A gain-of-function mutation in poxvirus encoded 2'o-methyltransferase: enhancing stability and translational efficiency of viral mRNA

Abstract

The present invention is related to method of preparing a gain-of-function mutation in poxvirus, like buffalopox virus (BPXV) by long-term propagating it in the presence of methyltransferase inhibitor like 3-Deazaneplanocin A (DZNep), which results in generation of viral 2'O-MTAse (VP39) mutant with a single point mutation (M236I). The resultant mutant has the following characteristics: (a) M236I mutation of 2'O-MTAse increases the cap-1 markings on viral mRNA; (b) The 2'O-MTAse mutant virus had hyperactive 2'O-MTAse and exhibited enhanced stability and translational efficiency of its mRNA; (c) The capping (cap1) performed by the mutant viral 2'O-MTAse (VP39) results in higher viral protein production, as compared to the wild type VP39 and (d) Mutant virus replicated at higher viral titer than the wild-type virus.

Description

[0001] A gain-of-function mutation in poxvirus encoded 2’O-methyltransferase: Enhancing stability and translational efficiency of viral mRNA

[0002] Field of the Invention:

[0003] Long-term propagation of buffalopox virus under selective pressure of a drug (that target methytransferase) resulted in generation of viral variant with a single point mutation in its VP39 protein (2’0-MTAse). The mutant viral mRNA exhibited higher stability and translational efficiency which allowed the virus to replicate much faster as compared to the wild type virus. The mutant 2’0-MTAse could be leveraged for diverse biotechnological applications including stability and translation enhancements for mRNA-based vaccines and therapeutics, as well as scaling up virus manufacturing.

[0004] Background of the Invention:

[0005] Poxvirus-encoded VP39, also known as the 2'0 methyltransferase enzyme, plays a critical role in the post-transcriptional modification of mRNA, specifically in the methylation of the 2'-hydroxyl group of the ribose at the first nucleotide of the mRNA cap structure (Cap 1). This modification is essential for the stability and translational efficiency of mRNA, contributing significantly to the effectiveness of viral gene expression.

[0006] In the context of therapeutics and vaccines, Cap 1 modification has gained significant attention for its role in enhancing the immunogenicity and stability of mRNA-based treatments. The VP39 enzyme's ability to catalyze this methylation process makes it a valuable tool in the design of mRNA vaccines and therapeutics, where precise control over mRNA stability and translation is crucial for effective outcomes. However, previous studies have shown that the wild-type VP39 exhibits relatively low catalytic efficiency, which compromises mRNA stability and expression efficiency. This limitation often necessitates higher doses of mRNA, which can lead to increased cytotoxicity.

[0007] In this invention, we discovered a particular mutation M236I in VP39 increases its catalytic activity, highlighting the enzyme's potential in optimizing mRNA-based interventions. The mutant VP39 modified GFP transcripts demonstrated sustained stability and yielded increased fluorescence signals than VP39 WT modified mRNA from 24 to 120 h post transfection in HeLa cells. By enhancing mRNA stability and translational efficiency, this mutated form of VP39 can be a promising candidate for the development of next-generation mRNA vaccines. Besides we demonstrated that overexpression of mutant VP39 significantly enhances Buffalopox virus titre as compared to the wild-type VP39. The VP39 is commonly present in all the poxviruses including monkeypox virus. Therefore, the overexpression of the mutant VP39 significantly enhances the yield of poxvirus in the target cells. Therefore, this overall strategy can economize the cost of the production of certain poxvirus vaccines.

[0008] Objectives of the Invention:

[0009] • The primary objective was to investigate the role of 2’0-MTAse in buffalopox virus replication and selection of viral mutant with increased replication capability.

[0010] • The secondary objective was to map the mutation(s) in viral genome responsible for increased virus replication and their functional analysis.

[0011] • The third objective was to exploit the mutant form of VP39 to modify viral mRNA for stability and translational efficiency.

[0012] Summary of the Invention In one aspect the present invention provides successful cloning of mutant VP39 enzyme in pDEST40 and pDEST17 vectors for the purpose of overexpression studies and enzyme (VP39) isolation for mRNA modification respectively. In second aspect, the present invention suggests that mutant VP39 with a single point mutation (M236I) enhances the stability and translational efficiency of viral mRNA in target cells. In third aspect mutant VP39 overexpression enhances the yield of Buffalopox virus in the target cells and hence this strategy economise the cost of viral, particular poxvirus vaccine production in the cell culture system.

[0013] Detailed Description of the Drawings:

[0014] The following drawings form part of the present specification and are included to further illustrate aspects of the present invention. The invention may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein. It is to be noted, however, that the appended drawings illustrate only typical embodiments of the present subject matter, and are therefore, not to be considered for limiting of its scope, for the subject matter may admit to other equally effective embodiments.

[0015] Figure 1: Schematic illustration of SAM cycle. Methyltransferases catalyses SAM for target methylation leading to SAH buildup which is promptly converted to homocysteine by SAH hydrolase. DZNep blocks SAH hydrolase activity and prevents the clearance of SAH, which in turn suppresses methyltransferase activity. Methionine synthetase further converts homocysteine to methionine. Finally, methionine adenosyl transferase converts the methionine back to SAM.

[0016] Figure 2: In vitro antiviral efficacy of DZNep against BPXV: Vero cells, in triplicates, were infected with BPXV at MOI of 0.1 in the presence of indicated concentrations of DZNep or DMSO. The quantity of infectious virus particles present in the cell culture supernatants after 72 hours post-infection (hpi) were determined using plaque assay

[0017] Figure 3: Selection of DZNep-resistant BPXV mutants. (A) Vero cells were infected with BPXV at MOI of 0.01 in the presence of either DZNep or DMSO. The progeny virus particles released in the supernatant was harvested either at 48-72 hpi or when -75% cells exhibited CPE. Fifty (50) such sequential passages were made. Thereafter, Vero cells, in triplicate, were infected with P0, P50-DZNep or P50-Control passaged viruses (BPXV) at MOI of 0.1 in the presence of either DZNep or DMSO and the progeny virus particles released in the supernatant at 72 hpi were quantified by plaque assay. Values are means ± SD and representative of the result of at least 3 independent experiments. (B) The identification of resistance-associated mutation in VP39 from whole genome sequencing of DZNep passaged BPXV variants.

[0018] Figure 4: Cloning of VP39 wild-type and mutant in pDONR221. (A, B) Schematic illustration of pDONR221 and VP39 cloned pDONR221 vector respectively. (C, D) Simulated and experimental digestion pattern of pDONR221 (Lane 1), and VP39 wildtype (lane 2) and VP39 mutant (lane 3) cloned in pDONR221 using restriction enzymes EcoRV and Hpal. (Ladder - Gene ruler 1 Kb) Figure 5: Cloning of VP39 wild-type and mutant in pcDNA DEST40 vector. (A, B) Schematic illustration of pcDNA DEST40 and VP39 cloned pcDNA DEST40 vector respectively. (C, D) Simulated and experimental digestion pattern of pcDNA DEST40 (Lane 1), and VP39 wild-type (lane 2) and VP39 mutant (lane 3) cloned in pcDNA DEST40 using restriction enzymes Ndel and Hpal. (Ladder - Generuler 1 Kb)

[0019] Figure 6: Cloning of VP39 wild-type and mutant in pcDNA DEST17 vector. (A, B) Schematic illustration of pcDNA DEST17 and VP39 cloned pcDNA DEST17 vector respectively. (C, D) Simulated and experimental digestion pattern of pcDNA DEST 17 (Lane 1), and VP39 wild-type (lane 2) and VP39 mutant (lane 3) cloned in pcDNA DEST 17 using restriction enzyme Kpn2I. (Ladder - Gene ruler 1 Kb)

[0020] Fig. 7: (A) NmSEER V2.0 prediction for 2’0-Me sites in BPXV M gene transcript. The M gene region covered by the primers designed for qRT-PCR were selected and uploaded online (http: / / www.rnanut.net / nmseer-v2 / ), and resultant sites are shown in tabular view.

[0021] (B) RTL-P assay: Vero cells in triplicates were infected with indicated BPXV variant at MOI of 5 followed by washing with PBS 5 times and supplementation of fresh DMEM at 1 hpi. Cells were scraped at 18 hpi and RNA were isolated using TRI reagent followed by cDNA preparation under low and high dNTP concentrations and quantified by qRT- PCR using primers directed towards BPXV M gene. Ct values were normalized with the P-actin housekeeping control gene and relative fold change was calculated by the AA Ct method.

[0022] (C) RNA IP assay: Vero cells were infected with BPXV at an MOI of 5. At 6 hours postinfection (hpi), the cells were treated with DZNep or DMSO. At 16 hpi, cell lysates were prepared following the procedure outlined for ChIP assay (as described in the materials and methods section). The clarified cell lysates were then incubated with a-m2A (reactive antibody), a-ERK (nonreactive antibody), or an equivalent volume of IP buffer (as beads control), followed by incubation with Protein A Sepharose slurry. Subsequently, the beads were washed five times in IP buffer. To reverse the cross -linking, the complexes were incubated with Proteinase K. Finally, the reaction mixtures were centrifuged, and the supernatant was used for cDNA preparation and quantification of BPXV RNA (Af gene) by qRT-PCR. Error bars indicate SD. Values are means ± SD and representative of the result of at least 3 independent experiments. Pair- wise statistical comparisons were performed using the Student’s t test (ns = non- significant, * = P<0.05, ** = P<0.01, *** = P<0.001).

[0023] Figure 8: VP39 overexpression implications in BPXV titres. HeLa cells were transfected with VP39_WT or VP39_mutant encoded pDEST40 plasmid constructs in triplicates, followed by BPXV infection after 48 h at 5 MOI. After 42 hpi supernatants were harvested and infectious progeny viruses produced in the supernatant were quantified using plaque assay in Vero cells. Error bars indicate SD. Values are means ± SD and representative of the result of at least 3 independent experiments. Pair-wise statistical comparisons were performed using the Student’s t test (ns = non-significant, * = P<0.05).

[0024] Figure 9: In vitro transcription and time-course analysis of GFP fluorescence. GFP transcripts either uncapped or capped with VP39_WT or VP39_mut were transfected in HeLa cells in triplicates for 12 h and fluorescence were measured using Spectramax i3x each day post transfection till day 5 in a well scan mode at LVLm = 472 / 512 nm. The measured fluorescence were normalized from the fluorescence obtained from unmodified mRNA.

[0025] Error bars indicate SD. Values are means ± SD and representative of the result of at least 3 independent experiments. Pair-wise statistical comparisons were performed using the Student’s t test (ns = non-significant, ** = P<0.01).

[0026] SUMMARY OF THE PRESENT INVENTION

[0027] 1. Inhibition of methyltransferase (MTase) activity leads to defective formation of the 5 ’-cap of buffalopox virus (BPXV) mRNA, which fails to interact with eIF4E, eventually resulting in abrogation of protein translation. Therefore, MTAse serve as a drug target against poxvirus.

[0028] 2. Long-term propagation of BPXV in the presence of methyltransferase inhibitor DZNep resulted in the generation of viral 2’0-MTAse (VP39) mutant (M236I) with following characteristics- a. M236I mutation of 2’ O-MTAse increases the cap- 1 markings on viral mRNA; b. The mutant virus had hyperactive 2’0-MTAse and exhibited enhanced stability and translational efficiency of its mRNA; c. The capping (capl) performed by the mutant viral 2’0-MTAse (VP39) results in higher viral protein production, as compared to the wild type VP39; d. The mutant virus replicates at higher viral titer than the wild-type virus

[0029] 3. Taken together, the mutant 2’0-MTAse selected through resistance evolution mechanism could be leveraged for diverse biotechnological applications including stability and translation enhancements for mRNA-based vaccines and therapeutics, as well as scaling up virus manufacturing.

[0030] Detailed Description of the Invention:

[0031] MATERIALS AND METHODS

[0032] Cells and viruses

[0033] Vero cells derived from African green monkey, available at the National Centre for Veterinary Type Cultures (NCVTC), Hisar were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with antibiotics and 10% foetal calf serum. Vero cell adapted BPXV (Accession Number, VTCC-AVA90) was available in our lab. Plaque assay was used to quantify BPXV titres and measured as plaque forming unit per millilitre (PFU / ml).

[0034] Cell Culture

[0035] Vero cell line was available in the lab were available at National Centre for Veterinary Type Cultures (NCVTC), Hisar. Cells were grown in Dulbeco Modified Essential Medium (DMEM) supplemented with 10% fetal calf serum (FBS) (Sigma, St. Louis, USA) and antibiotics solution (Penicillin-Streptomycin).

[0036] In vitro antiviral efficacy

[0037] Vero cells were infected with BPXV variants in triplicates at 0.1 MOI (multiplicity of infection) in the presence of 3-Deazaneplanocin A (DZNep) using concentration 1.85 pg / ml or 0.05% DMSO. After 72 hours post infection (hpi) progeny viruses released in supernatant were quantified by plaque assay. Reverse Transcription under Low dNTP conditions followed by PCR (RTL-P assay)

[0038] The RTL-P assay was performed according to previously defined protocol. Briefly, confluent monolayers of Vero cells in 24 well plate were infected with either BPXV- P50-Control or BPXV-P50-DZNep for 1 hr followed by 5 times washing with PBS and supplemented with fresh serum free DMEM. At 12 hpi, cells were harvested and total RNA was isolated using TRI reagent. For cDNA library preparation, 800 ng of RNA was taken and divided into two groups of low or high dNTPs. For low dNTPs stock dNTP (10 mM) was diluted to 1: 1000 in nuclease free water, while for high dNTP, stock lOmM concentration was used according to the manufacturer’s protocol (Fermentas, Hanover, USA). Oligo dT was utilized for gene specific library preparation followed by quantification of BPXV M gene in both groups using qRT-PCR. P-actin (house-keeping gene) was utilized for background normalization. RTP-L efficiency was calculated as mentioned in the protocol.

[0039] RNA immuno-precipitation (RNA-IP) assay

[0040] To evaluate the interaction between BPXV mRNA and eIF4E, and BPXV mRNA and m2A specific antibody, RNA-IP assay was performed as described previously. Briefly, confluent monolayers of Vero cells in triplicates, were infected with BPXV WT or P50 variants at 5 MOI followed by washing with PBS at 1 hpi. Cells were supplemented with fresh DMEM and DZNep (1.85 pg / ml) or vehicle control was added at 3 hpi. At 12 hpi cells were treated with 1% formaldehyde to covalently cross-link the interacting proteins and nucleic acid. After 10 min crosslinking was stopped using 125 mM glycine followed by ice-cold PBS washing. The cell lysates were prepared in immunoprecipitation buffer i.e. 150 mM NaCl, 50 mM Tris-HCl [pH 7.5], 5 mM EDTA, 0.5% NP-40, 1% Triton X- 100 in addition to protease and phosphatase inhibitor cocktail. Thereafter, the cell lysates were sonicated in a Qsonica Sonicator Q500 (Qsonica, Newtown, CT, USA) using parameters - 6 pulse; 15 sec each; amplitude 40%. The sonicated lysates were centrifuged at 12000 g for 10 min and clarified supernatants were mixed with 10 units of RiboLock RNase Inhibitor (Thermo Scientific, USA) and then incubated with m2A specific antibody. a-MNKl was taken as nonreactive antibody control and equivalent volume of IP buffer was taken as beads control. After 45 min, 40 pL (5 ng / pL) of Protein A Sepharose® slurry, prepared as per the instructions of the manufacturer (Abeam, USA) was incubated with each reaction at 4 °C on a rotary platform overnight. Using IP buffer the beads were then washed 5 times and crosslinking was reversed by using 20 mg / ml Proteinase K and incubating at 56 °C for 40 min. The reaction mixtures were then centrifuged at 12000 g for 1 min and RNA was isolated from the supernatant. Following that cDNA were prepared and BPXV M gene were quantified in each group using qRT- PCR and normalized to input RNA.

[0041] VP39 overexpression studies

[0042] The VP39 variants were initially cloned into the pDONR221 vector and subsequently transferred to the pDEST14 vector using GATEWAY cloning technology (Invitrogen), following the manufacturer's protocol. The cloned constructs were then transfected into HeLa cells at equimolar concentrations using the Lipofectamine transfection reagent.

[0043] At 12 hours post- transfection, the transfection reagent was removed from the cells and replaced with fresh DMEM. After 48 hours, the cells were infected with BPXV at an MOI of 5 for 1 hour. Following infection, the cells were extensively washed to remove any unattached virions, and fresh DMEM was added at 1 hour post-infection (hpi).The supernatants were harvested at 42 hpi for titration of infectious progeny virions using a plaque assay.

[0044] In vitro transcription and modification using VP39

[0045] The VP39_WT and VP39_mut constructs, initially cloned into the pDONR221 vector, were transferred to the pDEST17 vector using GATEWAY cloning for bacterial expression. Following this, the constructs were transformed into chemically competent DH5a E. coli cells. After selection on ampicillin plates, successful clones were propagated, isolated, and further transformed into chemically competent BL21-A1 E. coli cells. The transformed BL21-A1 cells were selected on ampicillin plates and propagated until an optical density (GD600) of 0.6 was reached. Induction of protein expression was achieved by adding 0.2% L-arabinose. Three hours post-induction, bacterial pellets were harvested and resuspended in Xtractor lysis buffer (Takara), supplemented with Benzonase nuclease, lysozyme, and an EDTA-free protease inhibitor cocktail. The resuspended cultures were lysed using a probe sonicator at 40% power, employing a 10- second start and pause cycle for a total of 60 seconds. Cell debris was then removed by centrifugation at 12,000 g for 30 minutes.

[0046] The clarified supernatant were incubated with Ni-NTA agarose beads (Qiagen) and loaded on polypropylene tubes (Qiagen) to remove non-specific proteins, followed by washing with His-select washing buffer and eluting using His-select elution buffer (Sigma Aldrich). The isolated proteins were visualized by resolving them on an 8% SDS- PAGE and probing with anti-His antibody.

[0047] For buffer exchange and protein concentration, Amicon Ultra Filters (30 kDa MWCO) were utilized, and the proteins were stored at -20°C in a storage buffer composed of 20 mM Tris-HCl, 100 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 0.1% Triton X-100, 50% glycerol, pH 8.0, to maintain enzymatic activity. Protein concentrations were measured using the Qubit Protein Assay Kit (Invitrogen). Unless otherwise stated all protein isolation procedures were performed at 4°C.

[0048] The EGFP-EC3 plasmid (Plasmid #11546, Addgene) was used as a template for amplification. A forward primer containing the T7 promoter and Kozak sequences at the 5' end, and a reverse primer with a T30 sequence at the 3' end, were used for the amplification. The resulting amplicons were purified using the Monarch PCR Cleanup Kit (NEB). Subsequently, mRNA constructs were synthesized by in vitro transcription (IVT) using the HiScribe T7 High Yield RNA Synthesis Kit (NEB), following the manufacturer's protocol.

[0049] The linear RNA was treated with DNase I and subsequently purified using the MEGAclear Transcription Clean-Up Kit (Thermo Fisher Scientific). The yield of the IVT was determined using the Qubit RNA Assay Kit (Invitrogen). The purified RNA was then capped with the 3'-O-Me-m7G(5')ppp(5') anti-reverse cap analog (NEB) and divided into two groups of 10 pg each for cap-1 modification, utilizing either VP39 WT or VP39_mut.The capping reaction was conducted for 1 hour at 37°C, incubating equimolar concentrations of the VP39 WT and VP39_mut variants in a capping buffer containing 50 mM Tris-HCl, 5 mM KC1, 1 mM MgC12, 1 mM DTT, pH 8.0. Following the capping reaction, the capped mRNAs were purified again using the MEGAclear Transcription Clean-Up Kit (Thermo Fisher Scientific) and quantified using the Qubit RNA Assay Kit (Invitrogen).

[0050] Time-Course analysis of GFP fluorescence in HeLa Cells following VP39 capping modifications

[0051] For GFP time-course experiment, HeLa cells were cultured in FluoroBrite DMEM (Invitrogen) supplemented with 10% FBS, L-Glutamine and antibiotic-antimycotic solution. For transfection HeLa cells were cultured in 96 well black-walled, clear-bottom plate (Costar). After 24 hours, 400 ng of mRNAs — either uncapped or capped with VP39 WT or VP39_mut — were encapsulated using Lipofectamine MessengerMAX (Invitrogen) according to the manufacturer’s protocol. Transfection was carried out in FluoroBrite DMEM supplemented with L- Glutamine for 12 hours, after which the transfection media was replaced with fresh FluoroBrite DMEM supplemented with L- Glutamine.

[0052] Fluorescence was measured using a Spectramax i3x (Molecular Devices) with excitation / emission wavelengths set at 472 / 512 nm in a well-scan mode, utilizing 30 dots per well and 200 flashes per read. The fluorescence readings were collected from the bottom of the wells and normalized by subtracting the fluorescence from wells transfected with uncapped mRNA.

[0053] RESULTS

[0054] DZNep treatment suppresses BPXV replication

[0055] The role of DZNep in suppressing the SAM cycle is depicted in Fig. 1. To evaluate the antiviral efficacy of DZNep, we performed the antiviral assay by infecting Vero cells with 0.1 MOI BPXV in the presence of 3 fold dilutions of DZNep from non-cytotoxic concentration (5 pM). The plaque assay of supernatant harvested at 96 hpi revealed dosedependent antiviral response (Fig. 2) with maximum virus suppression of 2.7 log 10 pfu / ml.

[0056] Selection of potential DZNep-resistant mutants under long-term in vitro culture

[0057] For the investigation of the possibility of potential emergence of drug-resistant virus variants under long term selection pressure, we sequentially passaged the BPXV for up to 50 times in the presence of either DZNep or vehicle control (DMSO). At passage 50 (P=50), the susceptibility of DZNep-passaged (BPXV-P50-DZNep) and controlpassaged (BPXV-P50-control) viruses to DZNep treatment was evaluated by titrating the infectious progeny virus present in the supernatant by plaque assay.

[0058] As illustrated in Fig. 3A, DZNep treatment suppressed the replication of both BPXV WT as well as BPXV-P50-control variants by approximately 2 log 10 pfu / ml compared to the DMSO treatment. However, the BPXV-P50-DZNep variant demonstrated remarkable resistance to DZNep treatment, with no significant difference in virus titres observed between the DMSO-treated and DZNep-treated groups, suggesting the acquisition of DZNep resistance.

[0059] Next, we performed whole genome sequencing of passaged variants and found resistance-associated mutation in DZNep passaged BPXV variants (Fig. 3B).

[0060] Mutant VP39 marks significantly more 2’O-methylation over viral transcripts

[0061] Although the vaccinia encoded VP39 is widely considered as a cap 1 modifier, previous studies have demonstrated that Trypanosoma protozoa encoded TbMT48 and TbMT57 MTases, which is sequence and structurally similar to VP39, modifies cap at position +2, +3 and +4 positions. Therefore, this led us to speculate that VP39 might 2’O-methylate at positions other than cap 1. Using NmSEER V2.0 online webserver, we first predicted the sites for 2’O-methylation in BPXV M gene region covered by our qRT-PCT primers. Data shown in Fig. 7A indicated 13 such sites. Next, we conducted RTL-P assay (reverse transcription at Low dNTP concentrations followed by PCR) to assess the capping efficiencies of VP39 variants using a previously defined protocol and found 0.10 and 0.16 fold of relative RT efficiency for BPXV-P50-DZNep compared to WT and P50- control variant respectively (Fig. 7B), suggesting higher 2’O-methylation by VP39_mut encoded by P50-DZNep variant. The was also supported by RNA-IP assay using m2A specific antibody (Fig. 7C). Notably, the m2A modification in P50-DZNep variant was unsusceptible to DZNep treatment as evidenced by significant m2A markings regardless of DZNep treatment. Taken together, these data suggest that RAM augmented VP39 in higher capping efficiency besides conferring resistance to DZNep treatment.

[0062] Mutant VP39 overexpression significantly increased the BPXV titres compared to

[0063] WT variant Strategies aimed at increasing viral titres are crucial for enhancing the output of the vaccine manufacturing process. Most of these approaches involve manipulating the host genome to elevate viral titres; for a comprehensive review, see Rodrigues, A.F., et al, 2015. This prompted us to overexpress variants of the VP39 protein in HeLa cells to evaluate their impact on viral replication.

[0064] For this, we cloned the wild-type VP39 (VP39 WT) and the VP39 DZN variant into the pDEST40 vector using GATEWAY cloning (Fig. 4, 5). HeLa cells were then transfected with these constructs and subsequently infected with BPXV at a multiplicity of infection (MOI) of 5, 24 hours post-transfection. Following extensive washing one hour postinfection (hpi), the cells were replenished with serum-free DMEM, and supernatants were collected at 42 hpi to titrate infectious progeny viruses using a plaque assay.

[0065] The data presented in Fig. 8 indicate that VP39 DZN significantly enhanced BPXV titres by 164% compared to mock-transfected HeLa cells and by 138% compared to HeLa cells transfected with VP39 WT. These findings suggest the potential application of VP39 DZN in vaccine production.

[0066] GFP mRNA with cap-1 modified from mutant GFP demonstrates higher translation compared to WT variant

[0067] To investigate the differential effects of VP39 variant-modified mRNAs, we first cloned the VP39 WT and _mut variants into the pDEST 17 vector using GATEWAY cloning (Fig. 6) and isolated the proteins through Ni-NTA affinity chromatography. Given that VP39 is a known cap-1 modifier, we initially performed in vitro transcription (IVT) of Green Fluorescent Protein (GFP) followed by cap-0 modification using ARCA. Subsequently, the cap-0 transcripts were incubated with equimolar concentrations of either VP39 WT or VP39 DZN for one hour. After purifying the transcripts, we transfected HeLa cells with these mRNA constructs using Lipofectamine messengerMAX for 12 hours followed by supplementing with fresh DMEM supplemented with L-Glutamine. We conducted a time-course study of GFP production over a 120-hour period and normalized the relative fluorescence intensity to uncapped mRNA. As shown in Fig. 9, the GFP transcripts modified by VP39_mut demonstrated stable expression levels up to day 5, while the expression from VP39 WT-modified transcripts gradually decreased. Notably, the expression levels were 3.03 fold higher at 24 hours post-transfection which further increased to 27.90 fold at 120 h. These results strongly indicate that VP39 DZN-mediated modifications significantly enhance mRNA stability and expression in an unprecedented manner.

Claims

AMENDED CLAIMS received by the International Bureau on 11 June 2025 (11 .06.2025)We Claim1 . A method of producing a mutant pox virus encoding a mutant 2'0-methyltransferase(2'0-MTAse) / VP39 protein comprising the M236I amino acid substitution, wherein the method comprises serial passaging of the pox virus in the presence of a methyltransferase inhibitor.

2. The method of claim 1 , wherein the methyltransferase inhibitor is 3-DeazaneplanocinA (DZNep).

3. A mutant 2'0-methyltransferase (2'0-MTAse) / VP39 protein from poxvirus comprising the M236I amino acid substitution.

4. The mutant 2'0-methyltransferase (2'0-MTAse) / VP39 protein of claim 3, wherein said mutant: a) increases the cap-1 markings on viral mRNA compared to wild-type VP39 b) confers enhanced stability and translational efficiency of modified transcripts; and c) enhances viral titers as compared to the wild-type virus.

5. A recombinant nucleic acid construct encoding the mutant 2'0-methyltransferase(2'0-MTAse) / VP39 protein of claim 1 .

6. A recombinant poxvirus comprising the M236I mutation in 2'0-methyltransferase, wherein said virus: a) replicates at higher titre than wild-type under identical conditions; and b) generates viral mRNA with increased cap-1 methylation.

7. A method for producing a stabilized mRNA, comprising: a) synthesizing an mRNA transcript encoding a target protein; and b) contacting said transcript with the mutant VP39 protein of claim 5 under conditions permitting cap-1 methylation, thereby generating a modified mRNA exhibiting >3-fold higher translational efficiency compared to wild-type VP39- modified mRNA.

8. The method of claim 7, wherein the target protein is a vaccine antigen or therapeutic protein.

9. A method for enhancingviral vaccine production, comprising:a) transfecting host cells with the recombinant nucleic acid construct of claim 5; b) infecting said cells with viruses; and c) culturing the cells.[0001][0002]STATEMENT UNDER ARTICLE 19 (1 )[0003]We herewith submit amended claims under Article 19(1) with respect to the International Search Report and the written opinion of the International Searching authority, with mailing date of 19thApril 2025.[0004]The International Searching Authority is requested to take the amended claims on record.[0005]A) Amendment under Article 19[0006]Pursuant to Article 19, we wish to amend the claims. A copy of the amended claims and a marked- up copy of claims are enclosed herewith.[0007]B) Statement of Article[0008]The present set of claims currently on file is now being replaced by the amended set of claims enclosed herewith. The amended set of claims forms the basis of further proceedings.[0009]It is stated that all the amended claims are within the scope of the originally filed patent specification and no new matter has been added.[0010]The ISA is requested to take the enclosed formal comments on record.

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