CCUGCUGCUGGGAAGAGAGGCGACUCCGGGCUGCUCCGGGUCUGCGCUGCCCUGAGC AUCCCUAAGCCAUCUGAAGCCGUGAGGCCCAGCCGGAUUCCUAGAGCCCCUAGAGUU CCCCGGGAGCCCCGGGUGCCCAGAGAACCAAGAGAACCUCGCGUGCCACGGUCCCCU CGAGAGCCACGGGUUCCUCGCAUCCCCCGGGAUCCCCGGCCUCCACGUCCACCCCGG GUGCCCAGGGAGCCACGGCCUCCAAGGGAGCCUCGGGCCACCAGAGGCCUGGCC Amino MASGVSPAHPQTPVGAGSRDLSLKGTPSDGMQPRGADTLEGHSLPTDGPPHRGGDHD 100 Acid PAAGKRGDSGLLRVCAALSIPKPSEAVRPSRIPRAPRVPREPRVPREPREPRVPRSP Sequence REPRVPRIPRDPRPPRPPRVPREPRPPREPRATRGLA

[0409] Herpes Simplex Virus (HSV) ISC34.5

[0410] DNA ATGTCCCGTCGGAGAGGCCCAAGACGGAGAGGCCCTCGGAGAAGGCCTAGGCCTGGT 22 Sequence GCTCCAGCCGTGCCCAGACCGGGCGCCCCTGCCGTTCCCCGGCCTGGGGCCCTGCCC ACCGCAGACTCCCAGATGGTGCCTGCCTATGACAGTGGCACAGCCGTGGAGAGCGCC CCCGCAGCCAGCAGCCTGCTACGACGCTGGCTGCTGGTGCCTCAAGCAGATGACTCT GATGATGCCGACTACGCTGGAAATGACGATGCAGAGTGGGCCAACAGCCCTCCTTCA GAAGGAGGAGGCAAAGCCCCAGAAGCCCCCCATGCCGCCCCGGCTGCTGCCTGCCCT CCCCCTCCACCCAGGAAGGAGAGGGGCCCCCAGAGGCCTCTGCCTCCACACCTGGCC CTCCGACTGCGCACCACCACAGAGTACCTGGCAAGACTGTCCCTGCGTCGGCGCAGA CCACCTGCCTCCCCGCCTGCTGATGCACCACGGGGGAAGGTGTGTTTCTCTCCTCGG GTCCAGGTGAGACACCTTGTGGCCTGGGAGACTGCCGCCAGACTCGCAAGGAGAGGA AGCTGGGCACGGGAAAGAGCTGACAGAGACAGATTTCGACGACGTGTGGCTGCCGCC GAGGCTGTCATCGGCCCCTGCCTGGAGCCAGAGGCCAGAGCCAGGGCCAGAGCACGC GCCCGAGCCCACGAAGATGGCGGCCCTGCGGAGGAGGAAGAAGCTGCTGCTGCAGCC CGTGGCAGCTCTGCAGCCGCTGGGCCCGGGCGCAGAGCGGTG RNA AUGUCCCGUCGGAGAGGCCCAAGACGGAGAGGCCCUCGGAGAAGGCCUAGGCCUGGU 63 Sequence GCUCCAGCCGUGCCCAGACCGGGCGCCCCUGCCGUUCCCCGGCCUGGGGCCCUGCCC ACCGCAGACUCCCAGAUGGUGCCUGCCUAUGACAGUGGCACAGCCGUGGAGAGCGCC CCCGCAGCCAGCAGCCUGCUACGACGCUGGCUGCUGGUGCCUCAAGCAGAUGACUCU GAUGAUGCCGACUACGCUGGAAAUGACGAUGCAGAGUGGGCCAACAGCCCUCCUUCA GAAGGAGGAGGCAAAGCCCCAGAAGCCCCCCAUGCCGCCCCGGCUGCUGCCUGCCCU CCCCCUCCACCCAGGAAGGAGAGGGGCCCCCAGAGGCCUCUGCCUCCACACCUGGCC CUCCGACUGCGCACCACCACAGAGUACCUGGCAAGACUGUCCCUGCGUCGGCGCAGA CCACCUGCCUCCCCGCCUGCUGAUGCACCACGGGGGAAGGUGUGUUUCUCUCCUCGG GUCCAGGUGAGACACCUUGUGGCCUGGGAGACUGCCGCCAGACUCGCAAGGAGAGGA AGCUGGGCACGGGAAAGAGCUGACAGAGACAGAUUUCGACGACGUGUGGCUGCCGCC GAGGCUGUCAUCGGCCCCUGCCUGGAGCCAGAGGCCAGAGCCAGGGCCAGAGCACGC GCCCGAGCCCACGAAGAUGGCGGCCCUGCGGAGGAGGAAGAAGCUGCUGCUGCAGCC CGUGGCAGCUCUGCAGCCGCUGGGCCCGGGCGCAGAGCGGUG

[0411] Amino MSRRRGPRRRGPRRRPRPGAPAVPRPGAPAVPRPGALPTADSQMVPAYDSGTAVESA 101 Acid PAASSLLRRWLLVPQADDSDDADYAGNDDAEWANSPPSEGGGKAPEAPHAAPAAACP Sequence PPPPRKERGPQRPLPPHLALRLRTTTEYLARLSLRRRRPPASPPADAPRGKVCFSPR VQVRHLVAWETAARLARRGSWARERADRDRFRRRVAAAEAVIGPCLEPEARARARAR ARAH E D GG P AE E E EAAAAARG S S AAAG P GRRAV

[0412] Herpes Simplex Virus (HSV) ICPO

[0413] DNA ATGGAACCCCGGCCAGGAACTAGCTCGCGGGCAGACCCAGGACCCGAACGGCCTCCG 23 Sequence CGGCAGACTCCTGGCACCCAGCCTGCAGCCCCACATGCCTGGGGCATGCTCAACGAT ATGCAGTGGCTGGCGAGCAGCGACTCCGAAGAGGAGACAGAAGTTGGCATCTCTGAT GACGACTTGCACAGAGACTCCACCAGTGAGGCAGGAAGCACAGACACTGAGATGTTT GAGGCCGGGCTGATGGATGCCGCTACTCCACCCGCGCGTCCTCCCGCCGAACGGCAA GGAAGTCCGACCCCAGCTGATGCACAAGGTTCTTGTGGGGGAGGTCCTGTCGGAGAG GAAGAGGCCGAAGCTGGCGGCGGAGGGGATGTGTGTGCTGTCTGCACAGATGAGATT

[0414]

[0415] GCGCCACCTCTGCGCTGCCAGTCCTTCCCATGCCTCCACCCTTTCTGCATCCCCTGC

[0416] 12673398.1 ATGAAAACCTGGATTCCCTTAAGAAATACCTGCCCGCTCTGCAACACACCTGTGGCC TACCTTATCGTTGGCGTGACTGCCTCTGGCTCCTTCTCTACCATCCCTATTGTGAAT GACCCTAGGACCCGAGTGGAGGCTGAAGCTGCCGTGCGGGCAGGAACCGCAGTGGAC TTCATCTGGACAGGAAACCCAAGGACCGCTCCCCGGTCACTGTCTCTGGGCGGCCAC ACAGTGCGTGCCCTGTCACCCACTCCGCCTTGGCCAGGCACAGATGATGAAGATGAT GACCTAGCAGATGTGGACTATGTGCCCCCCGCGCCCCGGCGGGCTCCTAGAAGGGGA GGTGGCGGGGCAGGCGCCACCCGAGGAACCAGTCAGCCAGCGGCAACCCGGCCCGCA CCCCCAGGCGCCCCGAGAAGCTCCAGTAGCGGGGGCGCTCCCCTGAGAGCAGGTGTC GGCTCTGGCTCGGGGGGGGGCCCTGCCGTGGCCGCCGTGGTCCCCCGCGTCGCTTCC CTGCCACCTGCAGCCGGAGGAGGTCGGGCCCAAGCTAGACGGGTGGGGGAGGACGCC GCCGCCGCAGAGGGCAGAACACCTCCTGCTCGCCAACCCAGGGCGGCTCAGGAACCA CCCATCGTGATAAGCGACAGCCCTCCCCCAAGCCCTAGACGGCCTGCCGGCCCGGGA CCACTGAGCTTTGTGTCTAGTAGTTCTGCTCAAGTGAGCAGCGGCCCTGGAGGCGGT GGCTTGCCGCAGTCGAGCGGACGTGCCGCGCGGCCTAGAGCCGCTGTGGCACCTCGT GTGAGAAGTCCCCCCCGGGCGGCAGCTGCTCCGGTGGTTTCCGCTTCAGCGGATGCC GCGGGCCCAGCACCTCCAGCTGTCCCTGTGGATGCCCACCGGGCCCCCAGGTCCAGA ATGACACAGGCCCAGACCGACACTCAGGCTCAGTCTCTCGGTCGAGCGGGGGCCACA GACGCCCGAGGCTCGGGAGGACCTGGCGCAGAGGGAGGGCCTGGGGTTCCTAGAGGA ACCAACACCCCTGGCGCCGCGCCTCACGCTGCCGAGGGAGCCGCGGCCAGACCCCGT AAGAGAAGAGGCAGTGACTCCGGCCCGGCTGCTTCCTCCAGCGCCAGCTCGTCGGCT GCCCCTCGGAGTCCTTTGGCCCCCCAGGGCGTGGGCGCAAAGAGGGCCGCCCCTCGA CGTGCGCCTGACTCGGATAGCGGGGATAGGGGCCATGGCCCGCTGGCCCCTGCATCT GCAGGTGCAGCCCCACCTTCAGCCTCTCCTAGCTCTCAAGCGGCGGTGGCTGCGGCC TCAAGCAGCAGCGCCAGCAGCTCGAGCGCCAGCTCTAGCTCCGCTTCCTCGAGCAGC GCTAGCTCGTCTAGCGCATCCAGCTCCAGCGCGTCCTCCTCCTCTGCAAGCAGCAGC GCCGGGGGAGCTGGTGGGAGCGTGGCGTCTGCCTCCGGGGCCGGCGAGAGGAGAGAA ACCTCCCTGGGCCCCAGGGCAGCTGCACCGAGAGGCCCAAGAAAATGTGCCCGGAAA ACACGCCACGCAGAGGGCGGTCCTGAGCCAGGCGCCCGGGACCCTGCCCCCGGCCTC ACCAGGTACCTGCCCATTGCAGGGGTTTCCTCAGTGGTGGCGCTGGCGCCCTATGTG AACAAGACTGTCACGGGAGACTGCCTACCTGTCCTGGACATGGAGACAGGCCATATC GGAGCCTACGTTGTCCTGGTGGATCAGACAGGAAATGTGGCAGACCTGCTGCGCGCC GCCGCCCCTGCTTGGTCAAGAAGAACCCTCCTCCCGGAGCATGCCCGCAACTGTGTA CGGCCCCCAGACTACCCGACCCCTCCGGCGTCTGAGTGGAATTCCCTGTGGATGACC CCCGTGGGCAACATGCTGTTTGACCAGGGCACGCTGGTCGGGGCACTGGATTTCCAT GGCCTCCGGTCCAGGCACCCCTGGAGCAGGGAGCAGGGGGCACCGGCCCCAGCTGGC GACGCCCCAGCCGGGCACGGAGAA RNA AUGGAACCCCGGCCAGGAACUAGCUCGCGGGCAGACCCAGGACCCGAACGGCCUCCG 64 Sequence CGGCAGACUCCUGGCACCCAGCCUGCAGCCCCACAUGCCUGGGGCAUGCUCAACGAU AUGCAGUGGCUGGCGAGCAGCGACUCCGAAGAGGAGACAGAAGUUGGCAUCUCUGAU GACGACUUGCACAGAGACUCCACCAGUGAGGCAGGAAGCACAGACACUGAGAUGUUU GAGGCCGGGCUGAUGGAUGCCGCUACUCCACCCGCGCGUCCUCCCGCCGAACGGCAA GGAAGUCCGACCCCAGCUGAUGCACAAGGUUCUUGUGGGGGAGGUCCUGUCGGAGAG GAAGAGGCCGAAGCUGGCGGCGGAGGGGAUGUGUGUGCUGUCUGCACAGAUGAGAUU GCGCCACCUCUGCGCUGCCAGUCCUUCCCAUGCCUCCACCCUUUCUGCAUCCCCUGC AUGAAAACCUGGAUUCCCUUAAGAAAUACCUGCCCGCUCUGCAACACACCUGUGGCC UACCUUAUCGUUGGCGUGACUGCCUCUGGCUCCUUCUCUACCAUCCCUAUUGUGAAU GACCCUAGGACCCGAGUGGAGGCUGAAGCUGCCGUGCGGGCAGGAACCGCAGUGGAC UUCAUCUGGACAGGAAACCCAAGGACCGCUCCCCGGUCACUGUCUCUGGGCGGCCAC ACAGUGCGUGCCCUGUCACCCACUCCGCCUUGGCCAGGCACAGAUGAUGAAGAUGAU GACCUAGCAGAUGUGGACUAUGUGCCCCCCGCGCCCCGGCGGGCUCCUAGAAGGGGA GGUGGCGGGGCAGGCGCCACCCGAGGAACCAGUCAGCCAGCGGCAACCCGGCCCGCA CCCCCAGGCGCCCCGAGAAGCUCCAGUAGCGGGGGCGCUCCCCUGAGAGCAGGUGUC GGCUCUGGCUCGGGGGGGGGCCCUGCCGUGGCCGCCGUGGUCCCCCGCGUCGCUUCC CUGCCACCUGCAGCCGGAGGAGGUCGGGCCCAAGCUAGACGGGUGGGGGAGGACGCC GCCGCCGCAGAGGGCAGAACACCUCCUGCUCGCCAACCCAGGGCGGCUCAGGAACCA CCCAUCGUGAUAAGCGACAGCCCUCCCCCAAGCCCUAGACGGCCUGCCGGCCCGGGA CCACUGAGCUUUGUGUCUAGUAGUUCUGCUCAAGUGAGCAGCGGCCCUGGAGGCGGU GGCUUGCCGCAGUCGAGCGGACGUGCCGCGCGGCCUAGAGCCGCUGUGGCACCUCGU GUGAGAAGUCCCCCCCGGGCGGCAGCUGCUCCGGUGGUUUCCGCUUCAGCGGAUGCC

[0417]

[0418] GCGGGCCCAGCACCUCCAGCUGUCCCUGUGGAUGCCCACCGGGCCCCCAGGUCCAGA 12673398.1 AUGACACAGGCCCAGACCGACACUCAGGCUCAGUCUCUCGGUCGAGCGGGGGCCACA GACGCCCGAGGCUCGGGAGGACCUGGCGCAGAGGGAGGGCCUGGGGUUCCUAGAGGA ACCAACACCCCUGGCGCCGCGCCUCACGCUGCCGAGGGAGCCGCGGCCAGACCCCGU AAGAGAAGAGGCAGUGACUCCGGCCCGGCUGCUUCCUCCAGCGCCAGCUCGUCGGCU GCCCCUCGGAGUCCUUUGGCCCCCCAGGGCGUGGGCGCAAAGAGGGCCGCCCCUCGA CGUGCGCCUGACUCGGAUAGCGGGGAUAGGGGCCAUGGCCCGCUGGCCCCUGCAUCU GCAGGUGCAGCCCCACCUUCAGCCUCUCCUAGCUCUCAAGCGGCGGUGGCUGCGGCC UCAAGCAGCAGCGCCAGCAGCUCGAGCGCCAGCUCUAGCUCCGCUUCCUCGAGCAGC GCUAGCUCGUCUAGCGCAUCCAGCUCCAGCGCGUCCUCCUCCUCUGCAAGCAGCAGC GCCGGGGGAGCUGGUGGGAGCGUGGCGUCUGCCUCCGGGGCCGGCGAGAGGAGAGAA ACCUCCCUGGGCCCCAGGGCAGCUGCACCGAGAGGCCCAAGAAAAUGUGCCCGGAAA ACACGCCACGCAGAGGGCGGUCCUGAGCCAGGCGCCCGGGACCCUGCCCCCGGCCUC ACCAGGUACCUGCCCAUUGCAGGGGUUUCCUCAGUGGUGGCGCUGGCGCCCUAUGUG AACAAGACUGUCACGGGAGACUGCCUACCUGUCCUGGACAUGGAGACAGGCCAUAUC GGAGCCUACGUUGUCCUGGUGGAUCAGACAGGAAAUGUGGCAGACCUGCUGCGCGCC GCCGCCCCUGCUUGGUCAAGAAGAACCCUCCUCCCGGAGCAUGCCCGCAACUGUGUA CGGCCCCCAGACUACCCGACCCCUCCGGCGUCUGAGUGGAAUUCCCUGUGGAUGACC CCCGUGGGCAACAUGCUGUUUGACCAGGGCACGCUGGUCGGGGCACUGGAUUUCCAU GGCCUCCGGUCCAGGCACCCCUGGAGCAGGGAGCAGGGGGCACCGGCCCCAGCUGGC GACGCCCCAGCCGGGCACGGAGAA

[0419] Amino MEPRPGTSSRADPGPERPPRQTPGTQPAAPHAWGMLNDMQWLASSDSEEETEVGISD 102 Acid DDLHRDSTSEAGSTDTEMFEAGLMDAATPPARPPAERQGSPTPADAQGSCGGGPVGE Sequence EEAEAGGGGDVCAVCTDEIAPPLRCQSFPCLHPFCIPCMKTWIPLRNTCPLCNTPVA YL I VGVT AS G S F S T I P I VN D P RT RVEAEAAVRAGT AVD F I WT GN P RT AP RS L S L GGH TVRALSPTPPWPGTDDEDDDLADVDYVPPAPRRAPRRGGGGAGATRGTSQPAATRPA PPGAPRSSSSGGAPLRAGVGSGSGGGPAVAAWPRVASLPPAAGGGRAQARRVGEDA AAAEGRTPPARQPRAAQEPPIVISDSPPPSPRRPAGPGPLSFVSSSSAQVSSGPGGG GL PQ S S GRAARP RAAVAP RVRS P P RAAAAP WS AS ADAAGPAP PAVP VDAHRAP RS R MTQAQTDTQAQSLGRAGATDARGSGGPGAEGGPGVPRGTNTPGAAPHAAEGAAARPR KRRGSDSGPAASSSASSSAAPRSPLAPQGVGAKRAAPRRAPDSDSGDRGHGPLAPAS AGAAPPSASPSSQAAVAAASSSSASSSSASSSSASSSSASSSSASSSSASSSSASSS AGGAGGSVASASGAGERRETSLGPRAAAPRGPRKCARKTRHAEGGPEPGARDPAPGL TRYLPIAGVSSWALAPYVNKTVTGDCLPVLDMETGHIGAYWLVDQTGNVADLLRA AAPAWSRRTLLPEHARNCVRPPDYPTPPASEWNSLWMTPVGNMLFDQGTLVGALDFH GLRSRHPWSREQGAPAPAGDAPAGHGE

[0420] Influenza Virus A / Puerto Rico / 8 / 34 (IAV PR8) Non-Structural Protein 1 (NS1)

[0421] DNA ATGGATCCCAACACAGTCTCTTCCTTCCAGGTGGACTGTTTCCTCTGGCATGTAAGG 24 Sequence AAGCGCGTAGCAGACCAGGAGTTGGGAGATGCCCCCTTCCTGGATCGGCTGAGACGG GACCAGAAAAGCCTGCGAGGCAGAGGCAGCACCCTGGGGCTGGACATAGAAACAGCC ACCCGGGCAGGGAAGCAGATCGTGGAGCGCATCCTTAAGGAGGAGTCTGATGAGGCT CTCAAGATGACCATGGCCTCAGTGCCTGCCAGCAGGTACCTGACAGACATGACACTG GAGGAAATGTCAAGAGACTGGAGCATGCTGATCCCTAAACAGAAGGTGGCGGGCCCT CTTTGCATCCGGATGGATCAAGCCATCATGGACAAGAACATTATCCTGAAAGCCAAC TTCAGCGTCATCTTTGACAGGCTGGAAACCCTCATCCTTCTGCGGGCCTTCACAGAA GAAGGAGCCATTGTGGGAGAGATCAGCCCACTGCCCAGTCTGCCTGGCCACACTGCA GAAGAT GT GAAGAAT GCT GT GGGCGT CTTAATT GGAGGCTT GGAGT GGAAT GACAAC ACTGTGAGAGTTAGTGAGACACTGCAGAGATTTGCCTGGAGGTCCAGCAATGAAAAC GGCCGGCCTCCCCTTACCCCAAAGCAAAAACGGGAAATGGCTGGCACCATCCGCTCA GAGGTG RNA AUGGAUCCCAACACAGUCUCUUCCUUCCAGGUGGACUGUUUCCUCUGGCAUGUAAGG 65 Sequence AAGCGCGUAGCAGACCAGGAGUUGGGAGAUGCCCCCUUCCUGGAUCGGCUGAGACGG GACCAGAAAAGCCUGCGAGGCAGAGGCAGCACCCUGGGGCUGGACAUAGAAACAGCC ACCCGGGCAGGGAAGCAGAUCGUGGAGCGCAUCCUUAAGGAGGAGUCUGAUGAGGCU CUCAAGAUGACCAUGGCCUCAGUGCCUGCCAGCAGGUACCUGACAGACAUGACACUG GAGGAAAUGUCAAGAGACUGGAGCAUGCUGAUCCCUAAACAGAAGGUGGCGGGCCCU CUUUGCAUCCGGAUGGAUCAAGCCAUCAUGGACAAGAACAUUAUCCUGAAAGCCAAC UUCAGCGUCAUCUUUGACAGGCUGGAAACCCUCAUCCUUCUGCGGGCCUUCACAGAA GAAGGAGCCAUUGUGGGAGAGAUCAGCCCACUGCCCAGUCUGCCUGGCCACACUGCA

[0422]

[0423] GAAGAUGUGAAGAAUGCUGUGGGCGUCUUAAUUGGAGGCUUGGAGUGGAAUGACAAC

[0424] 12673398.1 ACUGUGAGAGUUAGUGAGACACUGCAGAGAUUUGCCUGGAGGUCCAGCAAUGAAAAC GGCCGGCCUCCCCUUACCCCAAAGCAAAAACGGGAAAUGGCUGGCACCAUCCGCUCA GAGGUG

[0425] Amino MDPNTVSSFQVDCFLWHVRKRVADQELGDAPFLDRLRRDQKSLRGRGSTLGLDIETA 103 Acid TRAGKQIVERILKEESDEALKMTMASVPASRYLTDMTLEEMSRDWSMLIPKQKVAGP Sequence LCIRMDQAIMDKNIILKANFSVIFDRLETLILLRAFTEEGAIVGEISPLPSLPGHTA EDVKNAVGVLIGGLEWNDNTVRVSETLQRFAWRSSNENGRPPLTPKQKREMAGTIRS EV

[0426] Toscana Virus (TOSV) Nonstructural protein (NSs)

[0427] DNA ATGCAGAGTCGGGCTGTCATCCTTAAGTATAGAAGTGGCTCGGGCCACAAAAGAAGC 25 Sequence CTGCCACGCTTCTATATTGACTGTGACCTGGATACCTTTGACTTTGAGAAAGACTGC TCACTGATCGAAAATGAATTCCCCATCTACATCAACAACTATAAGGTGGTTTACAAA AGCAAACCTACACTCTCCCATTTCCTGATCGAAAAGGAGTTCCCTGCCGTGCTAGGG CCTGGTATGATCTCTGCTGTGAGAACCCGGCTGTATGAGCCTACCATGCGGGAGCTC TACCAGGAGAGCATTCACCAGCTAAAGAGAAGCAACAAGAAATACCTCCTGAGCGCC CTGCGCTGGCCAACTGGCATTCCTACGCTGGAGTTTATAGATTACTACTTTGAAGAA CTGCTGTTCCTGTCTGAATTTGACCCTGGAAGCATCCAGAGGTACCTGAAGTTACTG GT GAAGGCTAGCGGT CT GTACAACT CTACCAAT GAGGAGCAGATT GTAGAGAT CCAC AGGAGAGTGCTTATCGAAGGGAAGAAGCATGGCCTGACCGCCTTTGATCTGCCAGGA AATGATATTCTGGGAGACATCTGTGTGGTGCAAGCCGCTCGAGTTACTAGACTGGTG GCCAAGACTTTTTCCAAGATGACAAGAGACACACACCTGATGATTTACTTCTCGATC T CCCCAGT GGAGCT GGT GCT GAGCAAGCTAGACAAAAAGGGAGATAAAAGAGCAAAA GCCAAAGGCCTCATGAGCATGTCAGCCGCCAGAAGCTATGACTATTTCATGAGAACA GATTTGGGCTTCCGGGAGACAGCCCTGTCGACATTCTGGGCAAAGGACTGGCCCACC CCCCAGGAAACCATCCTATCTGATAAGCGCTGCTTGAAGGAAGATATGAGGGTCACC AAGTGGCTGCCCTCCCCCCCGCACTACCCACCGCTGTGA RNA AUGCAGAGUCGGGCUGUCAUCCUUAAGUAUAGAAGUGGCUCGGGCCACAAAAGAAGC 66 Sequence CUGCCACGCUUCUAUAUUGACUGUGACCUGGAUACCUUUGACUUUGAGAAAGACUGC U CACU GAU C GAAAAU GAAUU C C C CAU CUACAU CAACAACUAUAAGGU GGUUUACAAA AGCAAACCUACACUCUCCCAUUUCCUGAUCGAAAAGGAGUUCCCUGCCGUGCUAGGG CCUGGUAUGAUCUCUGCUGUGAGAACCCGGCUGUAUGAGCCUACCAUGCGGGAGCUC UACCAGGAGAGCAUUCACCAGCUAAAGAGAAGCAACAAGAAAUACCUCCUGAGCGCC CUGCGCUGGCCAACUGGCAUUCCUACGCUGGAGUUUAUAGAUUACUACUUUGAAGAA CUGCUGUUCCUGUCUGAAUUUGACCCUGGAAGCAUCCAGAGGUACCUGAAGUUACUG GUGAAGGCUAGCGGUCUGUACAACUCUACCAAUGAGGAGCAGAUUGUAGAGAUCCAC AGGAGAGUGCUUAUCGAAGGGAAGAAGCAUGGCCUGACCGCCUUUGAUCUGCCAGGA AAUGAUAUUCUGGGAGACAUCUGUGUGGUGCAAGCCGCUCGAGUUACUAGACUGGUG GC CAAGACUUUUU C CAAGAU GACAAGAGACACACAC CU GAU GAUUUACUU CU C GAU C UCCCCAGUGGAGCUGGUGCUGAGCAAGCUAGACAAAAAGGGAGAUAAAAGAGCAAAA GCCAAAGGCCUCAUGAGCAUGUCAGCCGCCAGAAGCUAUGACUAUUUCAUGAGAACA GAUUUGGGCUUCCGGGAGACAGCCCUGUCGACAUUCUGGGCAAAGGACUGGCCCACC CCCCAGGAAACCAUCCUAUCUGAUAAGCGCUGCUUGAAGGAAGAUAUGAGGGUCACC AAGUGGCUGCCCUCCCCCCCGCACUACCCACCGCUGUGA

[0428] Amino MQSRAVILKYRSGSGHKRSLPRFYIDCDLDTFDFEKDCSLIENEFPIYINNYKWYK 104 Acid SKPTLSHFLIEKEFPAVLGPGMISAVRTRLYEPTMRELYQESIHQLKRSNKKYLLSA Sequence LRWPTGIPTLEFIDYYFEELLFLSEFDPGSIQRYLKLLVKASGLYNSTNEEQIVEIH RRVLIEGKKHGLTAFDLPGNDILGDICWQAARVTRLVAKTFSKMTRDTHLMIYFSI SPVELVLSKLDKKGDKRAKAKGLMSMSAARSYDYFMRTDLGFRETALSTFWAKDWPT PQETILSDKRCLKEDMRVTKWLPSPPHYPPL

[0429] Encephalomyocarditis virus (EMCV) leader protease (Lpro)

[0430] DNA ATGGCCACGACCATGGAGCAGGAGACATGCGCCCACAGCCTGACCTTCGAGGAATGC 26 Sequence CCCAAATGCTCTGCTCTGCAGTACAGAAACGGCTTCTACCTGCTGAAGTATGATGAA GAGTGGTACCCTGAGGAGCTGCTCACAGATGGAGAGGATGACGTCTTTGACCCAGAG CT GGACAT GGAAGT GGT GTTT GAACTT CAA RNA AUGGCCACGACCAUGGAGCAGGAGACAUGCGCCCACAGCCUGACCUUCGAGGAAUGC 67 Sequence CCCAAAUGCUCUGCUCUGCAGUACAGAAACGGCUUCUACCUGCUGAAGUAUGAUGAA

[0431] GAGUGGUACCCUGAGGAGCUGCUCACAGAUGGAGAGGAUGACGUCUUUGACCCAGAG

[0432]

[0433] CU GGACAU GGAAGU GGU GUUU GAACUU CAA

[0434] 12673398.1 Amino MATTMEQETCAHSLTFEECPKCSALQYRNGFYLLKYDEEWYPEELLTDGEDDVFDPE 105 Acid LDMEWFELQ

[0435] Sequence

[0436] Vaccinia Virus (VACV) NIL

[0437] DNA ATGAGAACCCTGCTGATCAGATACATCCTGTGGAGGAATGATAATGACCAGACCTAC 27 Sequence TACAACGACAACTT CAAGAAACT GAT GCT GCTT GAT GAGCT GGT GGAT GAT GGGGAC

[0438] GT GT GCACACT CAT CAAGAACAT GAGGAT GACGCT GAGT GAT GGCCCT CTT CT GGAC CGTCTGAACCAGCCCGTCAACAACATTGAAGATGCCAAGAGAATGATCGCCATCTCT GCAAAAGTGGCTCGGGACATTGGAGAGAGAAGCGAAATCCGCTGGGAGGAGAGCTTC ACCATCCTGTTTCGCATGATCGAAACATACTTTGATGACCTAATGATTGACCTCTAT GGCGAGAAGtga

[0439] RNA AU GAGAAC C CU GCU GAU CAGAUACAU C CU GU GGAGGAAU GAUAAU GAC CAGAC CUAC 68 Sequence UACAACGACAACUUCAAGAAACUGAUGCUGCUUGAUGAGCUGGUGGAUGAUGGGGAC GUGUGCACACUCAUCAAGAACAUGAGGAUGACGCUGAGUGAUGGCCCUCUUCUGGAC C GU CU GAAC CAGC C C GU CAACAACAUU GAAGAU GC CAAGAGAAU GAU C GC CAU CU CU GCAAAAGUGGCUCGGGACAUUGGAGAGAGAAGCGAAAUCCGCUGGGAGGAGAGCUUC AC CAU C CU GUUU C GCAU GAU C GAAACAUACUUU GAU GAC CUAAU GAUU GAC CU CUAU GGCGAGAAGtga

[0440] Amino MRTLLIRYILWRNDNDQTYYNDNFKKLMLLDELVDDGDVCTLIKNMRMTLSDGPLLD 106 Acid RLNQPVNNIEDAKRMIAI SAKVARDIGERSEIRWEESFTILFRMIETYFDDLMIDLY

[0441]

[0442] Sequence GEK*

[0443] Table 6, Illustrative Linker Sequences

[0444] Linkers SEQID NO: Coxsackievirus B3 (CVB3) IRES

[0445] DNA CACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTAT 28 Sequence CAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACA AATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGTCATGACCAAAATCC CT T AAC GT GAGT TTTCGTTC CACT GAGC GT CAGAC C C C GT AGAAAAGAT CAAAGGA TCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACC ACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGA AGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCG TAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCT AATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGG ACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCG TGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCG TGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGG TAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCC TGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTT GTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTT TACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCC CCTGATTCTGTGGATAACCGTGCGGCCGCTCGGTCTCATATCTTAAAACAGCCTGT GGGTTGATCCCACCCACAGGCCCATTGGGCGCTAGCACTCTGGTATCACGGTACCT TTGTGCGCCTGTTTTATACCCCCTCCCCCAACTGTAACTTAGAAGTAACACACACC GATCAACAGTCAGCGTGGCACACCAGCCACGTTTTGATCAAGCACTTCTGTTACCC CGGACTGAGTATCAATAGACTGCTCACGCGGTTGAAGGAGAAAGCGTTCGTTATCC GGCCAACTACTTCGAAAAACCTAGTAACACCGTGGAAGTTGCAGAGTGTTTCGCTC AGCACTACCCCAGTGTAGATCAGGTCGATGAGTCACCGCATTCCCCACGGGCGACC GTGGCGGTGGCTGCGTTGGCGGCCTGCCCATGGGGAAACCCATGGGACGCTCTAAT ACAGACATGGTGCGAAGAGTCTATTGAGCTAGTTGGTAGTCCTCCGGCCCCTGAAT GCGGCTAATCCTAACTGCGGAGCACACACCCTCAAGCCAGAGGGCAGTGTGTCGTA ACGGGCAACTCTGCAGCGGAACCGACTACTTTGGGTGTCGTGTTTCATTTTATTCC TATACTGGCTGCTTATGGTGACAATTGAGAGATCGTTACCATATAGCTATTGGATT GGCCATCCGGTGACTAATAGAGCTATTATATATCCCTTTGTTGGGTTTATACCACT T AG C T T GAAAGAG GT T AAAAC AT T AC AAT T CAT TGTTAAGTT GAAT AC AG C AAAAT GGTGAGACCGCGGCCGCGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATT

[0446]

[0447] AAAAAT GAAGT T T T AAAT CAAT CT AAAGT AT AT AT GAGT AAACT T GGT CT GACAGT

[0448] 12673398.1 TACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATC CATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCAT CTGGCCCCAGTGCTGCAATGATACCGCGGGACCCACGCTCACCGGCTCCAGATTTA TCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTT ATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGC CAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGC TCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTAC ATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTG TCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAAT TCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAAC CAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAA TACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAA CGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGAT GTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTT CTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGA RNA GAG GGAAAU GUU GAAUACU CAUACU CUUCCUUUUU CAAUAUUAUU GAAGCAUUUAU 69 Sequence CAGGGUUAUU GU CU GAU GAGC GGAUACAUAUUU GAAU GUAUUUAGAAAAAUAAACA AAUAGGGGUUCCGCGCACAUUUCCCCGAAAAGUGCCACCUGUCAUGACCAAAAUCC CUUAAC GU GAGUUUU C GUU C CACU GAGC GU CAGAC C C C GUAGAAAAGAU CAAAGGA UCUUCUUGAGAUCCUUUUUUUCUGCGCGUAAUCUGCUGCUUGCAAACAAAAAAACC ACCGCUACCAGCGGUGGUUUGUUUGCCGGAUCAAGAGCUACCAACUCUUUUUCCGA AGGUAACUGGCUUCAGCAGAGCGCAGAUACCAAAUACUGUUCUUCUAGUGUAGCCG UAGUUAGGCCACCACUUCAAGAACUCUGUAGCACCGCCUACAUACCUCGCUCUGCU AAUCCUGUUACCAGUGGCUGCUGCCAGUGGCGAUAAGUCGUGUCUUACCGGGUUGG ACUCAAGACGAUAGUUACCGGAUAAGGCGCAGCGGUCGGGCUGAACGGGGGGUUCG UGCACACAGCCCAGCUUGGAGCGAACGACCUACACCGAACUGAGAUACCUACAGCG UGAGCUAUGAGAAAGCGCCACGCUUCCCGAAGGGAGAAAGGCGGACAGGUAUCCGG UAAGCGGCAGGGUCGGAACAGGAGAGCGCACGAGGGAGCUUCCAGGGGGAAACGCC UGGUAUCUUUAUAGUCCUGUCGGGUUUCGCCACCUCUGACUUGAGCGUCGAUUUUU GUGAUGCUCGUCAGGGGGGCGGAGCCUAUGGAAAAACGCCAGCAACGCGGCCUUUU UACGGUUCCUGGCCUUUUGCUGGCCUUUUGCUCACAUGUUCUUUCCUGCGUUAUCC CCUGAUUCUGUGGAUAACCGUGCGGCCGCUCGGUCUCAUAUCUUAAAACAGCCUGU GGGUUGAUCCCACCCACAGGCCCAUUGGGCGCUAGCACUCUGGUAUCACGGUACCU UUGUGCGCCU GUUUUAUAC CCCCUCCCC CAACU GUAACUUAGAAGUAACACACAC C GAUCAACAGUCAGCGUGGCACACCAGCCACGUUUUGAUCAAGCACUUCUGUUACCC CGGACUGAGUAUCAAUAGACUGCUCACGCGGUUGAAGGAGAAAGCGUUCGUUAUCC GGCCAACUACUUCGAAAAACCUAGUAACACCGUGGAAGUUGCAGAGUGUUUCGCUC AGCACUACCCCAGUGUAGAUCAGGUCGAUGAGUCACCGCAUUCCCCACGGGCGACC GUGGCGGUGGCUGCGUUGGCGGCCUGCCCAUGGGGAAACCCAUGGGACGCUCUAAU ACAGACAUGGUGCGAAGAGUCUAUUGAGCUAGUUGGUAGUCCUCCGGCCCCUGAAU GCGGCUAAUCCUAACUGCGGAGCACACACCCUCAAGCCAGAGGGCAGUGUGUCGUA ACGGGCAACUCUGCAGCGGAACCGACUACUUUGGGUGUCGUGUUUCAUUUUAUUCC UAUACUGGCUGCUUAUGGUGACAAUUGAGAGAUCGUUACCAUAUAGCUAUUGGAUU GGCCAUCCGGUGACUAAUAGAGCUAUUAUAUAUCCCUUUGUUGGGUUUAUACCACU UAGCUU GAAAGAGGUUAAAACAUUACAAUU CAUU GUUAAGUU GAAUACAGCAAAAU GGUGAGACCGCGGCCGCGAUUAUCAAAAAGGAUCUUCACCUAGAUCCUUUUAAAUU AAAAAU GAAGUUUUAAAU CAAU CUAAAGUAUAUAU GAGUAAACUU GGU CU GACAGU UAC CAAU GCUUAAU CAGU GAGGCAC CUAU CU CAGC GAU CU GU CUAUUU C GUU CAU C CAUAGUUGCCUGACUCCCCGUCGUGUAGAUAACUACGAUACGGGAGGGCUUACCAU CUGGCCCCAGUGCUGCAAUGAUACCGCGGGACCCACGCUCACCGGCUCCAGAUUUA UCAGCAAUAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGUGGUCCUGCAACUUU AUCCGCCUCCAUCCAGUCUAUUAAUUGUUGCCGGGAAGCUAGAGUAAGUAGUUCGC CAGUUAAUAGUUUGCGCAACGUUGUUGCCAUUGCUACAGGCAUCGUGGUGUCACGC UCGUCGUUUGGUAUGGCUUCAUUCAGCUCCGGUUCCCAACGAUCAAGGCGAGUUAC AUGAUCCCCCAUGUUGUGCAAAAAAGCGGUUAGCUCCUUCGGUCCUCCGAUCGUUG UCAGAAGUAAGUUGGCCGCAGUGUUAUCACUCAUGGUUAUGGCAGCACUGCAUAAU U CU CUUACU GU CAU GC CAU C C GUAAGAU GCUUUU CU GU GACU GGU GAGUACU CAAC CAAGUCAUUCUGAGAAUAGUGUAUGCGGCGACCGAGUUGCUCUUGCCCGGCGUCAA UACGGGAUAAUACCGCGCCACAUAGCAGAACUUUAAAAGUGCUCAUCAUUGGAAAA

[0449]

[0450] CGUUCUUCGGGGCGAAAACUCUCAAGGAUCUUACCGCUGUUGAGAUCCAGUUCGAU 12673398.1 GUAAC C CACU C GU GCAC C CAACU GAU CUU CAGCAU CUUUUACUUU GAG CAGC GUUU CUGGGUGAGCAAAAACAGGAAGGCAAAAUGCCGCAAAAAAGGGAAUAAGGGCGA

[0451] Encephalomyocarditis Virus (EMCV) IRES

[0452] DNA GTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGG 29 Sequence TCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAA CGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTC TGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTG CCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTAT TCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTG GGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAACGTCTAGGC CCCCCGAACCACGGGGACGTGGTTTTC RNA GUCUUCUUGACGAGCAUUCCUAGGGGUCUUUCCCCUCUCGCCAAAGGAAUGCAAGG 70 Sequence U CU GUU GAAU GU C GU GAAGGAAGCAGUU C CU CU GGAAGCUU CUU GAAGACAAACAA CGUCUGUAGCGACCCUUUGCAGGCAGCGGAACCCCCCACCUGGCGACAGGUGCCUC UGCGGCCAAAAGCCACGUGUAUAAGAUACACCUGCAAAGGCGGCACAACCCCAGUG CCACGUUGUGAGUUGGAUAGUUGUGGAAAGAGUCAAAUGGCUCUCCUCAAGCGUAU UCAACAAGGGGCUGAAGGAUGCCCAGAAGGUACCCCAUUGUAUGGGAUCUGAUCUG GGGCCUCGGUGCACAUGCUUUACAUGUGUUUAGUCGAGGUUAAAAAACGUCUAGGC CCCCCGAACCACGGGGACGUGGUUUUC

[0453] Porcine Kobuvirus (PKV) IRES

[0454] DNA TTTGAAAAGGGGGTGGGGGGGCCTCGGCCCCCTCACCCTCTTTTCCGGTGGCCACC 30 Sequence CGCCCGGGCCACCGTTACTCCACTCCACTCCTTCGGGACTGGTTTGGAGGAACATA ACAGGGCTTCCCATCCCTGTTTACCCTTACTCCACTCACCCTTCCCCTTGACCAAC CCTATCCACACCCCACTGACTGACTCCTTTGGATCTTGACCTCGGAATGCCTACTT GACCTCCCACTTGCCTCTCCCTTTTCGGATTGCCGGTGGTGCCTGGCGGAAAAAGC ACAAGTGTGTTGTTGGCTACCAAACTCCTACCCGACAAAGGTGCGTGTCCGCGTGC T GAGTAAT GGGATAGGAGAT GCCAATAACAGGCT CGCCCAT GAGTAGAGCAT GGAC TGCGGTGCATG RNA UUUGAAAAGGGGGUGGGGGGGCCUCGGCCCCCUCACCCUCUUUUCCGGUGGCCACC 71 Sequence CGCCCGGGCCACCGUUACUCCACUCCACUCCUUCGGGACUGGUUUGGAGGAACAUA ACAGGGCUU C C CAU C C CU GUUUAC C CUUACU C CACU CAC CCUUCCCCUU GAC CAAC CCUAUCCACACCCCACUGACUGACUCCUUUGGAUCUUGACCUCGGAAUGCCUACUU GACCUCCCACUUGCCUCUCCCUUUUCGGAUUGCCGGUGGUGCCUGGCGGAAAAAGC ACAAGUGUGUUGUUGGCUACCAAACUCCUACCCGACAAAGGUGCGUGUCCGCGUGC UGAGUAAUGGGAUAGGAGAUGCCAAUAACAGGCUCGCCCAUGAGUAGAGCAUGGAC UGCGGUGCAUG

[0455] Manhattan Parechovirus (MPV) IRES

[0456] DNA TTGAAAAGGGGGGGGTGGGGCTCATGCCTCACCCTCTTCCTTTGCTTCCGGGCTGG 31 Sequence GAAGCTTGGGGCGCACCCCTCCCTTTTCATGTCAAGTTGCACCTGCCACTCTCCTG GCATGTCTTTCTGTCCAAACTTTCTAGGAACCACATGCGTGCCTACCCCCATTCTC GTGGGAGACTGTCCTGTGCAATTTTTCTTTAACTTGTAACCACTAAGCTTGTTAGT CCTTGTTGTGCCTGATTGCCCCTGACCGAACCAGGGCTCGACCCCCTGTAGTAGGG CT GGACAAGCAT GAGGGATAT GCACAACGAAACT GCGGACGGT GAGT GACCTAT GC AGAATCTAATCCCAGGAGTACCTGGAGATCCTAGGGCCCGACCGGGGCTGATGTAA CATACCAGCCTAAACCGCTCGGTCAACCCTAGGGGGACACGTTCCAATTTTCTGCT ATTTGGGGAAGGGACGCCCCAGACCGGAGTAGCTTCCGGGGTCATACAGCGTGATA GCGTGGCGGCCAGGCCCTGTCTGCTTTTAAACTGGTAGGCCATATACTATG RNA UUGAAAAGGGGGGGGUGGGGCUCAUGCCUCACCCUCUUCCUUUGCUUCCGGGCUGG 72 Sequence GAAGCUUGGGGCGCACCCCUCCCUUUUCAUGUCAAGUUGCACCUGCCACUCUCCUG GCAU GUCUUUCUGUC CAAACUUU CUAGGAAC CACAU GC GU GC CUAC C C C CAUU CU C GU GGGAGACU GU C CU GU GCAAUUUUU CUUUAACUU GUAAC CACUAAGCUU GUUAGU CCUUGUUGUGCCUGAUUGCCCCUGACCGAACCAGGGCUCGACCCCCUGUAGUAGGG CUGGACAAGCAUGAGGGAUAUGCACAACGAAACUGCGGACGGUGAGUGACCUAUGC AGAAUCUAAUCCCAGGAGUACCUGGAGAUCCUAGGGCCCGACCGGGGCUGAUGUAA CAUACCAGCCUAAACCGCUCGGUCAACCCUAGGGGGACACGUUCCAAUUUUCUGCU AUUUGGGGAAGGGACGCCCCAGACCGGAGUAGCUUCCGGGGUCAUACAGCGUGAUA GCGUGGCGGCCAGGCCCUGUCUGCUUUUAAACUGGUAGGCCAUAUACUAUG

[0457]

[0458] Tortoise Rafivirus (TraV) IRES

[0459] 12673398.1 DNA TAAGTAGGGAACATATTCAATTCATATTGTTCATCTCACTGAACCCGCATGAAGGA 32 Sequence CTGCATTGCATATCCTGGACGAGGTGACGTGGAATATTTGGACATTTATGGATTGG ACACTATAACGCTTTGTGCCTCTACGGAGATGTAACCATAATCTTAAGTAGTAGTA CCCCAGCACAAGAGGATAAAGTGGCATACACGACAACGGGTGTTGCTCGCACCTTA GTAATGTGGATGTCCACCCTTGGAGCGTGCTGAAACTCTGTGGGTAAAGACACATA TTAGTACAAATGTGGGGGAACTCACTGAAAGGGCATGTCCCGTGTACTGGTGTGCC GGAAAGTGGGGGTCGCTTTCTGGAGAACTTAGTAGTTCTTGTTATTGGGTGATAGC CTTGCGGCGGATCAACCCACAGTTTTAATCCGTTGTTTTGCATATG RNA UAAGUAGGGAACAUAUU CAAUU CAUAUU GUU GAU CU CACU GAAC C C GCAU GAAGGA 73 Sequence CUGCAUUGCAUAUCCUGGACGAGGUGACGUGGAAUAUUUGGACAUUUAUGGAUUGG ACACUAUAAC GCUUU GU GC CU CUAC GGAGAU GUAAC CAUAAU CUUAAGUAGUAGUA CCCCAGCACAAGAGGAUAAAGUGGCAUACACGACAACGGGUGUUGCUCGCACCUUA GUAAUGUGGAUGUCCACCCUUGGAGCGUGCUGAAACUCUGUGGGUAAAGACACAUA UUAGUACAAAUGUGGGGGAACUCACUGAAAGGGCAUGUCCCGUGUACUGGUGUGCC GGAAAGUGGGGGUCGCUUUCUGGAGAACUUAGUAGUUCUUGUUAUUGGGUGAUAGC CUUGCGGCGGAUCAACCCACAGUUUUAAUCCGUUGUUUUGCAUAUG

[0460] T2A Peptide

[0461] DNA GGCTCCGGCGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCC 33 Sequence CGGCCCA

[0462] RNA GGCUCCGGCGAGGGCAGGGGAAGUCUUCUAACAUGCGGGGACGUGGAGGAAAAUCC 74 Sequence CGGCCCA

[0463] Amino Acid GSGEGRGSLLTCGDVEENPGP 107 Sequence

[0464] P2A Peptide

[0465] DNA GGCAGCGGCGCCACCAACTTCAGCCTGCTGAAGCAAGCCGGCGACGTGGAGGAGAA 34 Sequence CCCCGGCCCC

[0466] RNA GGCAGCGGCGCCACCAACUUCAGCCUGCUGAAGCAAGCCGGCGACGUGGAGGAGAA 75 Sequence CCCCGGCCCC

[0467] Amino Acid GSGATNFSLLKQAGDVEENPGP 108 Sequence

[0468] E2A Peptide

[0469] DNA GGCTCGGGCCAGTGTACTAATTATGCTCTCTTGAAATTGGCTGGAGATGTTGAGAG 35 Sequence CAACCCAGGTCCC

[0470] RNA GGCUCGGGCCAGUGUACUAAUUAUGCUCUCUUGAAAUUGGCUGGAGAUGUUGAGAG 76 Sequence CAACCCAGGUCCC

[0471] Amino Acid GSGQCTNYALLKLAGDVESNPGP 109 Sequence

[0472] Furin-T2A Peptide

[0473] DNA AGGAAGAGGAGGGGCTCCGGCGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGT 36 Sequence GGAGGAAAATCCCGGCCCA

[0474] RNA AGGAAGAGGAGGGGCUCCGGCGAGGGCAGGGGAAGUCUUCUAACAUGCGGGGACGU 77 Sequence GGAGGAAAAUCCCGGCCCA

[0475] Amino Acid RKRRGSGEGRGSLLTCGDVEENPGP 110 Sequence

[0476] Furin-P2A Peptide

[0477] DNA AGGAAGAGGAGGGGCAGCGGCGCCACCAACTTCAGCCTGCTGAAGCAAGCCGGCGA 37 Sequence CGTGGAGGAGAACCCCGGCCCC

[0478] RNA AGGAAGAGGAGGGGCAGCGGCGCCACCAACUUCAGCCUGCUGAAGCAAGCCGGCGA 78 Sequence CGUGGAGGAGAACCCCGGCCCC

[0479] Amino Acid RKRRGSGATNFSLLKQAGDVEENPGP 111 Sequence

[0480] EEEV 3 / 4J

[0481] DNA GTGCGGAGGCACTCGAATTGACGGTACGAAGCGGGTGCGTACATTTTCTCATCCGA 38 Sequence GACGGGACAAGGGCACCTGCAACAAAAATCTACGCGGCAATGCAAACTCCAGTATC CAATCCTGGAGCGTTCCGTCCATGAGAAATTTTACGCCCCGCGCCTCGATCTCGAG RNA GUGCGGAGGCACUCGAAUUGACGGUACGAAGCGGGUGCGUACAUUUUCUCAUCCGA 79 Sequence GACGGGACAAGGGCACCUGCAACAAAAAUCUACGCGGCAAUGCAAACUCCAGUAUC

[0482]

[0483] CAAUCCUGGAGCGUUCCGUCCAUGAGAAAUUUUACGCCCCGCGCCUCGAUCUCGAG

[0484] 12673398.1 Amino Acid VRRHSNRYEAGAYIFSSETGQGHLQQKSTRQCKLQYPILERSVHEKFYAPRLDLE 112 Sequence

[0485] (version 1)

[0486] Amino Acid RYEAGAYIFSSETGQGHLQQKSTRQCKLQYPILERSVHEKFYAPRLDLE 113 Sequence

[0487]

[0488] (version 2)

[0489] Table 7, Illustrative trRNA Sequences

[0490] trRNAs SEQID NO: oeSTR-Fluc

[0491] DNA ATAGAAGATGGCGGCGTAGTACACACTATTGAATCAAACAGCCGACCAATTGCACT 39 Sequence AC CAT CACAAC GGAGAAGC CAGT AGT AAAC GT AGAC GT AGAC C C C CAGAGT C C GT T TGTCGTGCAACTGCAAAAAAGCTTCCCGCAATTTGAGGTAGTAGCACAGGAGGTGA CTCCAAATCACCATCCTAATCCCAGAGCATTTTCGCATCTGGGCAGTAAACTAATC GAGCTGGAGGTTCCTACCACAGCGACGATCTTGGACATAGGCAGCGCACCGGCTCG TAGAACGATAAACCCCTAGTGCCACCATGGAGGATGCCAAAAACATCAAAAAGGGC CCTGCCCCCTTCTACCCACTGGAGGACGGCACCGCTGGCGAGCAGCTGCACAAAGC AATGAAGCGGTATGCACTCGTGCCAGGAACCATCGCTTTCACAGACGCCCACATCG AAGTGGATATTACTTACGCTGAGTACTTTGAGATGTCAGTGAGACTGGCTGAGGCC AT GAAAAGATACGGACT GAACACAAACCACAGGATT GT GGT CT GTT CT GAGAACAG CCTGCAGTTCTTCATGCCTGTGCTGGGCGCCTTGTTTATTGGAGTGGCAGTCGCCC CT GCAAAT GACATTTACAAT GAAAGGGAGCT GCT CAACAGCAT GGGGAT CT CT CAG CCTACAGTGGTCTTTGTCAGCAAAAAAGGCCTGCAGAAGATCCTGAATGTCCAGAA GAAGCTCCCAATCATCCAAAAGATTATTATCATGGATTCTAAGACAGACTACCAAG GATTCCAATCCATGTACACTTTCGTCACGTCCCACCTGCCCCCCGGCTTCAATGAG TATGACTTTGTCCCTGAGTCCTTTGATCGGGACAAAACCATTGCCCTGATAATGAA TTCCAGCGGCAGCACAGGCCTCCCTAAAGGAGTAGCCCTTCCCCACAGAACTGCCT GCGTTAGATTCTCCCATGCGCGGGACCCTATTTTCGGAAACCAAATCATACCAGAC ACAGCTATCCTCTCGGTGGTTCCTTTCCACCACGGCTTCGGGATGTTTACGACCCT GGGCTACCTCATTTGCGGCTTCCGTGTGGTACTCATGTATCGCTTCGAAGAGGAGC TGTTCCTGCGGTCCTTACAGGACTACAAAATCCAGTCAGCCCTGCTGGTGCCCACC CTATTCTCTTTCTTTGCCAAGTCCACCCTGATCGACAAGTATGATCTGTCTAACTT GCATGAAATCGCCTCAGGAGGAGCCCCCCTGAGCAAGGAAGTTGGCGAAGCAGTGG CAAAGAGGTTCCATCTGCCGGGGATCCGACAGGGCTATGGTCTGACTGAAACCACC TCAGCCATCCTCATCACACCTGAAGGCGACGACAAGCCAGGTGCCGTGGGTAAGGT GGTACCTTTCTTTGAAGCTAAGGTGGTGGACCTGGACACAGGCAAAACGCTGGGCG TCAACCAGAGAGGCGAGCTGTGTGTGAGAGGCCCCATGATTATGAGCGGCTACGTC AACAACCCTGAGGCCACCAACGCCCTGATTGATAAGGATGGCTGGCTGCACAGCGG AGACATCGCGTACTGGGATGAGGATGAACACTTCTTCATCGTAGACAGACTTAAGA GCCTGATCAAATACAAGGGGTACCAGGTCGCGCCGGCAGAGCTAGAGTCCATCCTT CTGCAGCATCCAAACATCTTTGACGCTGGCGTGGCCGGCCTGCCTGATGACGATGC TGGAGAACTCCCCGCGGCCGTTGTGGTGCTGGAGCACGGCAAGACCATGACTGAAA AGGAGATCGTCGACTATGTGGCCAGCCAGGTAACCACCGCCAAGAAGCTGAGAGGC GGCGTAGTGTTCGTGGATGAGGTGCCCAAAGGTCTTACCGGAAAGCTGGACGCCAG GAAGAT CCGGGAGAT CCT GAT CAAAGCTAAGAAGGGGGGCAAGATAGCT GT GT GAT AATAGCTCGAGGCGGCCGCAGGAGCTTAATTCGACGAATAATTGGATTTTTATTTT AT T T T G C AAT T G GT T T T T AAT AT T T C C AAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAA RNA AUAGAAGAUGGCGGCGUAGUACACACUAUUGAAUCAAACAGCCGACCAAUUGCACU 80 Sequence AC C AU CACAAC G GAGAAG C C AGU AGU AAAC GU AGAC GU AGAC C C C CAGAGU C C GUU UGUCGUGCAACUGCAAAAAAGCUUCCCGCAAUUUGAGGUAGUAGCACAGGAGGUGA CUCCAAAUCACCAUCCUAAUCCCAGAGCAUUUUCGCAUCUGGGCAGUAAACUAAUC GAGCUGGAGGUUCCUACCACAGCGACGAUCUUGGACAUAGGCAGCGCACCGGCUCG UAGAACGAUAAACCCCUAGUGCCACCAUGGAGGAUGCCAAAAACAUCAAAAAGGGC CCUGCCCCCUUCUACCCACUGGAGGACGGCACCGCUGGCGAGCAGCUGCACAAAGC AAUGAAGCGGUAUGCACUCGUGCCAGGAACCAUCGCUUUCACAGACGCCCACAUCG AAGUGGAUAUUACUUACGCUGAGUACUUUGAGAUGUCAGUGAGACUGGCUGAGGCC

[0492]

[0493] AU GAAAAGAUAC GGACU GAACACAAAC CACAGGAUU GU GGU CU GUU CU GAGAACAG

[0494] 12673398.1 CCUGCAGUUCUUCAUGCCUGUGCUGGGCGCCUUGUUUAUUGGAGUGGCAGUCGCCC CUGCAAAUGACAUUUACAAUGAAAGGGAGCUGCUCAACAGCAUGGGGAUCUCUCAG CCUACAGUGGUCUUUGUCAGCAAAAAAGGCCUGCAGAAGAUCCUGAAUGUCCAGAA GAAGCU C C CAAU GAU C CAAAAGAUUAUUAU GAU GGAUU CUAAGACAGACUAC CAAG GAUUCCAAUCCAUGUACACUUUCGUCACGUCCCACCUGCCCCCCGGCUUCAAUGAG UAU GACUUU GU C C CU GAGU C CUUU GAU C GGGACAAAAC CAUU GC C CU GAUAAU GAA UUCCAGCGGCAGCACAGGCCUCCCUAAAGGAGUAGCCCUUCCCCACAGAACUGCCU GCGUUAGAUUCUCCCAUGCGCGGGACCCUAUUUUCGGAAACCAAAUCAUACCAGAC ACAGCUAUCCUCUCGGUGGUUCCUUUCCACCACGGCUUCGGGAUGUUUACGACCCU GGGCUACCUCAUUUGCGGCUUCCGUGUGGUACUCAUGUAUCGCUUCGAAGAGGAGC UGUUCCUGCGGUCCUUACAGGACUACAAAAUCCAGUCAGCCCUGCUGGUGCCCACC CUAUUCUCUUUCUUUGCCAAGUCCACCCUGAUCGACAAGUAUGAUCUGUCUAACUU GCAUGAAAUCGCCUCAGGAGGAGCCCCCCUGAGCAAGGAAGUUGGCGAAGCAGUGG CAAAGAGGUUCCAUCUGCCGGGGAUCCGACAGGGCUAUGGUCUGACUGAAACCACC UCAGCCAUCCUCAUCACACCUGAAGGCGACGACAAGCCAGGUGCCGUGGGUAAGGU GGUACCUUUCUUUGAAGCUAAGGUGGUGGACCUGGACACAGGCAAAACGCUGGGCG UCAACCAGAGAGGCGAGCUGUGUGUGAGAGGCCCCAUGAUUAUGAGCGGCUACGUC AACAACCCUGAGGCCACCAACGCCCUGAUUGAUAAGGAUGGCUGGCUGCACAGCGG AGACAU C GC GUACU GGGAU GAGGAU GAACACUU CUU CAU C GUAGACAGACUUAAGA GCCUGAUCAAAUACAAGGGGUACCAGGUCGCGCCGGCAGAGCUAGAGUCCAUCCUU CUGCAGCAUCCAAACAUCUUUGACGCUGGCGUGGCCGGCCUGCCUGAUGACGAUGC UGGAGAACUCCCCGCGGCCGUUGUGGUGCUGGAGCACGGCAAGACCAUGACUGAAA AGGAGAUCGUCGACUAUGUGGCCAGCCAGGUAACCACCGCCAAGAAGCUGAGAGGC GGCGUAGUGUUCGUGGAUGAGGUGCCCAAAGGUCUUACCGGAAAGCUGGACGCCAG GAAGAUCCGGGAGAUCCUGAUCAAAGCUAAGAAGGGGGGCAAGAUAGCUGUGUGAU AAUAGCUCGAGGCGGCCGCAGGAGCUUAAUUCGACGAAUAAUUGGAUUUUUAUUUU AUUUU GCAAUU GGUUUUUAAUAUUU C CAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAA

[0495] E3L-IRES-F1UC

[0496] DNA ATGTACATCGACGAGCGGAGCAACGCCGAGATTGTGTGCGAGGCCATCAAGACCAT 40 Sequence CGGAATCGAAGGCGCCACAGCCGCTCAGCTGACCAGACAGCTGAACATGGAAAAGC GGGAAGTGAACAAGGCCCTGTACGACCTGCAGAGAAGCGCCATGGTGTACAGCAGC GACGACATCCCTCCTCGGTGGTTTATGACCACAGAGGCCGACAAGCCTGACGCCGA TGCTATGGCCGACGTGATCATCGACGACGTGTCCCGCGAGAAGTCCATGAGAGAGG ACCACAAGAGCTTCGACGATGTGATCCCCGCCAAGAAGATCATCGATTGGAAGGGC GCCAATCCTGTGACCGTGATCAACGAGTACTGCCAGATCACCAGAAGAGACTGGTC CTTCCGGATCGAGAGCGTGGGCCCTAGCAATAGCCCTACCTTCTACGCCTGCGTGG ACATCGACGGCAGAGTGTTCGATAAGGCCGACGGCAAGAGCAAGCGGGACGCCAAA AACAATGCCGCCAAGCTGGCCGTGGATAAGCTGCTGGGCTATGTGATCATCCGGTT CTGATAATAGTTAAAACAGCCTGTGGGTTGATCCCACCCACAGGCCCATTGGGCGC TAGCACTCTGGTATCACGGTACCTTTGTGCGCCTGTTTTATACCCCCTCCCCCAAC TGTAACTTAGAAGTAACACACACCGATCAACAGTCAGCGTGGCACACCAGCCACGT TTTGATCAAGCACTTCTGTTACCCCGGACTGAGTATCAATAGACTGCTCACGCGGT TGAAGGAGAAAGCGTTCGTTATCCGGCCAACTACTTCGAAAAACCTAGTAACACCG TGGAAGTTGCAGAGTGTTTCGCTCAGCACTACCCCAGTGTAGATCAGGTCGATGAG TCACCGCATTCCCCACGGGCGACCGTGGCGGTGGCTGCGTTGGCGGCCTGCCCATG GGGAAACCCATGGGACGCTCTAATACAGACATGGTGCGAAGAGTCTATTGAGCTAG TTGGTAGTCCTCCGGCCCCTGAATGCGGCTAATCCTAACTGCGGAGCACACACCCT CAAGCCAGAGGGCAGTGTGTCGTAACGGGCAACTCTGCAGCGGAACCGACTACTTT GGGTGTCCGTGTTTCATTTTATTCCTATACTGGCTGCTTATGGTGACAATTGAGAG ATCGTTACCATATAGCTATTGGATTGGCCATCCGGTGACTAATAGAGCTATTATAT ATCCCTTTGTTGGGTTTATACCACTTAGCTTGAAAGAGGTTAAAACATTACAATTC ATTGTTAAGTTGAATACAGCAAAGCCACCATGGAGGATGCCAAAAACATCAAAAAG GGCCCTGCCCCCTTCTACCCACTGGAGGACGGCACCGCTGGCGAGCAGCTGCACAA AGCAATGAAGCGGTATGCACTCGTGCCAGGAACCATCGCTTTCACAGACGCCCACA TCGAAGTGGATATTACTTACGCTGAGTACTTTGAGATGTCAGTGAGACTGGCTGAG GCCATGAAAAGATACGGACTGAACACAAACCACAGGATTGTGGTCTGTTCTGAGAA CAGCCTGCAGTTCTTCATGCCTGTGCTGGGCGCCTTGTTTATTGGAGTGGCAGTCG

[0497]

[0498] CCCCTGCAAATGACATTTACAATGAAAGGGAGCTGCTCAACAGCATGGGGATCTCT

[0499] 12673398.1 CAGCCTACAGTGGTCTTTGTCAGCAAAAAAGGCCTGCAGAAGATCCTGAATGTCCA GAAGAAGCTCCCAATCATCCAAAAGATTATTATCATGGATTCTAAGACAGACTACC AAGGATTCCAATCCATGTACACTTTCGTCACGTCCCACCTGCCCCCCGGCTTCAAT GAGTATGACTTTGTCCCTGAGTCCTTTGATCGGGACAAAACCATTGCCCTGATAAT GAATTCCAGCGGCAGCACAGGCCTCCCTAAAGGAGTAGCCCTTCCCCACAGAACTG CCTGCGTTAGATTCTCCCATGCGCGGGACCCTATTTTCGGAAACCAAATCATACCA GACACAGCTATCCTCTCGGTGGTTCCTTTCCACCACGGCTTCGGGATGTTTACGAC CCTGGGCTACCTCATTTGCGGCTTCCGTGTGGTACTCATGTATCGCTTCGAAGAGG AGCTGTTCCTGCGGTCCTTACAGGACTACAAAATCCAGTCAGCCCTGCTGGTGCCC ACCCTATTCTCTTTCTTTGCCAAGTCCACCCTGATCGACAAGTATGATCTGTCTAA CTTGCATGAAATCGCCTCAGGAGGAGCCCCCCTGAGCAAGGAAGTTGGCGAAGCAG TGGCAAAGAGGTTCCATCTGCCGGGGATCCGACAGGGCTATGGTCTGACTGAAACC ACCTCAGCCATCCTCATCACACCTGAAGGCGACGACAAGCCAGGTGCCGTGGGTAA GGTGGTACCTTTCTTTGAAGCTAAGGTGGTGGACCTGGACACAGGCAAAACGCTGG GCGTCAACCAGAGAGGCGAGCTGTGTGTGAGAGGCCCCATGATTATGAGCGGCTAC GTCAACAACCCTGAGGCCACCAACGCCCTGATTGATAAGGATGGCTGGCTGCACAG CGGAGACATCGCGTACTGGGATGAGGATGAACACTTCTTCATCGTAGACAGACTTA AGAGCCTGATCAAATACAAGGGGTACCAGGTCGCGCCGGCAGAGCTAGAGTCCATC CTTCTGCAGCATCCAAACATCTTTGACGCTGGCGTGGCCGGCCTGCCTGATGACGA TGCTGGAGAACTCCCCGCGGCCGTTGTGGTGCTGGAGCACGGCAAGACCATGACTG AAAAGGAGATCGTCGACTATGTGGCCAGCCAGGTAACCACCGCCAAGAAGCTGAGA GGCGGCGTAGTGTTCGTGGATGAGGTGCCCAAAGGTCTTACCGGAAAGCTGGACGC CAGGAAGAT CCGGGAGAT CCT GAT CAAAGCTAAGAAGGGGGGCAAGATAGCT GT G RNA AUGUACAUCGACGAGCGGAGCAACGCCGAGAUUGUGUGCGAGGCCAUCAAGACCAU 81 Sequence CGGAAUCGAAGGCGCCACAGCCGCUCAGCUGACCAGACAGCUGAACAUGGAAAAGC GGGAAGUGAACAAGGCCCUGUACGACCUGCAGAGAAGCGCCAUGGUGUACAGCAGC GACGACAUCCCUCCUCGGUGGUUUAUGACCACAGAGGCCGACAAGCCUGACGCCGA UGCUAUGGCCGACGUGAUCAUCGACGACGUGUCCCGCGAGAAGUCCAUGAGAGAGG ACCACAAGAGCUUCGACGAUGUGAUCCCCGCCAAGAAGAUCAUCGAUUGGAAGGGC G C C AAU CCUGUGACCGU GAU C AAC GAGU ACU G C C AGAU GAG C AGAAGAGACU G GU C CUUCCGGAUCGAGAGCGUGGGCCCUAGCAAUAGCCCUACCUUCUACGCCUGCGUGG ACAUCGACGGCAGAGUGUUCGAUAAGGCCGACGGCAAGAGCAAGCGGGACGCCAAA AACAAUGCCGCCAAGCUGGCCGUGGAUAAGCUGCUGGGCUAUGUGAUCAUCCGGUU CUGAUAAUAGUUAAAACAGCCUGUGGGUUGAUCCCACCCACAGGCCCAUUGGGCGC UAGCACUCUGGUAUCACGGUACCUUUGUGCGCCUGUUUUAUACCCCCUCCCCCAAC U GU AACUU AGAAGU AAC AC AC AC C GAU C AAC AGU C AG C GU G G C AC AC C AG C C AC GU UUUGAUCAAGCACUUCUGUUACCCCGGACUGAGUAUCAAUAGACUGCUCACGCGGU U GAAGGAGAAAGC GUU C GUUAU C C GGC CAACUACUU C GAAAAAC CUAGUAACAC C G UGGAAGUUGCAGAGUGUUUCGCUCAGCACUACCCCAGUGUAGAUCAGGUCGAUGAG UCACCGCAUUCCCCACGGGCGACCGUGGCGGUGGCUGCGUUGGCGGCCUGCCCAUG GGGAAACCCAUGGGACGCUCUAAUACAGACAUGGUGCGAAGAGUCUAUUGAGCUAG UUGGUAGUCCUCCGGCCCCUGAAUGCGGCUAAUCCUAACUGCGGAGCACACACCCU CAAGCCAGAGGGCAGUGUGUCGUAACGGGCAACUCUGCAGCGGAACCGACUACUUU GGGUGUCCGUGUUUCAUUUUAUUCCUAUACUGGCUGCUUAUGGUGACAAUUGAGAG AUCGUUACCAUAUAGCUAUUGGAUUGGCCAUCCGGUGACUAAUAGAGCUAUUAUAU AU C C CUUU GUU GGGUUUAUAC CACUUAGCUU GAAAGAGGUUAAAACAUUACAAUU C AUUGUUAAGUUGAAUACAGCAAAGCCACCAUGGAGGAUGCCAAAAACAUCAAAAAG GGCCCUGCCCCCUUCUACCCACUGGAGGACGGCACCGCUGGCGAGCAGCUGCACAA AGCAAUGAAGCGGUAUGCACUCGUGCCAGGAACCAUCGCUUUCACAGACGCCCACA U C GAAGU GGAUAUUACUUAC GCU GAGUACUUU GAGAU GU CAGU GAGACU GGCU GAG GC CAU GAAAAGAUAC GGACU GAACACAAAC CACAGGAUU GU GGU CU GUU CU GAGAA CAGCCUGCAGUUCUUCAUGCCUGUGCUGGGCGCCUUGUUUAUUGGAGUGGCAGUCG CCCCUGCAAAUGACAUUUACAAUGAAAGGGAGCUGCUCAACAGCAUGGGGAUCUCU CAGCCUACAGUGGUCUUUGUCAGCAAAAAAGGCCUGCAGAAGAUCCUGAAUGUCCA GAAGAAGCU C C CAAU CAU C CAAAAGAUUAUUAU CAU GGAUU CUAAGACAGACUAC C AAGGAUUCCAAUCCAUGUACACUUUCGUCACGUCCCACCUGCCCCCCGGCUUCAAU GAGU AU GACUUU GU C C CU GAGU C CUUU GAU C GGGACAAAAC CAUU GC C CU GAU AAU GAAUUCCAGCGGCAGCACAGGCCUCCCUAAAGGAGUAGCCCUUCCCCACAGAACUG CCUGCGUUAGAUUCUCCCAUGCGCGGGACCCUAUUUUCGGAAACCAAAUCAUACCA

[0500]

[0501] GACACAGCUAUCCUCUCGGUGGUUCCUUUCCACCACGGCUUCGGGAUGUUUACGAC 12673398.1 CCUGGGCUACCUCAUUUGCGGCUUCCGUGUGGUACUCAUGUAUCGCUUCGAAGAGG AGCUGUUCCUGCGGUCCUUACAGGACUACAAAAUCCAGUCAGCCCUGCUGGUGCCC AC C CUAUU CU CUUU CUUU GC CAAGU C GAG C CU GAU C GACAAGUAU GAU CU GU CUAA CUUGCAUGAAAUCGCCUCAGGAGGAGCCCCCCUGAGCAAGGAAGUUGGCGAAGCAG UGGCAAAGAGGUUCCAUCUGCCGGGGAUCCGACAGGGCUAUGGUCUGACUGAAACC ACCUCAGCCAUCCUCAUCACACCUGAAGGCGACGACAAGCCAGGUGCCGUGGGUAA GGUGGUACCUUUCUUUGAAGCUAAGGUGGUGGACCUGGACACAGGCAAAACGCUGG GCGUCAACCAGAGAGGCGAGCUGUGUGUGAGAGGCCCCAUGAUUAUGAGCGGCUAC GUCAACAACCCUGAGGCCACCAACGCCCUGAUUGAUAAGGAUGGCUGGCUGCACAG CGGAGACAUCGCGUACUGGGAUGAGGAUGAACACUUCUUCAUCGUAGACAGACUUA AGAGCCUGAUCAAAUACAAGGGGUACCAGGUCGCGCCGGCAGAGCUAGAGUCCAUC CUUCUGCAGCAUCCAAACAUCUUUGACGCUGGCGUGGCCGGCCUGCCUGAUGACGA UGCUGGAGAACUCCCCGCGGCCGUUGUGGUGCUGGAGCACGGCAAGACCAUGACUG AAAAGGAGAUCGUCGACUAUGUGGCCAGCCAGGUAACCACCGCCAAGAAGCUGAGA GGCGGCGUAGUGUUCGUGGAUGAGGUGCCCAAAGGUCUUACCGGAAAGCUGGACGC CAGGAAGAUCCGGGAGAUCCUGAUCAAAGCUAAGAAGGGGGGCAAGAUAGCUGUG

[0502] oeE3L-IRES-Fluc

[0503] DNA ATAGAAGATGGCGGCGTAGTACACACTATTGAATCAAACAGCCGACCAATTGCACT 41 Sequence AC CAT CACAAC GGAGAAGC CAGT AGT AAAC GT AGAC GT AGAC C C C CAGAGT C C GT T TGTCGTGCAACTGCAAAAAAGCTTCCCGCAATTTGAGGTAGTAGCACAGGAGGTGA CTCCAAATCACCATCCTAATCCCAGAGCATTTTCGCATCTGGGCAGTAAACTAATC GAGCTGGAGGTTCCTACCACAGCGACGATCTTGGACATAGGCAGCGCACCGGCTCG TAGAACGATAAACCCCTAGTGCCACCATGTACATCGACGAGCGGAGCAACGCCGAG ATTGTGTGCGAGGCCATCAAGACCATCGGAATCGAAGGCGCCACAGCCGCTCAGCT GACCAGACAGCTGAACATGGAAAAGCGGGAAGTGAACAAGGCCCTGTACGACCTGC AGAGAAGCGCCATGGTGTACAGCAGCGACGACATCCCTCCTCGGTGGTTTATGACC ACAGAGGCCGACAAGCCTGACGCCGATGCTATGGCCGACGTGATCATCGACGACGT GT C C C G C GAGAAGT C CAT GAGAGAG GAC C AC AAGAG C T T C GAC GAT GT GAT C C C C G CCA...

Claims

CLAIMSWhat is claimed is:

1. A trans-amplifying ribonucleic acid (RNA) (taRNA) comprising:a first RNA polynucleotide comprising a nucleic acid encoding an Eastern Equine Encephalitis Virus (EEEV) replicase; anda second RNA polynucleotide comprising a nucleic acid payload or a nucleic acid encoding a payload;wherein the first RNA polynucleotide and / or the second RNA polynucleotide further comprise a nucleic acid encoding an immune modulating protein (IMP).

2. The taRNA of claim 1 wherein, the first RNA polynucleotide comprises the nucleic acid encoding the IMP.

3. The taRNA of claim 1 wherein, the second RNA polynucleotide comprises the nucleic acid encoding the IMP.

4. The taRNA of any one of claims 1 to 3, wherein the IMP is a viral immune evasion protein (VIEP).

5. The taRNA of claim 4, wherein the VIEP is from vaccinia virus (VACV), herpes simplex virus (HSV), influenza virus, Toscana Virus (TOSV), or encephalomyocarditis virus (EMCV).

6. The taRNA of claim 5, wherein the VIEP is a VACV soluble IFN alpha / beta receptor B18 (B18R), VACV RNA-binding protein E3 (E3L), VACV F1L, VACV N1L, HSV infected cell protein 34.5 (ISC34.5), HSV unique short 1 (US1), HSV unique short 11 (US11), influenza virus non-structural protein 1 (NS1), TOSV non- structural (NSs) protein, or EMCVLpro.

7. The taRNA of claim 6, wherein the NS1 is from Influenza A / Puerto Rico / 8 / 34 influenza virus (PR8) (PR8 NS1).12673398.

18. The taRNA of claim 6, wherein the VIEP is PR8 NS 1.

9. The taRNA of claim 6, wherein the VIEP is TOSV NSs.

10. The taRNA of claim 6, wherein the VIEP is EMCV Lpro.

11. The taRNA of any one of claims 1 to 3, wherein the IMP antagonizes a mammalian antiviral factor.

12. The taRNA of claim 11, wherein the IMP that antagonizes a mammalian antiviral factor is dominant negative mitochondrial antiviral-signaling protein (dnMAVS), microprotein in antiviral immunity 1 (MAVI1), dominant negative protein kinase R (dnPKR), suppressor of cytokine signaling (SOCS), or dominant negative Zinc-finger Antiviral Protein (dnZAP).

13. The taRNA of claim 12, wherein the dnZAP is rat ZAP with cysteine-to-arginine mutation at position 88 (rZAPC88R).

14. The taRNA of claim 12, wherein the SOCS is SOCS1 or SOCS3.

15. The taRNA of any one of claims 1 to 14, wherein the first RNA polynucleotide comprises, from 5' to 3':(i) the nucleic acid encoding the EEEV replicase; and(ii) the nucleic acid encoding the IMP.

16. The taRNA of any one of claims 1 to 15, wherein the nucleic acid encoding the EEEV replicase and the nucleic acid encoding the IMP are joined by a linker.

17. The taRNA of claim 16, wherein the linker is a nucleic acid encoding a 2A peptide, or an internal ribosome entry site (IRES) element.

18. The taRNA of claim 17, wherein the 2A peptide is thosea asigna virus 2A (T2A), porcine teschovirus-1 2A (P2A), equine rhinitis A virus 2A (E2A), or Furin-2A.12673398.

119. The taRNA of claim 17, wherein the IRES element is an IRES from Coxsackievirus B3 (CVB3), encephalomyocarditis virus (EMCV), porcine kobuvirus (PKV), Manhattan Parechovirus (MPV), or tortoise rafivirus (TraV).

20. The taRNA of any one of claims 16 to 19, wherein the first RNA polynucleotide comprises, from 5' to 3':(i) the nucleic acid encoding the EEEV replicase;(ii) the linker; and(iii) the nucleic acid encoding the IMP.

21. The taRNA of any one of claims 16 to 19, wherein the first RNA polynucleotide comprises, from 5' to 3':(i) the nucleic acid encoding the IMP;(ii) the linker; and(iii) the nucleic acid encoding the EEEV replicase.

22. The taRNA of any one of claims 1 to 21, comprising a nucleic acid encoding a first IMP and a nucleic acid encoding a second IMP.

23. The taRNA of claim 22, wherein the first IMP and the second IMP are different IMPs.

24. The taRNA of claims 22 to 23, wherein the first RNA polynucleotide comprises, from 5' to 3':(i) the nucleic acid encoding the first IMP;(ii) a first linker;(iii) the nucleic acid encoding an EEEV replicase;(iv) a second linker; and(vi) the nucleic acid encoding the second IMP.

25. The taRNA of any one of claims 1 to 24, wherein the first RNA polynucleotide comprises, from 5' to 3':(i) the nucleic acid encoding the IMP, wherein the IMP comprises PR8 NS1 or TOSV NS;12673398.1- IOS-(ii) a nucleic acid encoding a 2A peptide; and(iii) the nucleic acid encoding the EEEV replicase.

26. The taRNA of any one of claims 1 to 24, wherein the first RNA polynucleotide comprises, from 5' to 3':(i) the nucleic acid the IMP, wherein the IMP comprises PR8 NS1 or TOSV NS; (ii) a nucleic acid encoding a 2A peptide;(iii) the nucleic acid encoding the EEEV replicase;(iv) a nucleic acid encoding a 2A peptide; and(v) a nucleic acid encoding an additional IMP, wherein the IMP comprises EMCV Lpro.

27. The taRNA of any one of claims 1 to 26, wherein the second RNA polynucleotide further comprises a 5' untranslated region (UTR).

28. The taRNA of claim 27, wherein the 5' UTR comprises a conserved sequence element (CSE) which is cognate to the EEEV replicase.

29. The taRNA of any one of claims 1 to 28, wherein the payload comprises an antigen.

30. The taRNA of any one of claims 1 to 29, wherein the EEEV replicase is a wildtype EEEV replicase.

31. The taRNA of any one of claims 1 to 29, wherein the EEEV replicase is an Opal-R EEEV replicase.

32. The taRNA of any one of claims 1 to 31, wherein the EEEV replicase comprises an amino acid sequence having at least 70% identity to the sequence set forth in SEQ ID NO: 83.

33. The taRNA of any one of claims 1 to 29, wherein the EEEV replicase comprises the amino acid sequence set forth in SEQ ID NO: 83.12673398.

134. The taRNA of any one of claims 1 to 29, wherein the EEEV replicase comprises the amino acid sequence set forth in SEQ ID NO: 117.

35. The taRNA of any one of claims 1 to 34, wherein the second RNA polynucleotide comprises a nucleotide sequence of any one of SEQ ID NO: 80-82.

36. A composition comprising a deoxyribonucleic acid (DNA) polynucleotide that can be transcribed to produce the first RNA polynucleotide of the taRNA of any one of claims 1 to 35.

37. The composition of claim 36, further comprising an additional DNA polynucleotide that can be transcribed to produce the second RNA polynucleotide of the taRNA of any one of claims 1 to 26.

38. A cell comprising the taRNA of any one of claims 1 to 37.

39. A method of expressing a payload in a cell, the method comprising: transfecting the cell with the taRNA of any one of claims 1 to 35 or contacting the cell with the composition of claim 36 or 37.

40. A method of expressing a payload in a subject, the method comprising: administering to a subject the taRNA of any one of claims 1 to 35 or the composition of claim 36 or 37.

41. The method of claim 40, wherein the subject is a mammal.

42. The method of claim 41, wherein the subject is a human.

43. A fusion protein comprising, from N-terminal to C-terminal: an Eastern Equine Encephalitis Virus (EEEV) replicase), a linker, and an immune modulating protein (IMP).

44. The fusion protein of claim 43, wherein the IMP is encephalomyocarditis virus (EMCV) leader protease (Lpro).

45. The fusion protein of claim 43, wherein the linker is a 2A peptide.12673398.

146. A fusion protein comprising, from N-terminal to C-terminal: a first immune modulating protein (IMP), a first linker, an Eastern Equine Encephalitis Virus (EEEV) replicase, a second linker, and a second IMP.

47. The fusion protein of claim 46, wherein the first IMP is Influenza Virus A / Puerto Rico / 8 / 34 nonstructural protein 1 (PR8 NS1) or Toscana Virus (TOSV) nonstructural protein (NSs).

48. The fusion protein of claim 46 or 47, wherein the second IMP is encephalomyocarditis virus (EMCV) leader protease (Lpro).

49. The fusion protein of any one of claims 43 to 48, wherein the EEEV replicase is a wildtype EEEV replicase.

50. The fusion protein of any one of claims 43 to 48, wherein the EEEV replicase is an Opal-R EEEV replicase.

51. A deoxyribonucleic acid (DNA) comprising nucleic acids encoding the fusion protein of any one of claims 43 to 50.

52. A ribonucleic acid (RNA) comprising nucleic acids encoding the fusion protein of any one of claims 43 to 50.

53. A method of amplifying a ribonucleic acid (RNA) polynucleotide comprising or encoding a payload in a subject in trans without inducing substantial cytotoxicity in the liver of the subject, the method comprising administering to the subject a / ra / .s-amplifying ribonucleic acid (RNA) (taRNA), the taRNA comprising:a first RNA polynucleotide comprising: a nucleic acid encoding an Eastern Equine Encephalitis Virus (EEEV) replicase, and a nucleic acid encoding an immune modulating protein (IMP); anda second RNA polynucleotide comprising: a nucleic acid encoding a payload, and a conserved sequence element (CSE) cognate to the EEEV replicase.12673398.1- 111-54. The method of claim 53, wherein the administering comprises intramuscular injection.

55. The method of claim 53, wherein the administering comprises intravenous injection.

56. The method of any one of claims 53 to 55, wherein the payload is an antigen.

57. The method of any one of claims 53 to 56, wherein the payload is a nucleic acid payload.

58. The method of any one of claims 53 to 57, wherein the nucleic acid encoding an IMP encodes a first IMP and a second IMP.

59. The method of any one of claims 53 to 58, wherein the nucleic acid encoding an IMP encodes encephalomyocarditis virus (EMCV) leader protease (Lpro).

60. The method of any one of claims 53 to 59, wherein the first RNA polynucleotide is the first RNA polynucleotide of any one of claims 1 to 34.

61. The method of claim 53, wherein administering the taRNA comprises administering the taRNA of any one of claims 1 to 35.12673398.1

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