Ribonucleic acid interactor group capture regimen and antiviral compositions found using regimen

Abstract

The present invention relates to a ribonucleic acid (RNA) interactome capture regimen and an antiviral composition found using the regimen. By using the antiviral composition and the pharmaceutical composition according to the present invention, viral infection can be effectively suppressed, and proliferation of viruses in vivo after infection can be suppressed.

Description

Technical Field

[0001] This application claims priority to Korean Patent Application No. 10-2020-0144787, filed on November 2, 2020, which is incorporated herein by reference in its entirety.

[0002] The present invention relates to a scheme for capturing a ribonucleic acid (RNA) interactor group and an antiviral composition discovered by using the scheme. Background Art

[0003] Coronavirus (CoV) is a group of membrane viruses with a non-segmented, single-stranded, positive-sense (+) RNA genome, belonging to the order Nidovirales, family Coronaviridae, subfamily Coronavirinae (Lai and Cavanagh, 1997). They are divided into four genera, including alphacoronavirus and betacoronavirus, which mainly infect mammals, and gammacoronavirus and gammacoronavirus, which mainly infect birds (Woo et al., 2012). Human coronaviruses such as alphacoronavirus HCoV-229E and betacoronavirus HCoV-OC43 have been known as etiologic agents of colds since 1960 (Hamre and Procknow, 1966). However, since the beginning of the 21st century, five new types of coronaviruses have emerged, including the highly pathogenic severe acute respiratory syndrome coronavirus (2002) (SARS-CoV) (Peiris et al., 2003), the Middle East respiratory syndrome coronavirus (2012) (MERs-CoV) (deGroot et al., 2013) and the 2019 novel coronavirus (SARS-CoV-2) (2019) (Zhou et al., 2020).

[0004] In the nucleus of coronaviruses, the RNA genome is encapsulated in the form of nucleocapsid (N) protein, surrounded by a viral membrane including spike (S), membrane (M) and outer membrane (E) proteins (Lai and Cavanagh, 1997). The coronavirus RNA genome is the longest among RNA viruses at ~30 kb and contains a 5′-cap and a 3′ poly (A) tail (Bouvet et al., 2010; Lai and Stohlman, 1981). Upon entry into the cell, the genomic RNA (gRNA) functions as a messenger RNA (mRNA) for the production of nonstructural proteins (nsps) required for viral RNA production (Perlman and Netland, 2009). ORF1a encodes polypeptide 1a (pp1a, 440-500 kDa) that is mediated by 11nsps (into 11nsps). The movement of the -1 ribosomal frame occurs immediately upstream of the stop codon of ORF1a, generating a large polypeptide (pp1ab, 740-810 kDa) cut at 15 nsps by allowing translation of downstream ORF1b. At the same time, 16 other nsp fragments are generated to form double-membrane vesicles (DMV), thereby regulating the subsequent steps of viral RNA synthesis.

[0005] After the above initial steps of viral translation, the genomic RNA is used as a template for the synthesis of negative-strand (-) RNA intermediates, and the product is reused as a template for the synthesis of positive-strand (+) RNA (Snijder et al., 2016; Sola et al., 2015). Ten different canonical (+) RNA species are generated in the SARS-CoV-2 genome, including 1 full-length genomic RNA and 9 subgenomic RNAs (sgRNA) (Kim et al., 2020a). All canonical viral (+) RNAs share a common 5′ terminal sequence and a 3′ terminal sequence called a guide sequence.

[0006] Subgenomic RNA is formed by inducing non-continuous transcription of a fusion between the “body” portion of the downstream open reading frame (Sola et al., 2015) encoding structural proteins (S, E, M and N) and accessory proteins (3a, 3c, 6, 7a, 7b, 8 and 9b) and the 5' guide sequence (Kim et al., 2020a).

[0007] To this end, coronaviruses use inherent strategies for evading, modulating, and exploiting host machinery (Fung and Liu, 2019). For example, genomic RNA molecules should maintain a complex balance between translation, transcription, and encapsulation by recruiting the correct host RNA binding proteins (RBPs) and forming specific ribonucleoprotein (RNP) complexes. In order to recognize and remove viral RNA, host cells activate RNA binding proteins such as RIG-I, MDA5, and TLR (Toll-like receptors) to respond to them. Subsequently, the virus should use its structural elements to evade the immune system in order to win the competition between virus and host. However, it has not yet been determined how this stealthy device is genetically encoded in the small RNA genome (Snijder et al., 2016). Therefore, the recognition of RNA binding proteins bound to viral transcripts (or SARS-CoV-2 RNA interactors) is particularly important in identifying the molecular rearrangement of viral gene regulation and the activation of antiviral defense systems.

[0008] With the development of biochemical techniques for studying RNA-protein interactions, protein-centric methods such as CLIP-seq (crosslinking immunoprecipitation sequencing) (Ule et al., 2018) have been developed (Ramanathan et al., 2019). In CLIP-seq experiments, to identify direct RNA-protein interactions, ribonucleoprotein complexes are crosslinked by irradiating ultraviolet (UV) into cells. To identify the associated RNAs, the protein of interest is immunoprecipitated (Lee and Ule, 2018; Van Nostrand et al., 2020). Recently, RNA-centric methods have been developed to analyze messenger RNA (mRNA) interactomes and ribonucleoprotein complexes (Roth and Diederichs, 2015). After UV irradiation, the RNA of interest is purified by oligonucleotide probes, and crosslinked proteins are identified by mass analysis. For example, RAP-MS showed very strong evidence in the very conclusive analysis of proteins bound to specific RNAs due to its combination of long probes and harsh denaturation conditions (Engreitz et al., 2013; McHugh et al., 2015). Summary of the invention

[0009] Technical issues

[0010] The inventors have made several efforts to develop a method for easily analyzing RNA viruses, more specifically, to easily develop the binding process of the genomic RNA of coronaviruses with the virus itself and / or host proteins. As a result, they have completed the development of a powerful ribonucleoprotein capture scheme for defining a library of viral and host proteins associated with coronavirus transcripts, and used it to identify new targets for inhibiting coronaviruses.

[0011] Therefore, an object of the present invention is to provide an antiviral composition.

[0012] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating coronavirus infection.

[0013] Another object of the present invention is to provide a method for screening coronavirus inhibitors.

[0014] Another object of the present invention is to provide a method for establishing a RNA interactome.

[0015] Technical Solution

[0016] According to one embodiment of the present invention, the present invention provides an antiviral composition comprising one or more proteins selected from the group consisting of LARP1, FUBP3, FUBP1, FAM120A, FAM120C, EIF4H, RPS3, RPS9, SND1, CELF1, RALY, CNBP, TRIM25, ZC3HAV1, PARP12, PPP1CA, HDLBP, CNBP, TIAL, UPF1 and SHFL as an active ingredient.

[0017] The inventors have made several efforts to develop a method for easily analyzing RNA viruses, more specifically, to easily develop the binding of the genomic RNA of coronaviruses to the virus itself and / or host proteins. As a result, they have completed the development of a powerful ribonucleoprotein capture scheme for defining a library of viral and host proteins associated with coronavirus transcripts, and used it to identify new targets for inhibiting coronaviruses.

[0018] A specific example of the ribonucleoprotein capture scheme proposed by the present invention for deriving components with antiviral activity and components with proviral activity includes the following steps: step (a), irradiating a sample containing target ribonucleic acid and protein with ultraviolet light to induce ribonucleic acid-protein cross-linking; step (b), denaturing by treating with deoxyribonuclease (DNase); step (c), using a biotinylated antisense oligonucleotide probe pool to capture the denatured ribonucleoprotein complex in a sequence-specific manner (in this step (b), the biotinylated antisense oligonucleotide contained in the above-mentioned probe pool is a target ribonucleoprotein complex). The method comprises the following steps: enumerating oligonucleotides of the same length selected from the starting position separated by a specified interval in a partially overlapping manner and within the range of 45 nt to 135 nt based on the nucleic acid sequence, more preferably, enumerating oligonucleotides of the same length selected from the range of 60 nt to 120 nt, the range of 75 nt to 105 nt, the range of 80 nt to 100 nt, or enumerating oligonucleotides each having a length of 90 nt); step (d), digestion by on-bead trypsin treatment; and step (e), obtaining interactor group information by decomposing the digestion products.

[0019] The inventors can obtain detailed information on the interactor group as a protein group that forms crosslinks with the genomic RNA of the coronavirus through a ribonucleoprotein capture scheme including the above steps. The term "interactor group" of the present invention refers to the totality of proteins that bind to the target RNA sequence, and more specifically, refers to the totality of proteins that bind to the genomic RNA sequence of the coronavirus. More specifically, for example, in the case of the "SARS-CoV-2 RNA interactor group", it refers to the 109 protein groups discovered by the ribonucleoprotein capture scheme.

[0020] In the case of the antiviral composition of the present invention, one or more proteins selected from the group consisting of LARP1, FUBP3, FUBP1, FAM120A, FAM120C, EIF4H, RPS3, RPS9, SND1, CELF1, RALY, CNBP, TRIM25, ZC3HAV1, PARP12, PPP1CA, HDLBP, CNBP, TIAL, UPF1 and sHFL are included as active ingredients. The inventors found through knock-down experiments that the depletion of the above proteins caused substantial upregulation of viral RNA, indicating that one or more proteins selected from the group consisting of the above proteins have antiviral function.

[0021] In the case of "LARP1" of the present invention, 5'TOP messenger RNA encoding ribosomal proteins and translation factors containing a 5' terminal oligopyrimidine (5'TOP) motif in the 5'UTR is stabilized, and the translation of 5'TOP messenger RNA is inhibited in response to metabolic stress in an mTORC1-dependent manner.

[0022] In the case of "FUBP3" of the present invention, its relevance to the life cycle of coronavirus is still unknown, but it is known that it is a nucleoprotein having four KH domains and binds to the 3'UTR of cellular messenger RNA that regulates the localization of messenger RNA.

[0023] In the case of "FUBP1" of the present invention, it contains a far upstream element (FUSE, Farupstream element) located upstream of c-myc, which is a single-stranded deoxyribonucleic acid (DNA) binding protein that binds to a variety of deoxyribonucleic acid (DNA) elements, but its correlation with the life cycle of the coronavirus is still unknown.

[0024] In the case of "FAM120A" of the present invention, it is expected to be involved in the transport of messenger RNA in the cytoplasm and is known to function as a scaffold protein capable of activating src series kinases and phosphorylating src series kinases and activating PI3-kinase.

[0025] "FAM120C" of the present invention is a potential transmembrane protein, mutations and deletions of which are known to be encoded by a gene located in a region associated with intellectual disability and autism.

[0026] In the case of "EIF4H" of the present invention, it is known that it functions as a cofactor of the RNA helicase EIF4A together with EIF4B, and that RNA particles are formed due to its depletion.

[0027] In the case of "RPS3" of the present invention, it is a structural element of the eukaryotic 40S ribosomal subunit and is known to be a multifunctional protein that regulates innate immune responses related to DNA recovery, apoptosis, and bacterial infection, but its relevance to virus inhibition is still unknown.

[0028] In the case of "RPS9" of the present invention, it is a structural element of the eukaryotic 40S subunit and has been reported to be expressed in a variety of ways in colon cancer, but the correlation between its expression level and the severity of the disease has not yet been determined, nor is its correlation with viral suppression.

[0029] The "SNDi" of the present invention is known to function as an oncogene in the course of various cancers, and is particularly known to promote tumor angiogenesis in hepatocellular carcinoma via a novel pathway involving NF-kappaB and miR-221.

[0030] In the case of "CELF1" of the present invention, it is known that ETS2 is targeted in colon cancer to enhance cell migration, invasion and chemoresistance, and although it corresponds to a RNA binding protein, its correlation with viral suppression is not known.

[0031] The "RALY" of the present invention is known as a ribonucleic acid binding protein. Anti-EBNA-1 antibodies that cross-react with various normal human proteins are produced in infectious mononucleosis, and RALY is one of such antigens and is known to be a member of the heterologous ribosomal protein gene family.

[0032] The "CNBP" of the present invention is found in multiple tissues of the body, especially a protein abundant in the heart and muscles used in exercise (skeletal muscle). It regulates the activities of other genes and is a protein that plays a role necessary for normal development before birth, especially normal development of muscles.

[0033] In the case of "TRIM25" of the present invention, it is known to be a protein that binds to the CARD domain of RIG-I.

[0034] The "ZC3HAV1 (ZAP / PARP13)" of the present invention is a protein reported to recognize CpG and recruit decay factors to decompose human acquired immunodeficiency virus ribonucleic acid (HIV RNA).

[0035] The "PARP12" of the present invention is a substance constituting the ADP-ribosyltransferase family, but is a protein whose function has not yet been clearly identified.

[0036] The "PPP1CA" of the present invention is associated with more than 200 regulatory proteins, and is a protein that dephosphorylates hundreds of biological targets with high specificity to form a holoenzyme.

[0037] The "HDLBP" of the present invention is a conserved protein comprising 14 KH domains, and is a protein associated with viral translation of dengue virus.

[0038] The "CNBP" of the present invention is a CCHC-type zinc finger nucleic acid binding protein, which is known to form related bindings with deoxyribonucleic acid and RNA, is known to play a role in the cap-dependent translation of ornithine decarboxylase messenger RNA, and is also known to play a role in sterol-mediated transcriptional regulation.

[0039] The "TIAL" of the present invention is a type of RNA-binding protein having three RNA recognition motifs (RRMs), which are known to correspond to proteins that bind to adenine- and uridine-rich elements of messenger RNA and precursor messenger RNA (pre-mRNA).

[0040] In the case of "UPF1" of the present invention, it is a protein that is a part of a multiprotein complex of the exon junction complex involved in the nuclear release of messenger RNA after splicing.

[0041] The "SHFL" of the present invention is known to be a protein associated with dengue virus, autoimmunity, peripheral neuropathy, and the like.

[0042] In the case of proteins, the relevance to the known and life cycle of coronaviruses is unknown.

[0043] According to another embodiment of the present invention, the present invention provides an antiviral composition, comprising as an active ingredient a nucleic acid encoding one or more proteins selected from the group consisting of LARP1, FUBP3, FUBP1, FAM120A, FAM120C, EIF4H, RPS3, RPS9, SND1, CELF1, RALY, CNBP, TRIM25, ZC3HAV1, PARP12, PPP1CA, HDLBP, CNBP, TIAL, UPF1 and SHFL.

[0044] It should be self-evident to those skilled in the art that the nucleotide sequence of the above-mentioned protein-encoding nucleic acid molecule of the present invention satisfies the nucleotide sequence encoding the amino acid sequence constituting the above-mentioned fusion protein, rather than being limited to a specific nucleotide sequence. This is because, even in the case of a nucleotide sequence mutation, there will be a situation where the mutated nucleotide sequence does not change in the protein sequence when expressed as a protein. It is called codon degeneration. Therefore, the above-mentioned nucleotide sequence includes a nucleotide sequence containing codons that are functionally equivalent or codons encoding the same amino acid (for example, through codon degeneration, there are six codons for arginine or serine) or codons encoding biologically equivalent amino acids.

[0045] The term "nucleic acids" in this specification has the meaning of including deoxyribonucleic acids (genomic deoxyribonucleic acid (gDNA) and complementary deoxyribonucleic acid (cDNA)) and ribonucleic acid molecules. The nucleotides, which are the basic structural units of nucleic acid molecules, include not only natural nucleotides but also analogues with modified sugar or base sites (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90: 543-584 (1990)).

[0046] Taking into account the above-mentioned variations with biologically equivalent activity, the nucleic acid molecules of the present invention encoding the amino acid sequences constituting the above-mentioned fusion proteins should also be interpreted as also including sequences that exhibit substantial identity therewith. The above-mentioned substantial identity means that when the above-mentioned sequence of the present invention is aligned with any other sequence in a maximum corresponding manner and the aligned sequences are analyzed using an algorithm commonly used in the technical field to which the present invention belongs, there is at least 60% homology, more specifically, more than 70% homology, more specifically, more than 80% homology, and further specifically, more than 90% homology, and most specifically, more than 95% homology. The alignment method used for sequence comparison is well known in the technical field to which the present invention belongs. Various methods and algorithms for alignment are described in Smith and Waterman, Adv. Appl. Math. 2: 482 (1981); Needleman and Wunsch, Mol. Bio. 48: 443 (1970); Pearson and Lipman, Methods in Mol. Biol. 24: 307-31 (1988); Higgins and Sharp, Gene 73: 237-44 (1988); Higgins and Sharp, CABIOS 5: 151-3 (1989); Corpet et al., Nuc. Acids Res. 16: 10881-90 (1988); Huang et al., Comp. Appl. BioSci. 8: 155-65 (1992) and Pearson et al., Comp. Appl. BioSci. 9: 156-113 (1993). al., Meth. Mol. Biol. 24: 307-31 (1994), but the present invention is not limited thereto.

[0047] In one embodiment of the present invention, the virus of the present invention is a coronavirus.

[0048] The term "coronavirus" of the present invention is a general term for RNA viruses belonging to the subfamily Coronavirus of the family Coronaviridae. Infections are induced in the respiratory and digestive systems of humans and animals, and are mainly easily transmitted through mucosal infection, droplet infection, etc. Recently, it has become a virus that causes fatal infections in humans. It is known that coronavirus is a pathogen that induces a considerable part of the common cold in humans, and can cause direct viral pneumonia, secondary bacterial pneumonia, and can also cause viral bronchitis and secondary bacterial bronchitis.

[0049] In one embodiment of the present invention, the coronavirus of the present invention can be selected from the group consisting of 229E, OC43, NL63, HKU1, MERS-CoV, SARS-CoV and SARS-CoV-2, but is not limited thereto. The above coronaviruses are equivalent to variant coronaviruses that can cause infection in humans, and are known to share a considerable portion of genomic RNA with genetic information. When the antiviral composition of the present invention is used, the proliferation and activation of the virus can be inhibited by inhibiting the replication of the genomic RNA and protein synthesis of the above coronavirus.

[0050] According to another embodiment of the present invention, the present invention provides an antiviral composition comprising a recombinant vector, wherein the recombinant vector comprises a nucleic acid molecule encoding one or more proteins selected from the group consisting of LARP1, FUBP3, FUBP1, FAM120A, FAM120C, EIF4H, RPS3, RPS9, SND1, CELF1, RALY, CNBP, TRIM25, ZC3HAV1, PARP12, PPP1CA, HDLBP, CNBP, TIAL, UPF1 and SHFL.

[0051] The term "vector" in this specification is a tool for expressing a target gene in a host cell, and includes plasmid vectors, cosmid vectors, and viral vectors such as phage vectors, adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors.

[0052] According to a specific example of the present invention, in the vector of the present invention, the nucleic acid molecule encoding one or more proteins selected from the group consisting of LARP1, FUBP3, FUBP1, FAM120A, FAM120C, EIF4H, RPS3, RPS9, SND1, CELF1, RALY, CNBP, TRIM25, ZC3HAV1, PARP12, PPP1CA, HDLBP, CNBP, TIAL, UPF1 and SHFL is operatively linked to the promoter of the above-mentioned vector.

[0053] In this specification, the term "operable binding" refers to the functional binding between a nucleic acid expression regulatory sequence (such as a promoter, a signal sequence sequence or an array of transcription regulatory factor binding epitopes) and other nucleic acid sequences, by which the above-mentioned regulatory sequence regulates the transcription and / or interpretation of the above-mentioned other nucleic acid sequences.

[0054] The recombinant vector system of the present invention can be constructed by various methods known in the technical field to which the present invention belongs. Specific methods therefor are disclosed in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001), which is incorporated herein by reference.

[0055] The vector of the present invention can be constructed as a vector for typical gene cloning or a vector for protein expression. In addition, the vector of the present invention can be constructed using prokaryotic cells or eukaryotic cells as hosts.

[0056] For example, when the vector of the present invention is an expression vector and a eukaryotic cell is used as a host, a promoter derived from the genome of a mammalian cell (e.g., a metallothionein promoter, a β-actin promoter, a human hemoglobin promoter, and a human muscle creatine promoter) or a promoter derived from a mammalian virus (e.g., an adenovirus late promoter, a vaccinia virus 7.5K promoter, an SV40 promoter, a cytomegalovirus promoter, a tk promoter of herpes simplex virus (HSV), a mouse mammary tumor virus (MMTV) promoter, an LTR promoter of a human acquired immunodeficiency virus, a promoter of a moloney virus, a promoter of an Epstein-Barr virus (EBV), and a promoter of a respiratory syncytial virus (RSV)) can be used. They usually have a polyadenylation sequence as a transcription termination sequence.

[0057] The vector of the present invention can be fused with other sequences for the purpose of facilitating the purification of the polypeptide or protein expressed therefrom. The fused sequences include, for example, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA) and hexahistidine (6xHis; Quiagen, USA).

[0058] On the other hand, the expression vector of the present invention comprises an antibiotic resistance gene commonly used in the technical field to which the present invention belongs as a selection marker, for example, genes resistant to ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and tetracycline.

[0059] According to another embodiment of the present invention, the present invention provides an antiviral composition comprising an expression inhibitor or activity inhibitor of one or more proteins selected from the group consisting of EIF3A, EIF3D, CSDE1, HNRNPM, YBX3, FMR1, RTCB and NUFIP2.

[0060] The "EIF3A" of the present invention is a RNA-binding component of the mammalian EIF3 complex and is a protein that has been evolutionarily conserved together with EIF3B and EIF3C.

[0061] The "EIF3D" of the present invention is known to interact with the cap of messenger RNA and acts as a specialized protein required for the initiation of translation.

[0062] The "CSDE1 (Unr)" of the present invention is known to be a protein required for IRES-dependent translation in human rhinovirus (Picornaviridae) and poliovirus (Picornaviridae).

[0063] The "HNRNPM" of the present invention is a protein associated with the spliceosome machinery by interacting with the downstream complex of the CDC5L / PLRG1 spliceosome. It is known that the HNRNPM complex with nuclear IMP-3 plays an important role in the efficient synthesis of CCND1, D3 and G1 and the proliferation of human cancer cells.

[0064] The "YBX3" of the present invention is known to inhibit influenza A virus by interacting with the viral ribonucleoprotein complex and impairing its function. However, its relevance to the inhibition of coronavirus has not been found.

[0065] The "FMR1" of the present invention is a protein that binds to RNA and associates with a polymer, and is known to correspond to a protein that associates with messenger RNA that can move from the nucleus to the cytoplasm.

[0066] The "RTCB" of the present invention is a ribonucleoside ligase component of the E. coli ribonucleoside recovery operon, and is known to be a protein that can catalyze the splicing of transfer RNA (tRNA) and HAC1 messenger RNA in vivo.

[0067] The "NUFIP2" of the present invention is a ribonucleic acid binding protein that exhibits cell cycle-dependent intracellular localization.

[0068] In one embodiment of the present invention, the protein expression inhibitor of the present invention is selected from the group consisting of microRNA (miRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), antisense oligonucleotide, CRISPRi and their combinations that are complementary to the nucleic acid sequence encoding the above protein.

[0069] The terms "microRNA (miRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA)" of the present invention refer to nucleic acid molecules that bind to messenger RNA transcribed mainly from a target gene to inhibit the translation of the above messenger RNA in order to mediate RNA blocking or gene silencing. Since the above-mentioned small interfering RNA or short hairpin RNA can always target the expression of a gene, it can be effectively used in a gene knockdown method or a gene therapy method. The purpose of the present invention is to use it in the expression inhibition of one or more proteins selected from the group consisting of EIF3A, EIF3D, CSDE1, HNRNPM, YBX3, FMR1, RTCB and NUFIP2.

[0070] In one specific example of the present invention, the specific examples of the small interfering RNA of the present invention for EIF3A, EIF3D, CSDE1, HNRNPM, YBX3, FMR1, RTCB and NUFIP2 are as follows, and one or more small interfering RNAs selected from the following small interfering RNA sequences can be used to inhibit protein expression.

[0071] siEIF3A: 5′-GAGAAGACUUGGCGAUAGU-3′ (sequence 1) / 5′-AGACAGAGAUUUUAUUCGUA-3′ (sequence 2) / 5′-GAAGAAAGGCAUAAUCGAU-3′ (sequence 3) / 5′-UCACAAAGGUUCUAAAUUG-3′ (sequence 4)

[0072] siEIF3D: 5′-GUUCAAGCCUAAUGAGUUU-3′ (SEQ ID NO: 5) / 5′-CAAGAUAAGAGGUACACAA-3′ (SEQ ID NO: 6) / 5′-AGACAAAGAUCGUCGGAAC-3′ (SEQ ID NO: 7) / 5′-CGAAUGAGAUUUGCCCAGA-3′ (SEQ ID NO: 8)

[0073] siCSDE1: 5′-AGACCGACGUGACAAAUUA-3′ (sequence 9) / 5′-UCAAAUAGGAGAUAAGGUU-3′ (sequence 10) / 5′-GAAAGAAAGAUCCGUCAAG-3′ (sequence 11) / 5′-GGUGAGGUCUAUCCAUUUG-3′ (sequence 12)

[0074] siHNRNPM: 5′-UGAAAUGGCAGUCACUUAA-3′ (SEQ ID NO: 13) / 5′-GAAUGGAAGGCAUAGGAUU-3′ (SEQ ID NO: 14) / 5′-GAUGAGAGGGCCUUACCAA-3′ (SEQ ID NO: 15) / 5′-GAGAUUGACGUUCGAAUUG-3′ (SEQ ID NO: 16)

[0075] siYBX3: 5′-GGAAGGGAGUCGUUACGCU-3′ (SEQ ID NO: 17) / 5′-GAUGAAGGAUGGAGUCCCA-3′ (SEQ ID NO: 18) / 5′-GCAAUAAAGUGGAAGACUA-3′ (SEQ ID NO: 19) / 5′-GCACGGAAGACAAGAGAGU-3′ (SEQ ID NO: 20)

[0076] siFMR1: 5′-CCAAAGAGGCGGCACAUAA-3′ (SEQ ID NO: 21) / 5′-GAUGAUAAAGGGUGAGUUU-3′ (SEQ ID NO: 22) / 5′-CACCAAAUCGUACAGAUAA-3′ (SEQ ID NO: 23) / 5′-AAAGCUAUGUGACUGAUGA-3′ (SEQ ID NO: 24)

[0077] siRTCB: 5′-GGAAUUGUUCAUCGAUCUA-3′ (SEQ ID NO: 25) / 5′-GAACGGACACUGUUAGUAC-3′ (SEQ ID NO: 26) / 5′-GGUGUCCGCUUGCUAAGAA-3′ (SEQ ID NO: 27) / 5′-GUCAACCGCUCUUCCAUGA-3′ (SEQ ID NO: 28)

[0078] siNUFIP2: 5′-AGGAAAGCUAGGCGCAAUA-3′ (SEQ ID NO: 29) / 5′-GGGUGAUAUGCUUCGGAAA-3′ (SEQ ID NO: 30) / 5′-AAUUAAGCCCUGCGAGAAU-3′ (SEQ ID NO: 31) / 5′-AUGGUGAACUAAACGGUAA-3′ (SEQ ID NO: 32)

[0079] The term "antisense oligonucleotide" of the present invention refers to a deoxyribonucleic acid, a ribonucleic acid or a derivative thereof containing a complementary nucleic acid sequence to a specific messenger RNA sequence, and exhibits the effect of binding to a complementary sequence within the messenger RNA to inhibit the translation of the messenger RNA into protein. The purpose of the present invention is that it can be used to inhibit the expression of one or more proteins selected from the group consisting of EIF3A, EIF3D, CSDE1, HNRNPM, YBX3, FMR1, RTCB and NUFIP2.

[0080] In the case of the term "CRISPERi (Clustered regularly interspaced short palindromic repeats interference)" of the present invention, in order to use the CRISPR-Cas system for the purpose of regulating gene expression, it is a system that can induce gene suppression at a target location by utilizing dCas that causes the lack (deletion) of the deoxyribonuclease activity of Cas. CRISPR is a protein associated with the adaptive immune system in eubacteria (Eubacteria) and archaea (Archaea), and Cas and subgenomic RNA can be used as a gene editing tool that can easily and quickly form gene insertions and deformations at a target location. In this case, since mutations may occur at a location that is not the target location, in order to replace gene editing with the regulation of gene expression for the purpose of solving the above-mentioned problem, CRISPRi (CRISPR interference) is proposed as a system that can induce gene suppression at a target location by utilizing dCas that causes the lack (deletion) of the deoxyribonuclease activity of Cas. By using CRISPRi, gene expression at the target location can be inhibited without causing mutation, and genes can be regulated without causing gene expression. It is an effective tool for deoxyribonucleic acid sequences used to specifically regulate gene expression in a variety of organisms. The purpose of the present invention is that it can be used to inhibit the expression of one or more proteins selected from the group consisting of EIF3A, EIF3D, CSDE1, HNRNPM, YBX3, FMR1, RTCB and NUFIP2.

[0081] In one embodiment of the present invention, the activity inhibitor of the protein of the present invention is selected from the group consisting of antibodies, aptamers, small molecule compounds and combinations thereof that can complementarily bind to the above protein.

[0082] The term "antibody" of the present invention refers to a protein molecule that can bind to the antigenic site of a protein or peptide molecule. Such an antibody can be prepared from the obtained protein species by conventional methods after obtaining the protein encoded by the above-mentioned marker gene by cloning each gene island expression vector according to conventional methods.

[0083] In the present invention, the above-mentioned antibodies can be used as a tool capable of maintaining the activity of the above-mentioned proteins by binding to EIF3A, EIF3D, CSDE1, HNRNPM, YBX3, FMR1, RTCB or NUFIP2 proteins activated in individuals suspected of being infected with coronavirus. As a specific example, it includes not only polyclonal antibodies and monoclonal antibodies that can specifically bind to EIF3A, EIF3D, CSDE1, HNRNPM, YBX3, FMR1, RTCB or NUFIP2, or, as long as it has antigen binding, a part of it is also included in the above-mentioned weight of the present invention, but also all immunoglobulin antibodies, and special antibodies such as humanized antibodies can also be included. At the same time, the above-mentioned antibodies can be not only in a complete form with 2 full-length light chains and 2 full-length heavy chains, but also in a form containing a functional fragment of an antibody molecule. The functional fragment of an antibody molecule refers to a fragment that at least retains the antigen binding function, which can be Fab, F(ab'), F(ab')2 and Fv, etc.

[0084] The term "aptamer" of the present invention refers to a nucleic acid molecule having binding activity to a specified target molecule. The above-mentioned aptamer can be ribonucleic acid, deoxyribonucleic acid, a modified nucleic acid or a mixture thereof, and can be in a linear or cyclic shape. Generally speaking, it is known that the shorter the sequence of the nucleotides constituting the above-mentioned aptamer, the easier it is to chemically synthesize and mass-produce, the more cost-effective it is, the easier it is to chemically deform, the better the stability in the body, and the lower the toxicity. In the present invention, the above-mentioned aptamer can be used as a tool capable of inhibiting the activity of the above-mentioned protein by binding to the activated EIF3A, EIF3D, CSDE1, HNRNPM, YBX3, FMR1, RTCB or NUFIP2 protein.

[0085] The term "small molecules" in the present invention refers to organic compounds with small molecular weight, which are molecules that regulate the function of biological macromolecules such as proteins by binding to them. They can be of natural origin or artificial synthesis, and can inhibit the function of proteins or inhibit protein-protein interactions, but are not limited to these.

[0086] Based on the purpose of the present invention, the above-mentioned small molecule compounds can include without limitation any molecules that can inhibit the activity of activated EIF3A, EIF3D, CSDE1, HNRNPM, YBX3, FMR1, RTCB or NUFIP2 proteins. As a specific example, it refers to a molecule that inhibits the activity by binding to activated EIF3A, EIF3D, CSDE1, HNRNPM, YBX3, FMR1, RTCB or NUFIP2, but is not limited to this.

[0087] According to another embodiment of the present invention, the present invention provides a pharmaceutical composition for preventing or treating coronavirus infection, comprising the antiviral composition described in the above another embodiment.

[0088] The term "coronavirus infection" of the present invention refers to the symptoms or disease state exhibited by the coronavirus infection in the group consisting of 229E, 0C43, NL63, HKU1, MERS-CoV, SARS-CoV and SARS-CoV-2. Specifically, it is known that coronavirus is a pathogen that induces a considerable part of the common cold in humans, and can induce direct viral pneumonia, secondary bacterial pneumonia, viral bronchitis, secondary bacterial bronchitis, etc., and the morbidity of the above-mentioned diseases can be prevented by preventing coronavirus infection. In addition, for the cold, viral pneumonia or viral bronchitis that are the direct cause of coronavirus, the effect of treating the above-mentioned diseases can be obtained by suppressing viral proliferation.

[0089] The term "prevention" in the present specification refers to all actions to inhibit or delay the onset of viral infection itself or diseases associated with viral infection by administering the pharmaceutical composition of the present invention.

[0090] The term "treatment" in this specification refers to all actions to improve or cure symptoms or diseases that may be caused by viral infection by administering the pharmaceutical composition of the present invention to inhibit viral proliferation.

[0091] In addition to the active ingredients, the pharmaceutical composition of the present invention also includes a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier included in the pharmaceutical composition of the present invention is a carrier commonly used during preparation, including lactose, glucose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, albendazole, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talcum powder, magnesium stearate and mineral oil, but is not limited thereto. In addition to the above-mentioned ingredients, the pharmaceutical composition of the present invention may also include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. Appropriate pharmaceutically acceptable carriers and preparations are recorded in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0092] The appropriate dosage of the pharmaceutical composition of the present invention is variously prescribed according to the preparation method, administration method, patient's age, weight, sex, disease state, diet, administration route, metabolic rate and reaction sensitivity, etc. On the other hand, preferably, the dosage of the pharmaceutical composition of the present invention is 0.001 mg / kg (body weight)-1000 mg / kg (body weight) per day.

[0093] The pharmaceutical composition of the present invention can be administered orally or parenterally. In the case of parenteral administration, it can be administered by topical application on the skin, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, etc.

[0094] Considering that the coronavirus of the present invention causes damage to the bronchi, lungs, etc. through mucosal infection, droplet infection, etc., preferably, the pharmaceutical composition of the present invention can be administered by local application to the end of the year, oral administration, intravenous injection, subcutaneous injection, etc.

[0095] The pharmaceutical composition of the present invention is prepared by using pharmaceutically acceptable carriers and / or excipients in a unit dose form or in a multi-dose container according to a method that can be easily implemented by a person skilled in the art to which the present invention belongs. In this case, the dosage form can be a solution, suspension or emulsion in an oily or sober solvent, or can also be in the form of an extractor, powder, granule, tablet or capsule, and can also contain a dispersant or stabilizer.

[0096] According to another embodiment of the present invention, the present invention provides a method for screening coronavirus inhibitors comprising the following steps:

[0097] Step (a), contacting a coronavirus inhibitor candidate substance with cells infected with coronavirus; and

[0098] Step (b) confirms whether the expression of one or more proteins selected from the group consisting of EIF3A, EIF3D, CSDE1, HNRNPM, YBX3, FMR1, RTCB and NUFIP2 is reduced compared to the control group of the untreated candidate substance. If the expression of the above protein is reduced, the candidate substance is judged to be a coronavirus inhibitor.

[0099] The EIF3A, EIF3D, CSDE1, HNRNPM, YBX3, FMR1, RTCB and NUFIP2 proteins of the present invention have proviral activity and can be used as targets for newly discovered drug compositions. Coronavirus inhibitors can be discovered by treating drug candidate substances in cells transformed by coronavirus and confirming the expression level of one or more of the above proteins.

[0100] According to another embodiment of the present invention, the present invention provides a method for establishing a set of RNA interactors comprising the following steps:

[0101] Step (a), irradiating a sample containing target RNA and protein with ultraviolet light to induce RNA-protein cross-linking;

[0102] step (b), denaturation by treatment with deoxyribonuclease;

[0103] Step (c), using a biotinylated antisense oligonucleotide probe pool to capture the denatured ribonucleoprotein complex in a sequence-specific manner, wherein the biotinylated antisense oligonucleotides contained in the probe pool are oligonucleotides of the same length selected from the starting positions separated by a specified interval in a partially overlapping manner and within the range of 45 nt to 135 nt based on the target ribonucleic acid sequence;

[0104] step (d), dissociation by on-bead trypsin treatment; and

[0105] Step (e), obtaining information on the interactor group by decomposition product analysis.

[0106] In one embodiment of the present invention, the target RNA of the present invention is the genomic RNA of RNA viruses.

[0107] In one example of the present invention, the probe pool of the present invention comprises a first probe pool that specifically hybridizes to genomic RNA molecules and a second probe pool that hybridizes to both genomic RNA and small guide RNA.

[0108] Effects of the Invention

[0109] Features and advantages of the present invention are summarized as follows.

[0110] (a) The present invention provides an antiviral composition.

[0111] (b) The present invention provides a pharmaceutical composition for preventing or treating coronavirus infection.

[0112] (c) The present invention provides a method for screening coronavirus inhibitors.

[0113] (d) The present invention provides a method for establishing a group of RNA interactors.

[0114] (e) When the antiviral composition and pharmaceutical composition of the present invention are used, viral infection can be effectively inhibited, and viral proliferation in vivo after viral infection can be inhibited. BRIEF DESCRIPTION OF THE DRAWINGS

[0115] Figure 1a to Figure 1g Shown are generalized recognition host and viral proteins that directly interact with SARS-CoV-2 RNA.

[0116] Figure 1a A schematic diagram showing the modified RAP-MS protocol in Vero cells infected with SARS-CoV-2.

[0117] Figure 1bA schematic diagram of two separate pools of 90-nt antisense oligonucleotides and their SARS-CoV-2 RNA coverage is shown. The first probe set "Probe I" consists of 707 oligonucleotides covering the intrinsic region of genomic RNA, and the second probe set "Probe II" consists of 275 oligonucleotides covering the common region of genomic RNA and subgenomic RNA.

[0118] Figure 1c Spectral coefficient ratios for probe I (x-axis) and probe II (y-axis) experiments relative to no-probe controls in Vero cells infected with SARS-CoV-2 (n=3 technical replicates) are shown. Grey circles represent host proteins, while viral proteins (n=9) are represented and labeled in black. The average spectral coefficient ratios for the probe I and probe II experiments are represented by vertical and horizontal dashed lines, respectively.

[0119] Figure 1d Statistical analysis of the amount of viral protein relative to a no-probe control group (i.e., probe binding) is shown. The adjusted p-values ​​for ProbeI and ProbeII experiments are indicated in yellow and green, respectively. The critical value for statistical significance (adj. p-value < 0.01) is indicated by a horizontal dashed line.

[0120] Figure 1e Figure 2. Comparison of spectral coefficient ratios from Probe I (x-axis) and Probe II (y-axis) experiments in Vero cells infected with SARS-CoV-2 with ribonucleoprotein capture experiments in uninfected cells (n=3 technical replicates). Statistically significant host proteins in both Probe I and Probe II experiments (n=37, adj.P value < 0.05) are represented by black circles. Representative host proteins are labeled. The average spectral coefficient ratios for Probe I and Probe II experiments are represented by vertical and horizontal dashed lines, respectively.

[0121] Figure 1f Statistical analysis of host proteins that were enhanced in both Probe I and Probe II experiments (ie, probe enhanced) is shown. The adjusted p-values ​​for Probe I and Probe II experiments are shown in yellow and green, respectively.

[0122] Figure 1g Statistical analysis of host proteins enriched only in probe I experiments is shown. Figure 1h Statistical analysis of host proteins that were only enhanced in the Probe II experiment is shown.

[0123] Figure 2a to Figure 2fComparison results of SARS-CoV-2 and HCoV-OC43 RNA interactors groups are shown.

[0124] Figure 2a A simplified diagram showing the ribonucleoprotein capture experiment over time in HCT-8 cells infected with HCoV-OC43.

[0125] Figure 2b The LFQ intensity of the enriched viral proteins confirmed in the HCoV-OC43 ribonucleoprotein capture experiment is shown at 12 hours, 24 hours, 36 hours and 48 hours after infection (hours post-infection; hpi).

[0126] Figure 2c Figure 2 shows the normalized LFQ intensity (nLFQ) heat map of HCoV-OC43 assays for host proteins that were enhanced in both SARS-CoV-2 Probe I and Probe II assays (left), enhanced only in SARS-CoV-2 Probe I assays (middle), and enhanced only in SARS-CoV-2 Probe II assays (right). nLFQ is the log10 fold change for the median LFQ intensity across each probe set (i.e., Probe I or Probe II). To account for missing values, 1×10 6 A pseudo-value.

[0127] Figure 2d Spectral count ratios of probe I (x-axis) and probe II (y-axis) experiments in HCT-8 cells infected with HCoV-OC43 at 36 hpi relative to a no-probe control group are shown. Host proteins are represented by gray circles, while viral proteins (n=14) are represented and labeled in black. The average spectral count ratios of probe I and probe II experiments are represented by vertical and horizontal dashed lines, respectively.

[0128] Figure 2e The ratio of the spectral coefficients of the probe I (x-axis) and probe II (y-axis) experiments in HCT-8 cells infected with HCoV-OC43 compared with cells not infected at 36 hpi is shown. Host proteins that are statistically significant in both probe I and probe II experiments (n=38, adj.P value <0.05) are represented by black circles. Representative host proteins are marked. The average spectral coefficient ratios of the probe I and probe II experiments are represented by vertical and horizontal dashed lines, respectively.

[0129] Figure 2fUpset plots showing proteins enhanced by HCoV-OC43 probe I (n=67), proteins enhanced by HCoV-OC43 probe II (n=70), proteins enhanced by SARS-CoV-2 probe I (n=73), and proteins enhanced by SARS-CoV-2 probe II (n=72). A total of 14 host proteins were enhanced in 4 experiments.

[0130] Figure 3a to Figure 3c Molecular network showing the SARS-CoV-2 RNA interactome.

[0131] Figure 3a The physical interaction network of the SARS-CoV-2 RNA interactome is shown (except for ribosomal proteins and EIF3 proteins). Host factors are represented in red. Host and viral proteins are represented by circles and diamonds, respectively. The size of the network nodes is proportional to the number of network edges (i.e., degree) of the node. The largest connected structural elements in the network are selected for visualization and interpretation.

[0132] Figure 3b Show Figure 2a Gene Ontology Biological Process term enrichment analysis of the proteins retrieved.

[0133] Figure 3c The intracellular localization of the SARS-CoV-2 RNA interactor group and the cellular messenger RNA interactor group is shown. The number of proteins with localization information is shown in brackets.

[0134] Figures 4a to 4d Modulation results of the SARS-CoV-2 RNA interactome are shown.

[0135] Figure 4a Results of differential expression analysis of ACE2-expressing A549 cells (A549-ACE2) after infection with SARS-CoV-2 (MOI = 2.0) are shown. The fold change (FC) of mRNAs of -1 and 1 is indicated by vertical dashed lines. 1% FDR is indicated by horizontal dashed lines. In cases where host genes of the SARS-CoV-2 RNA interactor set are differentially expressed (FDR < 1% and |FC| > 1), they are indicated in red, otherwise they are indicated in black.

[0136] Figure 4bComparison of mRNA fold changes in A549-ACE2 cells infected with SARS-CoV-2 (MOI=2.0) after treatment with ruxolitinib. Host genes of the SARS-CoV-2 RNA interactor set are shown in black.

[0137] Figure 4c Figure 2 shows the results of differential expression analysis of normal human bronchial epithelial (NHBE) cells after treatment with interferon β (INFβ). Figure 4d The same diagram notation is used to illustrate.

[0138] Figure 4d MA plot of publicly available mRNA-seq data showing postmortem lung samples from COVID-19 patients versus healthy lung tissue from uninfected individuals. The mean fold change is indicated by the dashed line. In cases where host genes in the SARS-CoV-2 RNA interactome are differentially expressed (FDR < 5%), they are indicated in red, otherwise they are indicated in black. Other differentially expressed genes are indicated in dark grey.

[0139] Figure 5a to Figure 5c Shown is the effect of RNA binding protein depletion on viral RNA abundance.

[0140] Figure 5a A simplified diagram showing knockdown of small interfering RNA in Calu-3 cells infected with SARS-CoV-2.

[0141] Figure 5b The viability of cells treated with small interfering RNA as determined by MTT assay is shown. Data are presented as mean±sem (n=3 independent experiments). siNC was used as a negative control group.

[0142] Figure 5c The changes in the levels of genomic RNA (amplicon nsp12, dark gray) and N subgenomic RNA (amplicon N, light gray) after depletion of small interfering RNA were measured by reverse transcription fluorescence quantitative polymerase chain reaction (RT-qPCR). Data are expressed as mean ± standard error (sem) (n = 3 or 6 independent experiments). The RNA levels of cells treated with siNC were used as negative control. 18S ribosomal RNA (rRNA) was used for normalization. *P < 0.05, **P < 0.01; two-tailed Student's t-test.

[0143] Figures 6a to 6f The experimental and statistical test results of RAP-MS experiments on the deformation of SARS-CoV-2 RNA are shown.

[0144] Figure 6a Results of antisense oligonucleotide-based RNA-protein (RNP) capture in Vero cells infected with SARS-CoV-2 are shown by silver staining and immunoblotting. SARS-CoV-2 nucleocapsid and RPS7 were used as controls for ribosomal RNA and SARS-CoV-2 RNA (probe I and probe II) ribonucleoprotein capture experiments. CL: cross-linked.

[0145] Figure 6b The RNA yield of antisense oligonucleotide-based RNA-protein (RNP) capture (ribosomal RNA, probe I and probe II) measured by reverse transcription fluorescence quantitative polymerase chain reaction (n=2 technical replicates) is shown. The fluorescence quantitative polymerase chain reaction (qPCR) primers for SARS-CoV-2N subgenomic RNA target the common region of genomic RNA and subgenomic RNA, and the genomic RNA fluorescence quantitative polymerase chain reaction primer targets nsp12.

[0146] Figure 6c The results of the protein domain enhancement analysis of host factors confirmed by statistical comparison with the no-probe control experiment (i.e., probe I and probe II combined) are shown. The critical value for statistical significance (P-value < 0.05) is indicated by a vertical dotted line.

[0147] Figure 6d Figure 2. Results of ribonucleoprotein capture tested by silver staining in Vero cells infected and not infected with SARS-CoV-2.

[0148] Figure 6e Shown are the length distributions of peptides with “on-bead” resolution in Vero cells infected and not infected with SARS-CoV-2 (n=3 technical replicates).

[0149] Figure 6f Gene Ontology (GO) Biological Process (BP) defined enrichment analysis of 37 host proteins that were statistically enriched in both Probe I and Probe II experiments is shown.

[0150] Figures 7a to 7f The experimental and statistical test results of ribonucleoprotein capture of HCoV-OC43 RNA are shown.

[0151] Figure 7aThe relative HCoV-OC43 genomic RNA (amplicon nsp12) levels determined by reverse transcription fluorescence quantitative polymerase chain reaction are shown. Figure 7b HCoV-OC43 N protein levels measured by immunoblotting for multiple virus loads (1×, 5×, and 25×) at time points 12, 24, 36, and 48 hours post-infection are shown. RNA levels are compared to genomic RNA levels at 12 hours post-infection with 1× virus load.

[0152] Figure 7c The results of confirming ribonucleoprotein capture by silver staining in HCT-8 cells infected with HCoV-OC43 are shown.

[0153] Figure 7d The protein structure and enhancement analysis results of host factors confirmed by statistical comparison of ribonucleoprotein capture at each time point after infection relative to a control experiment without probes (i.e., probe I and probe II binding). Statistical significance (P-value < 0.05) is indicated by a vertical dashed line.

[0154] Figure 7e Show Figure 2b LFQ intensity of viral proteins confirmed in HCoV-OC43 ribonucleoprotein capture at 12 hours, 24 hours, 36 hours and 48 hours post infection (hpi) not shown in FIG.

[0155] Figure 7f The results of Gene Ontology (GO) and Biological Process (BP) defined enrichment analysis of 38 host proteins that were statistically enriched in both OC43 Probe I and Probe II experiments are shown. txn: transcription.

[0156] Figures 8a to 8c The SARS-CoV-2 RNA interactome is shown in the context of physical interaction networks and gene-regulatory networks.

[0157] Figure 8a The physical interaction network of host and viral proteins enriched in both SARS-CoV-2 probe I and probe II experiments is shown in red. Host and viral proteins are represented by circles and diamonds, respectively. The size of the network node is proportional to the number of network edges (i.e., degree) of the node. The largest connected structural elements in the network are selected for visualization and interpretation.

[0158] Figure 8b for Figure 8aThe Gene Ontology Biological Process (GO BP) definitions of the retrieved host proteins enhanced the analysis results.

[0159] Figure 8c Results of differential expression analysis of the published proteome data of HeLa cells after IFN-β stimulation for 24 hours are shown. The fold change (FC) of protein levels between -1 and 1 is indicated by vertical dashed lines. The 1% FDR is indicated by horizontal dashed lines. In cases where host proteins of the SARS-CoV-2 RNA interactome are differentially expressed (FDR < 1% and |FC| > 1), they are indicated in red, otherwise they are indicated in black.

[0160] Figure 9a and Figure 9b The results of the small interfering RNA knockdown experiment in Calu-3 cells are shown.

[0161] Figure 9a The results of confirming the knockdown of small interfering RNA in Calu-3 cells by reverse transcription polymerase chain reaction (RT-PCR) are shown. Figure 9b Results showing knockdown of small interfering RNA in Calu-3 cells infected with SARS-CoV-2 at 24 hours post infection at 0.05 MOI. Data are presented as mean ± standard error (n = 3 independent experiments). 18s ribosomal RNA was used for normalization. DETAILED DESCRIPTION

[0162] The present invention is described in more detail below by way of examples. However, it should be self-evident to those skilled in the art that these examples are only used to more specifically describe the present invention, and according to the gist of the present invention, the scope of the present invention is not limited to these examples.

[0163] Example

[0164] Reagents and Resources

[0165]

[0166]

[0167]

[0168]

[0169] Experimental methods

[0170] Example 1: Preparation of antisense oligonucleotide template

[0171] The genomic RNA of SARS-CoV-2 (NCBI RefSeq accession NC_045512.2) and HCoV-OC43 (GenBank accession AY391777.1) was scanned from head to tail, and the 90nt tail was enumerated except for the partially folded tail. The above tails were designed in a 30nt interval, and adjacent tails had a total of 60nt subsequences. In order to avoid ambiguous targeting, bowtie 2 (Langmead and Salzberg, 2012) was used to align the tails with the transcriptome (October 14, 2019 version) and delete the multiple mapped sequences. In order to prepare a large number of biotinylated antisense oligonucleotides (ASOs), sequence elements for in vitro transcription (IVT), reverse transcription (RT) and polymerase chain reaction (PCR) were added to the 90nt tail. T7 promoter (5′-TAA TAC GACTCA CTA TAG GG-3′) and RT start pad (5′-TGG AAT TCT CGG GTG CCA AGG-3′) were added to the head and tail of each tail. For each viral genome, the inventors divided the AOS into two groups: "Probe I" targets the region inherent to the genomic RNA ([265:21553] of NC_045512.2; [:21506] of AY391777.1), and "Probe II" targets the genomic and subgenomic RNA ([21562:] of NC_045512.2; [21506:] of AY391777.1). The templates of the four ASO groups have clear polymerase chain reaction primer binding sites on both sides. Therefore, each ASO group can be selectively amplified in a single mixture. The final AOS template (167 nt) was prepared by the oligonucleotide pool synthesis service of Genscript and stored at -80°C. Table 2 shows the ASO templates used in the present invention.

[0172] Example 2: Mass production of biotin-labeled ASO

[0173] ASO templates were amplified using KAPA HiFi HotStart ReadyMix (Roche) and polymerase chain reaction primers for the ASO pool. The polymerase chain reaction products were purified using the QIAquick PCR purification kit (QIAGEN). Then, the RNA intermediates were transcribed using the 5X MEGAscript T7 transcription kit (Invitrogen) and the deoxyribonuclease templates were decomposed by TURBO deoxyribonuclease (Invitrogen). In order to purify the enzyme and other reagents, polyethylene glycol was added to the 1.8× reaction volume of AMPure XP (Beckman) to a final 20%. Size selection was performed according to the instructions of the production company. Biotinylated ASOs were synthesized by RevertAid reverse transcriptase (ReverseTranscriptase) (Thermo Scientific) and 5′ biotin-TEG primers. The RNA intermediates were hydrolyzed in 0.1M sodium hydroxide (NaOH) and neutralized with acetic acid. Finally, ASO purification was performed in the same manner as for IVT RNA selection. The primer sequences used for polymerase chain reaction and reverse transcription are shown in Table 6.

[0174] Table 6

[0175]

[0176]

[0177]

[0178] Example 3: Editing of proteome database

[0179] Uniprot reference proteomes for humans (UP000005640, canonical, SwissProt) and vervet monkeys (Chlorocebus sabaeus; UP000029965, canonical, SwissProt and TrEMBL) were used to identify host proteins in each quality analysis experiment (version 03 / 21 / 20) (UniProt Concortium, 2019). The reference proteome was extensively manually screened for the reference proteome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) based on the NCBI reference sequence (NC_045512.2) and relevant literature for accessory proteins (e.g., ORF3b, ORF9b, and ORF9c). The reference proteome for human coronavirus OC43 (HCoV-OC43) was compiled based on the Uniprot Swiss-Prot protein for HCoV-OC43 (taxonomy: 31631) after excluding HCoV-OC43 protein I separated into protein Ia and protein Ib (or N2) (Vijgen et al., 2005).

[0180] Example 4: Cell culture, transfection and viral infection

[0181] Virus experiments were performed according to the biosafety guidelines of the Korea Centers for Disease Control and Prevention (KCDC). The Seoul National University Biosafety Committee (SNUIBC) approved the protocols used in the present invention, including SNUIBC-R200331-1-1 for BL2 experiments and SNUIBC-200508-1 for BL3 experiments. Vero and HCT-8 cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) (Welgene) and RPMI 1640 (Welgene) containing 1X antibiotic-antimycotic (Gibco) and 10% fetal bovine serum (FBS) (Gibco), respectively, at a temperature of 37°C in the presence of 5% CO 2 CO 2 In the case of SARS-CoV-2 infection, 7 × 10 cells were plated in a T-175 flask 24 hours before infection. 6Vero cells. After washing the cells with serum-free medium, they were incubated with 5 mL of virus dilution medium at 0.1 MOI for 30 minutes according to the results confirmed in the plaque assay. After infection, the virus-containing medium was replaced with serum-reduced medium (2% fetal bovine serum) and cultured until harvest. In the case of HCoV-OC43 infection, a similar protocol was used except that the culture temperature was lowered to 33°C. For siRNA transfection, the cells were cultured at 37°C in an atmosphere containing 5% CO 2 CO 2 The incubator will maintain 3.5 x 10 cells of Dulbecco's modified Eagle's medium containing 1X antibiotic-antimycotic and 10% fetal bovine serum (FBS). 5 Calu-3 cells were placed in 12-well culture plates and reverse-transfected with 50 nM siRNA using Lipofectamine RNAiMAX (Invitrogen) and ON-TARGETplus SMARTpool siRNA (Horizon Discovery). The cell survival rate after siRNA knockdown was 1 / 100 of that of uninfected cells in 96-well culture plates at 48 hours after transfection. th The cell number was determined by MTT assay (Promega) 4 hours after the addition of tetrazolium dye.

[0182] Example 5: RNA purification and reverse transcription fluorescence quantitative polymerase chain reaction

[0183] To purify total RNA from virus-infected cells, 1 mL of TRIzol LS (Invitrogen) was added to the cell monolayer from which the culture medium was removed from a single well of a 12-well culture plate, followed by on-column DNA digestion and purification (Zymo Research). To purify RNA from ribonucleoprotein-captured samples, 100 ng of Proteinase K (PCR grade, Roche) was used to digest the RNA captured by the beads and incubated at 37°C for 1 hour before RNA was separated using TRIzol LS using GlycoBlue (Invitrogen). 1 μg to 5 μg of RNA was reverse transcribed using RevertAid transcriptase (Thermo Fisher Scientific) and random hexamer. Fluorescent quantitative polymerase chain reaction was performed using the primer pairs listed in Table S6 and PowerSYBR Green (Applied Biosystems), and analyzed using QuantStudio 5 (Thermo Fisher Scientific).

[0184] Example 6: Reverse purification of modified RNA by combined mass spectrometry (RAP-MS)

[0185] The virus-infected cells were separated from the culture vessel by trypsin and the cell pellet was resuspended in ice-cold phosphate buffered saline (PBS). 12 mL of the cell suspension was distributed in a 150 mm culture dish and the SARS-CoV-2 was detected by BIO-LINK BLx-254 at 2.5 J / cm 2Alternatively, HCoV-OC43 was irradiated with 254 nm ultraviolet light at 0.8 J / cm2 using a spectrolinker XL-1500. After the ultraviolet-crosslinked cells were pelleted by centrifugation, they were resuspended in a TURBO DNase solution (150 units per flask) and incubated at 37°C for 30 minutes. The cells from which the DNA was separated were supplemented with the same volume of preheated 2x lysis buffer (containing 40 mM Tris-Cl, 1 M lithium chloride (LiCl), 1% LDs, 2 mM ethylenediaminetetraacetic acid (EDTA), 10 mM dithiothreitol (DTT), and 8 M urea (Urea) at pH 7.5) and denatured by incubation at 68°C for 30 minutes. For each replicate, cell lysate from one flask (T-175) was mixed with 20 μg of biotin probe pool (probe I or probe II) to a final volume of 1 mL and incubated at 68°C for 30 minutes for hybridization. Streptavidin beads (1 mL per replicate, New England Biolabs) were added to the biotin-labeled ribonucleoprotein lysate and captured by rotating overnight at room temperature. The enriched probe ribonucleoprotein beads were washed twice with 1X lysis buffer and transferred to a new tube for a final wash with detergent-free wash buffer (containing 20 mM Tris-Cl, 0.5 M lithium chloride, and 1 mM ethylenediaminetetraacetic acid at pH 7.5). 1 / 10 was placed alone. th 1 / 10th of the beads were used for RNA content assessment by reverse transcription fluorescence quantitative polymerase chain reaction, and another 1 / 10th of the beads were used in silver staining (KOMA biotech). The remaining 8 / 10th of the beads were decomposed using 100 units of Benzonase nuclease (Millipore) at 37°C. th For peptide decomposition on the beads, the nuclease-treated beads were resuspended with a final 8M urea, reduced with 10mM dithiothreitol, alkylated with 40mM iodoacetamide (IAA) at 37°C for 1 hour, diluted with 50mM ammonium bicarbonate (ABC) and finally diluted with 1M urea. 300ng of trypsin (Thermo Fisher, MS grade) and 1mM calcium chloride (CaCl) were added to the above bead suspension. 2 ) and then decomposed at 37°C overnight. After separating the peptide solution in the magnetic beads, it was treated with HiPPR detergent removal spin column (Thermo Fisher Scientific) and then desalted using reverse phase C18 ziptip (Millipore). After washing and drying, the sample was reconstituted with 20 μL of 25 mM ammonium bicarbonate buffer for LC-MS / MS analysis.

[0186] Example 7: LC-MS / MS analysis

[0187] LC-MS / MS analysis was performed using an Orbitrap FusionLumos Tribrid MS (Thermo Fisher Scientific) coupled to a nanoAcquity UPLC system (Waters). Both the analytical capillary column (100 cm x 75 μm id) and the trapping column (3 cm x 150 μm id) were packaged in-house with 3 μm Jupiter C18 particles (Phenomenex, Torrance). The long analytical column was placed in a column heater (Analytical Sales and Services) regulated at 45°C. The LC flow rate was 300 nL / min, with a 100 min linear gradient from 95% solvent A (containing 0.1% formic acid (Merk)) to water (H2O). 2 The range of solvent B (0) to 40% solvent B (100% acetonitrile containing 0.1% formic acid) was 40%. Precursor ions were obtained at a resolution of 120K in m / z 200, and precursor separation for MS / MS analysis was performed at 1.4Th. High-energy collisional dissociation (HCD) with a collision energy of 30% was used in sequencing with a target value of 1E5 ions determined by automatic gain control. The resolution of the obtained MS2 spectra was set to 30K in m / z 200 with a maximum injection time of 120ms.

[0188] The raw data files of mass analysis were processed for label-free quantification using MaxQuant (version 1.6.15.0) (Cox and Mann, 2008) with the built-in Andromeda search engine (Cox et al., 2011) with default settings, except for a few exceptions. Briefly, for peptide-spectrum match searches, cysteine ​​carbamidomethylation was set as a fixed modification, and methionine oxidation and N-terminal acetylation were set as variable modifications. The acceptable errors (Tolerance) for the first and main peptide-spectrum matches were 20 ppm and 4.5 ppm, respectively. The contaminant database provided by MaxQuant was used to identify peptides that commonly contaminate proteins. A 1% FDR cutoff (treshold) was used for all peptide and protein levels. The Match-between-runs option was activated with default parameters in the identification step. Finally, LFQ was performed for cases with a minimum scale factor of 1.

[0189] Example 8: Statistical analysis for ribonucleoprotein capture experiments

[0190] In order to identify host and viral proteins that interact with a particular RNA species of interest (e.g., subgenomic RNA or genomic RNA), the results of "bead only" and "probe only" samples are used as technical background. In particular, "bead only (or no-probe)" experiments are used in infected cells to count non-specific interactions and biotin-containing carboxylases (e.g., PCCA, ACACA, and ACACB) to determine the group of host and viral proteins that bind to RNA in a broad sense, referred to as "probe I / probe II" binding proteins in the present invention. Probe experiments in uninfected cells (i.e., "probe only") are used as technical background for target RNA-independent interactions to determine the group of host proteins that are enriched for the target RNA, referred to as proteins "enhanced" by probe I / probe II in the present invention.

[0191] To achieve this, we treat protein spectral count data as a multinomial distribution and use statistical tests based on spectral count enhancement. pIn a case experiment (e.g., a probe I experiment in infected cells) the number of spectral counts identified in the protein group P, M p The number of individual counts was used as in control experiments (eg no-probe experiments in infected cells). For each protein i with Ni≥1, the statistical significance of the enrichment was as follows.

[0192]

[0193]

[0194] In N = ∑ i In, N i is the total spectrum count, θ i is the background probability, B(k;n,p) is the binomial distribution of k successes in a trail of n binomial probabilities with success probability p. Finally, the Benjamini-Hochberg method was used to adjust the p-value and control the false discovery rate.

[0195] Example 9: Gene Ontology (GO) enrichment analysis

[0196] We perform a gene ontology-enhanced analysis of the functions of dozens of host proteins identified in ribonucleoprotein capture experiments by means of summaries. Fisher's correct test is often used in the process of inferring the statistical significance of the association (i.e., contingency) between a particular gene ontology definition and a group of genes of interest.

[0197] To improve the power of this analysis, the inventors used the weight01 algorithm in the topGO R package (Alexa et al., 2006) that accounts for the structure of the Gene Ontology graph and reduces local dependencies between Gene Ontology definitions.

[0198] Gene ontology details were obtained from the GO.db R package (version 3.8.2), and gene annotations of the gene ontology were obtained from the org.Hs.eg.db R package (version 3.8.2).

[0199] Example 10: Analysis of protein-protein interaction networks

[0200] Protein-protein interaction data were integrated in the BioGRID database (Release 3.5.187) (Stark et al., 2006) to search for other proteins that do not necessarily bind to SARS-CoV-2 RNA but form temporary or stable interactions with host proteins confirmed by ribonucleoprotein capture experiments. Specifically, we only considered protein-protein interactions that: (1) were found in at least two different types of experiments; and (2) were reported in at least three published records of human protein-protein interactions that resulted in a total of 65,625 interactions covering 12,143 human proteins. The interactions between SARS-CoV-2 proteins and human proteins were confirmed by affinity capture and mass analysis in cells expressing SARS-CoV-2 proteins (Gordon et al., 2020). For visualization of the graphs, the network R package and the ggnet2 function of the GGally R package were used.

[0201] Example 11: Protein domain enrichment analysis

[0202] The Pfam database (version 33.1) (El-Gebali et al., 2019) was used in the protein structure and enhancement analysis. The ribonucleoprotein capture results of HCoV-OC43 and SARS-CoV-2 were analyzed using Taxon 9606 (human) and Taxon 60711 (black-faced green monkey) protein domain annotations, respectively. In order to infer the specific protein structure and statistical enhancement of a specific genome (such as SARS-CoV-2 probe I binding protein), a one-sided Fisher's exact test was used. For all proteins identified in the ribonucleoprotein capture experiment and the above proteins, the group of all annotated protein domains was used as a statistical background in the enhancement analysis.

[0203] Example 12: Cell location analysis

[0204] To investigate the subcellular localizations of the SARS-CoV-2 interactome, protein subcellular localization information from the Human Cell Atlas database vi (Go et al., 2019) was used. Although information from the SAFE algorithm was primarily used, it was supplemented by information from the NMF algorithm in cases with “no prediction” or “-” localization. Although the localization definitions of the NMF algorithm are generally consistent with those of the SAFE algorithm, a few of them are mapped to the superordinate definitions of the SAFE algorithm. For example, the definition of “cell junction” from the NMF algorithm was merged into “cell junction, plasma membrane” from the SAFE algorithm.

[0205] Experimental Results

[0206] 1. Purification of SARS-CoV-2 Ribonucleoprotein

[0207] To identify viral and host proteins that directly interact with genomic and subgenomic RNAs of SARS-CoV-2, we performed a reverse RNA purification coupled to a mass spectrometry (RAP-MS) protocol developed for the analysis of proteins interacting with specific RNA species (McHugh and Guttman, 2018) ( Figure 1a ).

[0208] In brief, cells are separated from the culture vessel and then irradiated with 254nm ultraviolet light to induce RNA-protein crosslinking while maintaining the integrity of RNA. After treating the crosslinked cells with deoxyribonuclease and homogenizing the high concentration of protein, cleavage is performed under optimized buffer conditions for denaturation. A large-scale pool of biotinylated antisense 90-nt probes is used to capture denatured ribonucleoprotein complexes in a sequence-specific manner. After rigorous washing and removal of detergent, the ribonucleoprotein complex is released and decomposed by continuous Benzonase and trypsin treatment on beads. Such a modification to the RAP-MS protocol makes it possible to make strong and sensitive identification of proteins directly bound to the RNA target of interest.

[0209] To maintain an unbiased view of the SARS-CoV-2 RNA interactome, we designed two separate pools of densely overlapping 90-nt antisense probes ( Figure 1b and Table 1).

[0210] Table 1

[0211]

[0212]

[0213]

[0214]

[0215]

[0216]

[0217]

[0218]

[0219]

[0220]

[0221]

[0222]

[0223]

[0224]

[0225]

[0226]

[0227]

[0228]

[0229]

[0230]

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239]

[0240]

[0241]

[0242]

[0243]

[0244]

[0245]

[0246]

[0247]

[0248]

[0249]

[0250]

[0251]

[0252]

[0253]

[0254]

[0255]

[0256]

[0257]

[0258]

[0259]

[0260]

[0261]

[0262]

[0263]

[0264]

[0265]

[0266]

[0267]

[0268]

[0269]

[0270]

[0271]

[0272]

[0273]

[0274]

[0275]

[0276]

[0277]

[0278]

[0279]

[0280]

[0281]

[0282]

[0283]

[0284]

[0285]

[0286]

[0287]

[0288]

[0289]

[0290]

[0291]

[0292]

[0293]

[0294]

[0295]

[0296]

[0297]

[0298]

[0299]

[0300]

[0301]

[0302]

[0303]

[0304]

[0305]

[0306]

[0307]

[0308]

[0309]

[0310] The SARS-CoV-2 transcriptome consists of the following components: (1) genomic RNA encoding 16 nonstructural proteins (nsps); and (2) multiple subgenomic RNA encoding structural and accessory proteins (Sola et al., 2015). Subgenomic RNA is more abundant than genomic RNA (Kim et al., 2020a). The first pool (“probe I”) was composed of 707 oligonucleotide tails of every 30 nucleotides through the ORF1ab region (266:21553, NC_045512.2), which specifically hybridizes with genomic RNA molecules ( Figure 1b The second pool of 275 oligonucleotides ("Probe II") included the remaining region common to both genomic and subgenomic RNA (21563:29872, NC_045512.2).

[0311] First, to confirm whether our method specifically captures viral ribonucleoprotein complexes, the purification results of Vero cells infected with SARS-CoV-2 (BetaCoV / Korea / KCDC03 / 2020) were compared at MOI 0.1 using probe I and probe II for 24 hours (Kim et al., 2020b). We used no probe (“no probe” control group) as a negative control or used control probes (18S or 28S rRNA) for pull-down. The protein composition of each ribonucleoprotein sample was as expected, as shown in the silver staining and immunoblotting of SARS-CoV-2N protein, which was significantly associated with probe I and probe II. Figure 6a ) are used to distinguish the results shown in the figure. The enhancement of SARS-CoV-2 RNA was performed by reverse transcription fluorescence quantitative polymerase chain reaction ( Figure 6b ) to confirm, which shows that our protocol purifies specific ribonucleoprotein complexes. SARS-CoV-2 genomic RNA was not enhanced in the Probe II experiment, which suggests that under our culture conditions, the amount of subgenomic RNA is greater than that of genomic RNA.

[0312] 2. Comprehensive identification of proteins binding to the SARS-CoV-2 transcriptome

[0313] We used tandem mass spectrometry (LC-MS / MS) coupled with liquid chromatography for label-free quantification (LFQ) and identified a total of 429 host proteins and 9 viral proteins ( Figure 1c). Since there will be a lot of proteins that are not specifically co-precipitated during the ribonucleoprotein capture experiment, we used the above protein background as a multinomial distribution to statistically model, and evaluated the probability (i.e., p-value) of the protein background confirmed in the ribonucleoprotein capture experiment (e.g., probe I) compared with the protein background experiment of the control group (e.g., the object probe). The results of the spectral coefficient analysis without applying weighted values ​​generated 199 and 220 proteins that were over-represented in each probe I and probe II sample (FDR < 10%, Table 2). Protein structure and enhancement analysis showed that such proteins actually retain RNA binding domains such as RNA recognition motif (RRM) domain and K homology (KH) domain ( Figure 6c ). As a reference, unlike the panel of cellular mRNA interactors (Castello et al., 2012; Gerstberger et al., 2014), the RNA-binding repertoire of SARS-CoV-2 RNA exhibited a depletion of the DEAD / DEAH box helicase domain and an enrichment of the KH domain.

[0314] Table 2

[0315]

[0316]

[0317]

[0318]

[0319]

[0320]

[0321] In the case of viral proteins, it is expected that the N protein is very strongly enhanced ( Figure 1d ). nsp1 protein was also statistically enriched in both probe I and probe II experiments.

[0322] Compared with probe II, Nsp12, S, M and nsp9 were detected more in probe I. Coronavirus nsp9 is a single-stranded RNA binding protein essential for viral replication (Egloff et al., 2004; Sutton et al., 2004)(Miknis et al., 2009). Nsp1 is one of the main toxic factors that inhibit host translation by binding to the 40S ribosomal subunit (Thoms et al., 2020). Nsp1 has been mainly studied in the context of host gene expression (Narayanan et al., 2008), but our results suggest a direct role of NSP1 in the transcriptome of SARS-CoV-2.

[0323] To illustrate the host proteins that are enhanced in the SARS-CoV-2 ribonucleoprotein complex, a negative control experiment using uninfected cells was performed ( Figure 1e In fact, as shown by staining, the above control group provides a conservative background of host proteins ( Figure 6d The distribution of peptide lengths was consistent across technical replicates ( Figure 6e ), showing the robustness of the "on-bead" decomposition step. Spectral coefficient analysis for the "uninfected probe control" yielded 74 and 72 proteins enriched in samples infected with probe I and probe II, respectively (FDR < 5%, Figure 1f-1h ). In the combination, we defined the above 109 proteins as the "SARS-CoV-2 RNA interactor group". 37 host proteins including CSDE1 (Unr), EIF4H, FUBP3, G3BP2, PABPC1, ZC3HAV1, etc. were enhanced in both probe I and probe II ribonucleoprotein capture experiments of infected cells, identifying a robust group of "nuclear SARS-CoV-2 RNA interactor group". Gene ontology definition enhancement analysis showed that such host factors are related to RNA stability control, messenger RNA function and viral processes ( Figure 6f ).

[0324] 3. Protection of the β-coronavirus ribonucleoprotein complex

[0325] To investigate the evolutionary conservation of RNA-protein interactions in coronaviruses, we performed ribonucleoprotein capture on HCoV-OC43, which belongs to the A system of the betacoronavirus genus. HCoV-OC43 shares 54.2% nucleotide homology with SARS-CoV-2, which belongs to the B system. The inventors analyzed the HCoV-OC43 ribonucleoprotein complex at multiple time points, including 12 hours, 24 hours, 36 hours, and 48 hours post-infection (hpi). Figure 2a , Figure 7a and Figure 7a ). Two antisense probe sets (i.e., OC43 probe I and OC43 probe II) were designed in a similar manner to the SARS-CoV-2 experiment. OC43 probe I hybridized only to the genomic RNA of HCoV-OC43, whereas probe II captured genomic and subgenomic RNA molecules. A control group without probe for infected cells (no probe) and a probe control group for uninfected cells were used as negative controls and confirmed by silver staining ( Figure 7c ).

[0326] Unweighted spectral coefficient analysis of the no-probe control group showed 133, 167, 192 and 160 proteins overrepresented in the OC43 probe I experiment at 12, 24, 36 and 48 hours post-infection, respectively (FDR < 10%, Table 3).

[0327] Table 3

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334]

[0335]

[0336]

[0337]

[0338]

[0339] In the case of the OC43 probe II experiment, 119, 189, 194 and 185 proteins were over-represented (FDR < 10%, Table 4) at the 12, 24, 36 and 48 hour infection time points.

[0340] Table 4

[0341]

[0342]

[0343]

[0344]

[0345]

[0346]

[0347]

[0348]

[0349]

[0350]

[0351]

[0352] All eight ribonucleoprotein capture assays analyzed had enhancement of proteins containing canonical RNA binding domains such as RRM and KH domains ( Figure 7d ), which shows the specificity of the co-precipitated RNA-binding proteins.

[0353] Fourteen viral proteins were detected in the HCoV-OC43 ribonucleoprotein complex ( Figure 2b , Figure 7e In particular, the LFQ intensity of viral structural proteins N, M, and S increased more clearly over time in the genomic RNA-hybridization probe I experiment ( Figure 2b). The accessory protein HCoV-OC43 2a, which is inherent to the β coronavirus A system, was also detected and increased over time, indicating that the above protein, whose function is unknown, can also act as an RNA binding protein. RdRP nsp12 and papain-like protease nsp3 also increased along with other nsp confirmed in the experiment. HCoV-OC43 nsp1 was detected in only a very small amount ( Figure 7e ), which indicates the functional spread of nsp1 in both the A and B systems of the beta coronavirus.

[0354] Next, the inventors compared the host factors that form the viral RNA interactors of HCoV-OC43 and SARS-CoV-2. All 107 proteins of the SARS-CoV-2 interactor group, except RBMS1 and DDX3Y ( Figure 2c ), was also detected in the HCoV-OC43 interactome at multiple infection time points, indicating that many of the same host proteins interact with all RNAs of HCoV-OC43 and SARS-CoV-2.

[0355] To identify protected nuclear host factors in the coronavirus RNA interactome, spectral coefficient analysis of the HCoV-OC43 experiment at 36 hpi was used ( Figure 7a ), compared with the no-probe control group ( Figure 2d , FDR<10%) and uninfected probe control group ( Figure 2e Statistical analysis was performed using comparisons of FDR < 5%.

[0356] For HCoV-OC43 probe I and probe II experiments, the inventors confirmed 67 and 70 host proteins, respectively (Table 5).

[0357] Table 5

[0358]

[0359]

[0360]

[0361]

[0362] Thirty-eight proteins were statistically enriched in both probe sets. Gene ontology-defined enrichment analysis showed that these 38 host proteins were associated with transcriptional regulation, RNA processing, and RNA stability control ( Figure 7f). 14 host proteins (CELF1, EIF4H, ELAVL1, FAM120A, FUBP3, IGF2BP3, MATR3, MOV10, NONO, PABPC1, PABPC4, PTBP3, RALY, SND1, and ZC3HAV1) among the 38 common proteins were also found in the SARS-CoV-2 experiment ( Figure 2f ), and therefore is a protected host structural element for betacoronavirus RNA interaction.

[0363] 4. Regulatory environment of the SARS-CoV-2 RNA interactome

[0364] To understand the regulatory significance of the SARS-CoV-2 RNA interactome, the inventors generated a list of "neighboring" proteins known to physically interact with the elements identified in this study. In particular, an interaction network was generated centered on (or seeded with) the nuclear SARS-CoV-2 interactome ( Figure 8a ). Network analysis revealed ribosomal subunits and EIF3 complexes as two network modules that were highly connected to multiple network hubs (e.g., NPM1 and PABPC1). Gene ontology-defined enhanced analysis revealed biological processes related to translation caused by overrepresentation of EIF3 complex subunits and ribosomal proteins that reflect the active translation state of viral mRNPs. Figure 8b ).

[0365] To achieve a deeper functional view of the RNA interactome, the physical interaction network with the SARS-CoV-2 RNA interactome was reconstructed, excluding ribosomal proteins and EIF3 proteins ( Figure 3a ). This analysis identified additional hub proteins such as TRIM25, SQSTM1, and KHDRBS1. Gene ontology-defined enrichment analysis revealed mRNA splicing, mRNA release, mRNA stability, and stress granule assembly ( Figure 3b) and other messenger RNA life cycle, which enlightens such messenger RNA regulators to be selected (co-opted) to assist the virus life cycle. Interestingly, the inventors have also found gene ontology definitions related to viral processes and innate immune responses. From the perspective of intracellular localization, the SARS-CoV-2 RNA interactor group is compared with the cell messenger RNA interactor group to be strengthened by proteins located in paraspeckles and cytoplasmic ribonucleoprotein particles (such as stress granules) ( Figure 3c )(Baltz et al., 2012; Castello et al., 2012). Such observations suggest regulatory mechanisms of viral RNA that are distinct from host mRNA, including activation of host antiviral machinery and sequestration of viral RNA.

[0366] Another approach to determine the regulatory functionality of the SARS-CoV-2 RNA interactome is to investigate its regulatory function in the context of transcriptional responses to viral infection. For example, infected cells recognize non-cellular RNA through multiple cytoplasmic sensors such as RIG-I (DDX58) and MDA5 (IFIH1) and ultimately induce interferons that upregulate interferon-stimulated genes (ISGs) through the JAK-STAT pathway (Sa Ribero et al., 2020). In such studies, aberrant type I / III interferon responses have been reported in SARS-CoV-2 infected cells and animal model systems (Blanco-Melo et al., 2020) and in blood biopsies of COVID-19 patients (Hadj adj et al.), supporting the therapeutic potential of active immune evasion by SARS-CoV-2 and appropriately timed interferon therapy (Sa Ribero et al., 2020).

[0367] To investigate the regulation of the SARS-CoV-2 RNA interactome, the inventors utilized publicly available transcriptome data of cells infected with SARS-CoV-2 (Blanco-Melo et al., 2020). Transcriptome analysis of A549 cells expressing ACE2 identified host factors that were differentially expressed in the SARS-CoV-2 RNA interactome after infection ( Figure 4aIn particular, PARP12, SHFL, CELF1, and TRIM25 were upregulated upon infection. When infected cells were treated with ruxolitinib, an inhibitor of JAK1 and JAK2, the expression of five host factors (MOV10, PARP12, SHFL, TRIM25, and ZC3HAV1) was suppressed ( Figure 4b TRIM25 and PARP12 are part of 62 vertebrate nuclear ISGs (Shaw et al., 2017).

[0368] Interferon-β treatment of normal human bronchial epithelial (NHBE) cells induced PARP12, SHFL and TRIM25 ( Figure 4c Consistently, proteomic analysis of IFN-β treated cells (Kerr et al., 2020) showed upregulation of PARP12, TRIM25, and ZC3HAV1 ( Figure 8c ), showing that all JAK-STAT signaling pathways regulate part of the SARS-CoV-2 RNA interactome. Furthermore, reanalysis of mRNA-seq data from postmortem lung samples of COVID-19 patients (Blanco-Melo et al., 2020) showed upregulation of SHFL and ZC3HAV1 ( Figure 4d ), which emphasizes the physiological relevance of the SARS-CoV-2 ribonucleoprotein in natural human infection with SARS-CoV-2.

[0369] 5. Host factors and functional modules regulating SARS-CoV-2 RNA

[0370] To investigate the effects of host proteins on coronavirus RNA, the inventors performed knockdown experiments and infected Calu-3 cells with SARS-CoV-2 ( Figure 5a and Figure 5b ). Calu-3 cells are human lung epithelial cells used as a model system for various coronavirus infections (Sims et al., 2008). Strategically, the inventors selected a broad range of SARS-CoV-2 RNA interactor groups covering JAK-STAT signaling, mRNA delivery, mRNA stability and translation, as well as the broad functional modules identified above. Knockdown of host factors stimulated by SARS-CoV-2 infection or interferon beta treatment, i.e., PARP12, TRIM25, ZC3HAV1, CELF1, and SHFL, increased viral RNA ( Figure 5c ). The above results suggest that these RNA-binding proteins that directly recognize viral RNA are induced by interferon and JAK-STAT signaling pathways to inhibit coronavirus.

[0371] ZC3HAV1 (ZAP / PARP13) is an ISG known to restrict the replication of multiple RNA viruses, including HIV-1 (Retroviridae), Sindbis virus (Togaviridae), and Ebola virus (Filoviridae) (Goodier et al., 2015). ZC3HAV1 was previously reported to be a protein that recognizes CpG and recruits decay factors to degrade human acquired immunodeficiency virus RNA (Takata et al., 2017). ZC3HAV1 mainly functions as a cofactor of TRIM25, an E3 ubiquitin ligase that promotes antiviral signaling through RIG-I (Choudhury et al., 2020; Gack et al., 2007). Our knockdown results indicate that both ZC3HAV1 and TRIM25 act as antiviral factors against SARS-CoV-2 ( Figure 5c ). The N protein of SARS-CoV was previously thought to inhibit RIG-I activation by interacting with the SPRY domain of TRIM25 (Hu et al., 2017).

[0372] PARP12, a cytoplasmic mono-ADP-ribosylation (\ARylation) enzyme, is also known to have cellular RNA translation blocking (Atasheva et al., 2014; Welsby et al., 2014) activity. ADP ribosyltransferases are evolutionarily ancient tools used in host-pathogen interactions (Fehr et al., 2020). For reference, in order to reverse the activity of the PARP enzyme, the coronavirus nsp3 has a protected macrodomain capable of eliminating ADP-ribose (Fehr et al., 2015). Knockdown of PARP12 and PARP14 showed increased replication of mouse hepatitis virus (MHV) lacking the macrodomain of the A system belonging to the genus β coronavirus (Grunewald et al., 2019), which is consistent with the knockdown results of the present invention ( Figure 5c Based on the RNA interactome data of the present invention, the inventors hypothesize that the RNA binding activity of PARP12 and its role in viral RNA recognition can explain the basic molecular mechanism of antiviral activity against the SARS-CoV-2 transcriptome.

[0373] Other interferon-stimulated RNA binding proteins may also be involved in host defense against SARS-CoV-2. CELF1 (CUGBP1 Elav-like protein family 1) mediates alternative splicing and controls messenger RNA stability and translation (Konieczny et al., 2014). Although CELF1 is required for IFNβ-mediated inhibition of simian immunodeficiency virus (Retroviridae) (Dudaronek et al., 2007), no other associations with viral infection are known. SHFL (Shiftless / RyDEN) is significantly induced during viral infection and interferon treatment and is inhibited by JAK inhibitors (Figure 4s). SHFL inhibits the translation of a variety of RNA viruses including dengue virus (Flaviviridae) and HIV (Retroviridae) (Balinsky et al., 2017; Suzuki et al., 2016; Wang et al., 2019). Under the experimental conditions of the present invention, upregulation of viral RNA was common in CELF1-depleted cells and SHFL-depleted cells, but since the knockdown efficiency of ISGs was low in infected cells, additional experiments were required ( Figure 9b ), perhaps this is because the virus induces the silencing of genes that damage the interferon response.

[0374] In addition to the above-mentioned RNA binding proteins, the present inventors have also confirmed previously undescribed host factors in the context of viral infection. In particular, LARP1 depletion leads to substantial upregulation of viral RNA ( Figure 5c ), which indicates that LARP1 may have antiviral function. LARP1 stabilizes 5′TOP messenger RNA encoding ribosomal proteins and translation factors containing a 5′ terminal oligopyrimidine (5′TOP) motif in the 5′UTR (Philippe et al., 2018). LARP1 also inhibits the translation of 5′TOP messenger RNA in response to metabolic stress in an mTORC1-dependent manner (Hong et al., 2017; Lahr et al., 2017). Little is known about the role of LARP1 in viral infection. Based on the ribonucleoprotein capture and knockdown results of the present invention, it can be considered that LARP1 can directly inhibit viral translation through viral RNA interactions.

[0375] In fact, the SARS-CoV-2 RNA interactome includes specific structural elements of the 40S and 60S ribosomal subunits and translation initiation factors ( Figure 1f -h). Knockdown experiments showed that ribosomal proteins (RPS9 and RPS3) and the translation initiation factor EIF4H can have antiviral activity ( Figure 5c ). EIF4H, together with EIF4B, is a cofactor of the RNA helicase EIF4A (Rogers et al., 2001), and its depletion forms RNA particles (Tauber et al., 2020). Both EIF4H and EIF4B have been confirmed by the nuclear SARS-CoV-2 RNA interactor group ( Figure 1f ). EIF4H also interacts with SARS-CoV-2nsp9 (Gordon et al., 2020), so it will be interesting to investigate the functional consequences of the EIF4H-nsp9 interaction. At the same time, the observations in the present invention suggest that SARS-CoV-2 infection may be closely linked to the regulation of ribosome biogenesis, metabolic rewiring, and global translational control.

[0376] Other translation factors EIF3A, EIF3D and CSDE1 showed proviral effects ( Figure 5c ).

[0377] EIF3A is the RNA binding component of the mammalian EIF3 complex and is evolutionarily conserved along with EIF3B and EIF3C (Masutani et al., 2007). EIF3D is known to interact with the messenger RNA cap and is required for specialized translation initiation (Lee et al., 2016). CSDE1 (Unr) is required for IRES-dependent translation in human rhinovirus (Picornaviridae) and poliovirus (Picornaviridae) (Anderson et al., 2007; Boussadia et al., 2003). In general, the present invention shows that SARS-CoV-2 can recruit EIF3D and CSDE1 to regulate the initiation of cap-dependent translation and IRES-dependent translation of SARS-CoV-2 genomic and subgenomic RNAs, respectively (Lee et al., 2017).

[0378] Finally, unlike the mRNA interactome, the coronavirus RNA interactome is enriched in RNA-binding proteins with KH domains ( Figure 6d and Figure 7b Depletion of FUBP3 (MARTA2) and HDLBP (Vigilin) ​​increases viral RNA levels ( Figure 5c), suggesting that proteins containing KH domains have potential antiviral effects. HDLBP is a protected protein containing 14 KH domains and is associated with viral translation of dengue virus (Ooi et al., 2019). FUBP3 was enriched in all four ribonucleoprotein capture experiments (i.e., SARS-CoV-2 probe I / probe II and OC43 probe I / probe II) ( Figure 2d FUBP3 is a nuclear protein with four KH domains that binds to the 3′UTR of cellular messenger RNA, which regulates the localization of messenger RNA (Blichenberg et al., 1999; Mukherjee et al., 2019). However, its relevance to the life cycle of coronaviruses is still unknown.

[0379] The inventors discovered a broad range of antiviral factors such as TRIM25, ZC3HAV1, PARP12 and SHFL, as well as many RNA binding proteins with unknown functions in the context of viral infection such as LARP1, FUBP3, FAM120A / C, EIF4H, RPS3, RPS9, SND1, CELF1, RALY, CNBP, EIF3A, EIF3D and CSDE1. This protein list reflects the ongoing host-pathogen interaction and is associated with unknown mechanisms of viral life cycle and immune deficiency.

[0380] As with proteins that regulate RNA, it will also be interesting to consider the functionality of "riboregulation" (Hentze et al., 2018) that regulates proteins that interact with RNA. For example, dengue virus uses a subgenomic RNA called flavivirus subgenomic RNA (sfRNA) to sequester TRIM25 (Chapman et al., 2014). The ratio of subgenomic RNA to genomic RNA is an important factor in determining the epidemic potential of dengue virus (Manokaran et al., 2015). In particular, coronaviruses including SARS-CoV-2 produce considerable amounts of noncanonical subgenomic RNA that can act as noncoding decoys that interact with host RNA binding proteins in order to modulate host immune responses (Kim et al., 2020a).

[0381] As of September 2020, there are no effective antiviral agents or vaccines that can prevent the current pandemic, although more than 3,200 human clinical studies for the treatment of COVID-19 are listed (ClinicalTrials.gov). This unmet medical need highlights the inadequate state of knowledge about SARS-CoV-2. Therefore, consideration of redefining antiviral strategies should take precedence over frequent drug repurposing efforts. To date, large-scale and multidisciplinary datasets of viral transcriptome (Kim et al., 2020a), host transcriptional responses (Blanco-Melo et al., 2020), ribosome profiling (Finkel et al., 2020), total proteomics (Bojkova et al., 2020), protein-protein interactions through co-IP (Gordon et al., 2020) and adjacent annotation (St-Germain et al., 2020), phosphoproteomics (Bouhaddou et al., 2020), RNA structure (Lan et al., 2020), whole-genome CRISPR screens (Wei et al., 2020), and direct off-label drug screening (Chen et al., 2020) have provided valuable insights into the basic biology of the novel human coronavirus. Following such efforts, the inventors have proposed a panel of SARS-CoV-2 RNA interactors that provide insights into host-virus interactions that regulate the life cycle of coronaviruses. Data interpretation in the context of publicly available orthogonal information enables the identification of proviral and antiviral protein candidates. It is expected that efforts to generate and integrate system-level data will lead to new strategies for ascertaining the pathogenicity of SARS-CoV-2 and eliminating COVID-19.

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[0471] Zhou, P., Yang, X.-L., Wang, X.-G., Hu, B., Zhang, L., Zhang, W., Si, H.-R., Zhu, Y., Li, B., Huang, C.-L., et al. (2020). A pneumonia outbreak associated with a new coronayirus of probable bat origin. Nature 579, 270-273. Sequence Listing <110> Seoul National University Industry-Academic Cooperation Group <120> RNA interactor group capture scheme and antiviral composition discovered using the scheme <130> PP210083 <150> KR 10-2020-0144787 <151> 2020-11-02 <160> 32 <170> KoPatentIn 3.0 <210> 1 <211> 19 <212> RNA <213> Artificial sequence <220> <223> siEIF3A <400> 1 gagaagacuu ggcgauagu 19 <210> 2 <211> 19 <212> RNA <213> Artificial sequence <220> <223> siEIF3A <400> 2 agacagagau uuauucgua 19 <210> 3 <211> 19 <212> RNA <213> Artificial sequence <220> <223> siEIF3A <400> 3 gaagaaaggc auaaucgau 19 <210> 4 <211> 19 <212> RNA <213> Artificial sequence <220> <223> siEIF3A <400> 4 ucacaaaggu ucuaaauug 19 <210> 5 <211> 19 <212> RNA <213> Artificial sequence <220> <223> siEIF3D <400> 5 guucaagccu aaugaguuu 19 <210> 6 <211> 19 <212> RNA <213> Artificial sequence <220> <223> siEIF3D <400> 6 caagauaaga gguacacaa 19 <210> 7 <211> 19 <212> RNA <213> Artificial sequence <220> <223> siEIF3D <400> 7 agacaaagau cgucggaac 19 <210> 8 <211> 19 <212> RNA <213> Artificial sequence <220> <223> siEIF3D <400> 8 cgaaugagau uugcccaga 19 <210> 9 <211> 19 <212> RNA <213> Artificial sequence <220> <223> siCSDE1 <400> 9 agaccgacgu gacaaauua 19 <210> 10 <211> 19 <212> RNA <213> Artificial sequence <220> <223> siCSDE1 <400> 10 ucaaauagga gauaagguu 19 <210> 11 <211> 19 <212> RNA <213> Artificial sequence <220> <223> siCSDE1 <400> 11 gaaagaaaga uccgucaag 19 <210> 12 <211> 19 <212> RNA <213> Artificial sequence <220> <223> siCSDE1 <400> 12 ggugaggucu auccauuug 19 <210> 13 <211> 19 <212> RNA <213> Artificial sequence <220> <223> siHNRNPM <400> 13 ugaaauggca gucacuuaa 19 <210> 14 <211> 19 <212> RNA <213> Artificial sequence <220> <223> siHNRNPM <400> 14 gaauggaagg cauaggauu 19 <210> 15 <211> 19 <212> RNA <213> Artificial sequence <220> <223> siHNRNPM <400> 15 gaugagaggg ccuuaccaa 19 <210> 16 <211> 19 <212> RNA <213> Artificial sequence <220> <223> siHNRNPM <400> 16 gagauugacg uucgaauug 19 <210> 17 <211> 19 <212> RNA <213> Artificial sequence <220> <223> ikB <400> 17 ggaagggagu cguuacgcu 19 <210> 18 <211> 19 <212> RNA <213> Artificial sequence <220> <223> ikB <400> 18 gaugaaggau ggaguccca 19 <210> 19 <211> 19 <212> RNA <213> Artificial sequence <220> <223> ikB <400> 19 gcaauaaagu ggaagacua 19 <210> 20 <211> 19 <212> RNA <213> Artificial sequence <220> <223> ikB <400> 20 gcacggaaga caagagagu 19 <210> twenty one <211> 19 <212> RNA <213> Artificial sequence <220> <223> siFMR1 <400> twenty one ccaaagaggc ggcacauaa 19 <210> twenty two <211> 19 <212> RNA <213> Artificial sequence <220> <223> siFMR1 <400> twenty two gaugauaaag ggugaguuu 19 <210> twenty three <211> 19 <212> RNA <213> Artificial sequence <220> <223> siFMR1 <400> twenty three caccaaaucg uacagauaa 19 <210> twenty four <211> 19 <212> RNA <213> Artificial sequence <220> <223> siFMR1 <400> twenty four aaagcuaugu gacugauga 19 <210> 25 <211> 19 <212> RNA <213> Artificial sequence <220> <223> siRTCB <400> 25 ggaauuguuc aucgaucua 19 <210> 26 <211> 19 <212> RNA <213> Artificial sequence <220> <223> siRTCB <400> 26 gaacggacac uguuaguac 19 <210> 27 <211> 19 <212> RNA <213> Artificial sequence <220> <223> siRTCB <400> 27 gguguccgcu ugcuaagaa 19 <210> 28 <211> 19 <212> RNA <213> Artificial sequence <220> <223> siRTCB <400> 28 gucaaccgcu cuuccauga 19 <210> 29 <211> 19 <212> RNA <213> Artificial sequence <220> <223> siNUFIP2 <400> 29 aggaaagcua ggcgcaaua 19 <210> 30 <211> 19 <212> RNA <213> Artificial sequence <220> <223> siNUFIP2 <400> 30 gggugauaug cuucggaaa 19 <210> 31 <211> 19 <212> RNA <213> Artificial sequence <220> <223> siNUFIP2 <400> 31 aauuaagccc ugcgagaau 19 <210> 32 <211> 19 <212> RNA <213> Artificial sequence <220> <223> siNUFIP2 <400> 32 auggugaacu aaacgguaa

Claims

1. An antiviral composition, characterized in that: The invention comprises one or more proteins selected from the group consisting of LARP1, FUBP3, FUBP1, FAM120A, FAM120C, EIF4H, RPS3, RPS9, SND1, CELF1, RALY, CNBP, TRIM25, ZC3HAV1, PARP12, PPP1CA, HDLBP, CNBP, TIAL, UPF1 and SHFL as an active ingredient.

2. An antiviral composition, characterized in that: The present invention comprises as an active ingredient a nucleic acid encoding one or more proteins selected from the group consisting of LARP1, FUBP3, FUBP1, FAM120A, FAM120C, EIF4H, RPS3, RPS9, SND1, CELF1, RALY, CNBP, TRIM25, ZC3HAV1, PARP12, PPP1CA, HDLBP, CNBP, TIAL, UPF1 and SHFL.

3. The antiviral composition according to claim 1, characterized in that The above virus is a coronavirus.

4. The antiviral composition according to claim 3, characterized in that The above coronavirus is selected from the group consisting of 229E, OC43, NL63, HKU1, MERS-CoV, SARS-CoV and SARS-CoV-2.

5. An antiviral composition, characterized in that: A recombinant vector comprising a nucleic acid encoding one or more proteins selected from the group consisting of LARP1, FUBP3, FUBP1, FAM120A, FAM120C, EIF4H, RPS3, RPS9, SND1, CELF1, RALY, CNBP, TRIM25, ZC3HAV1, PARP12, PPP1CA, HDLBP, CNBP, TIAL, UPF1 and SHFL.

6. An antiviral composition, characterized in that: An expression inhibitor or activity inhibitor comprising one or more proteins selected from the group consisting of EIF3A, EIF3D, CSDE1, HNRNPM, YBX3, FMR1, RTCB and NUFIP2.

7. The antiviral composition according to claim 6, characterized in that The protein expression inhibitor is selected from the group consisting of microRNAs, small interfering RNAs, short hairpin RNAs, antisense oligonucleotides, CRISPRi and combinations thereof that complementarily bind to the nucleic acid sequence encoding the protein.

8. The antiviral composition according to claim 6, characterized in that The activity inhibitor of the above protein is selected from the group consisting of antibodies, aptamers, small molecule compounds and combinations thereof that can complementarily bind to the above protein.

9. A pharmaceutical composition for preventing or treating coronavirus infection, characterized in that: An antiviral composition comprising the composition according to any one of claims 1 to 8.

10. A method for screening coronavirus inhibitors, characterized in that: include: Step (a), contacting a coronavirus inhibitor candidate substance with cells infected with coronavirus; and Step (b), confirming whether the expression level of one or more proteins selected from the group consisting of EIF3A, EIF3D, CSDE1, HNRNPM, YBX3, FMR1, RTCB and NUFIP2 is reduced compared with a control group not treated with the candidate substance, If the expression level of the above protein decreases, the candidate substance is judged to be a coronavirus inhibitor.

11. A method for establishing a set of RNA interactors, characterized in that: include: Step (a), irradiating a sample containing target RNA and protein with ultraviolet light to induce RNA-protein cross-linking; step (b), denaturation by treatment with deoxyribonuclease; Step (c), using a biotinylated antisense oligonucleotide probe pool to capture the denatured ribonucleoprotein complex in a sequence-specific manner, wherein the biotinylated antisense oligonucleotides contained in the probe pool are oligonucleotides of the same length selected from the starting positions separated by a specified interval in a partially overlapping manner and within the range of 45 nt to 135 nt based on the target ribonucleic acid sequence; step (d), dissociation by on-bead trypsin treatment; as well as Step (e), obtaining information on the interactor group by decomposition product analysis.

12. The method for establishing a set of RNA interactors according to claim 11, characterized in that: The target RNA is the genomic RNA of RNA viruses.

13. The method for establishing a set of RNA interactors according to claim 11, characterized in that: The probe pool comprises a first probe pool that specifically hybridizes with genomic RNA molecules and a second probe pool that hybridizes with both genomic RNA and small guide RNA.

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