BPR3P0128, a non-nucleoside RNA-dependent RNA polymerase inhibitor, inhibits SARS-CoV-2 variants of concern and exerts synergistic antiviral activity in combination with remdesivir.

Viral RNA-dependent RNA polymerase (RdRp), a highly conserved molecule in RNA viruses, has recently emerged as a promising drug target for broad-acting inhibitors. Through a Vero E6-based anti-cytopathic effect assay, we found that BPR3P0128, which incorporates a quinoline core similar to hydroxychloroquine, outperformed the adenosine analog remdesivir in inhibiting RdRp activity (EC50 = 0.66 µM and 3 µM, respectively). BPR3P0128 demonstrated broad-spectrum activity against various severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern. When introduced after viral adsorption, BPR3P0128 significantly decreased SARS-CoV-2 replication; however, it did not affect the early entry stage, as evidenced by a time-of-drug-addition assay. This suggests that BPR3P0128's primary action takes place during viral replication. We also found that BPR3P0128 effectively reduced the expression of proinflammatory cytokines in human lung epithelial Calu-3 cells infected with SARS-CoV-2. Molecular docking analysis showed that BPR3P0128 targets the RdRp channel, inhibiting substrate entry, which implies it operates differently-but complementary-with remdesivir. Utilizing an optimized cell-based minigenome RdRp reporter assay, we confirmed that BPR3P0128 exhibited potent inhibitory activity. However, an enzyme-based RdRp assay employing purified recombinant nsp12/nsp7/nsp8 failed to corroborate this inhibitory activity. This suggests that BPR3P0128 may inhibit activity by targeting host-related RdRp-associated factors. Moreover, we discovered that a combination of BPR3P0128 and remdesivir had a synergistic effect-a result likely due to both drugs interacting with separate domains of the RdRp. This novel synergy between the two drugs reinforces the potential clinical value of the BPR3P0128-remdesivir combination in combating various SARS-CoV-2 variants of concern.

A Marine Natural Product, Harzianopyridone, as an Anti-ZIKV Agent by Targeting RNA-Dependent RNA Polymerase.

The Zika virus (ZIKV) is a mosquito-borne virus that already poses a danger to worldwide human health. Patients infected with ZIKV generally have mild symptoms like a low-grade fever and joint pain. However, severe symptoms can also occur, such as Guillain-Barré syndrome, neuropathy, and myelitis. Pregnant women infected with ZIKV may also cause microcephaly in newborns. To date, we still lack conventional antiviral drugs to treat ZIKV infections. Marine natural products have novel structures and diverse biological activities. They have been discovered to have antibacterial, antiviral, anticancer, and other therapeutic effects. Therefore, marine products are important resources for compounds for innovative medicines. In this study, we identified a marine natural product, harzianopyridone (HAR), that could inhibit ZIKV replication with EC50 values from 0.46 to 2.63 µM while not showing obvious cytotoxicity in multiple cellular models (CC50 > 45 µM). Further, it also reduced the expression of viral proteins and protected cells from viral infection. More importantly, we found that HAR directly bound to the ZIKV RNA-dependent RNA polymerase (RdRp) and suppressed its polymerase activity. Collectively, our findings provide HAR as an option for the development of anti-ZIKV drugs.

Dicer-Like Protein 4 and RNA-Dependent RNA Polymerase 6 Are Involved in Tomato Torrado Virus Pathogenesis in Nicotiana benthamiana.

Tomato torrado virus (ToTV) is a type member of the Torradovirus genus in the Secoviridae family known to cause severe necrosis in susceptible tomato varieties. ToTV also infects other Solanaceae plants, including Nicotiana benthamiana, where it induces distinctive disease symptoms: plant growth drop with the emergence of spoon-like malformed systemic leaves. Virus-induced post-transcriptional gene silencing (PTGS) is significant among plant defense mechanisms activated upon virus invasion. The PTGS, however, can be counteracted by suppressors of RNA silencing commonly found in viruses, which efficiently disrupt the antiviral defense of their host. Here, we addressed the question of PTGS antiviral activity and its suppression in N. benthamiana during ToTV infection-a phenomenon not described for any representative from the Torradovirus genus so far. First, we showed that neither the Vp26-a necrosis-inducing pathogenicity determinant of ToTV-nor other structural viral proteins limited the locally induced PTGS similar to p19, a well-characterized potent suppressor of RNA silencing of tombusviruses. Moreover, by employing wild-type and transgenic lines of N. benthamiana with suppressed Dicer-like 2 (DCL2), Dicer-like 4 (DCL4), Argonaute 2 and RNA-dependent RNA polymerase 6 (RDR6) proteins, we proved their involvement in anti-ToTV defense. Additionally, we identified DCL4 as the major processor of ToTV-derived siRNA. More importantly, our results indicate the essential role of the Suppressor of Gene Silencing 3 (SGS3)/RDR6 pathway in anti-ToTV defense. Finally, we conclude that ToTV might not require a potent RNA silencing suppressor during infection of the model plant N. benthamiana.

Characterization of a novel endornavirus isolated from the phytopathogenic fungus Rhizoctonia solani.

Rhizoctonia solani endornavirus 8 (RsEV8) was isolated from strain XY175 of Rhizoctonia solani AG-1 IA. The full-length genome of RsEV8 is 16,147 nucleotides (nt) in length and contains a single open reading frame that encodes a large polyprotein of 5227 amino acids. The polyprotein contains four conserved domains: viral methyltransferase, putative DEAH box helicase, viral helicase, and RNA-dependent RNA polymerase (RdRp). RsEV8 has a shorter 3'-UTR (58 nt) and a longer 5'-UTR (404 nt). A multiple sequence alignment indicated that the RdRp of RsEV8 possesses eight typical RdRp motifs. According to a BLASTp analysis, RsEV8 shares 39.31% sequence identity with Rhizoctonia cerealis endornavirus-1084-7. Phylogenetic analysis demonstrated that RsEV8 clusters with members of the genus Betaendornavirus.

Favipiravir, an antiviral drug, in combination with tamoxifen exerts synergistic effect in tamoxifen-resistant breast cancer cells via hTERT inhibition.

Tamoxifen (TAM) is one of the most successful treatments for breast cancer; however, TAM resistance continues to be a significant barrier. TAM resistance has been reported to be associated with increased expression of human telomerase reverse transcriptase (hTERT). This enzyme shares structural similarity with RNA-dependent RNA polymerase (RdRp) enzyme of RNA viruses, suggesting that RdRp inhibitors may also inhibit hTERT. Favipiravir (FAV) is an antiviral drug that inhibits RdRp of RNA viruses. Thus, we propose that FAV may also elicit an antitumor effect by suppressing hTERT. This study aimed to investigate the effect of FAV and TAM on TAM-resistant breast cancer (TAMR-1). The cell viabilities were determined. The levels of CDK1/ hTERT, in addition to regulators of hTERT-targeted signaling pathways were measured. Apoptosis, migration, and cell cycle distribution were also determined. Our data revealed that the combination of TAM and FAV suppressed cell proliferation synergistically (CI < 1) and resulted in a significant change in cell migration and apoptosis. Indeed, this was associated with reduced levels of hTERT and CDK1 and shift in the cell cycle distribution. Our findings suggest that the TAM/FAV combination exhibits synergistic effects against TAMR-1 human breast cancer cells by targeting hTERT.

VP1 codon deoptimization and high-fidelity substitutions in 3D polymerase as potential vaccine strategies for eliciting immune responses against enterovirus A71.

Enterovirus A71 (EV-A71) can induce severe neurological complications and even fatal encephalitis in children, and it has caused several large outbreaks in Taiwan since 1998. We previously generated VP1 codon-deoptimized (VP1-CD) reverse genetics (rg) EV-A71 viruses (rgEV-A71s) that harbor a high-fidelity (HF) 3D polymerase. These VP1-CD-HF rgEV-A71s showed lower replication kinetics in vitro and decreased virulence in an Institute of Cancer Research (ICR) mouse model of EV-A71 infection, while still retaining their antigenicity in comparison to the wild-type virus. In this study, we aimed to further investigate the humoral and cellular immune responses elicited by VP1-CD-HF rgEV-A71s to assess the potential efficacy of these EV-A71 vaccine candidates. Following intraperitoneal (i.p.) injection of VP1-CD-HF rgEV-A71s in mice, we observed a robust induction of EV-A71-specific neutralizing IgG antibodies in the antisera after 21 days. Splenocytes isolated from VP1-CD-HF rgEV-A71s-immunized mice exhibited enhanced proliferative activities and cytokine production (IL-2, IFN-γ, IL-4, IL-6, and TNF-α) upon re-stimulation with VP1-CD-HF rgEV-A71, as compared to control mice treated with adjuvant only. Importantly, administration of antisera from VP1-CD-HF rgEV-A71s-immunized mice protected against lethal EV-A71 challenge in neonatal mice. These findings highlight that our generated VP1-CD-HF rgEV-A71 viruses are capable of inducing both cellular and humoral immune responses, supporting their potential as next-generation EV-A71 vaccines for combating EV-A71 infection.IMPORTANCEEV-A71 can cause severe neurological diseases and cause death in young children. Here, we report the development of synthetic rgEV-A71s with the combination of codon deoptimization and high-fidelity (HF) substitutions that generate genetically stable reverse genetics (rg) viruses as potential attenuated vaccine candidates. Our work provides insight into the development of low-virulence candidate vaccines through a series of viral genetic editing for maintaining antigenicity and genome stability and suggests a strategy for the development of an innovative next-generation vaccine against EV-A71.

Utilization of Bacteriophage phi6 for the Production of High-Quality Double-Stranded RNA Molecules.

Double-stranded RNA (dsRNA) molecules are mediators of RNA interference (RNAi) in eukaryotic cells. RNAi is a conserved mechanism of post-transcriptional silencing of genes cognate to the sequences of the applied dsRNA. RNAi-based therapeutics for the treatment of rare hereditary diseases have recently emerged, and the first sprayable dsRNA biopesticide has been proposed for registration. The range of applications of dsRNA molecules will likely expand in the future. Therefore, cost-effective methods for the efficient large-scale production of high-quality dsRNA are in demand. Conventional approaches to dsRNA production rely on the chemical or enzymatic synthesis of single-stranded (ss)RNA molecules with a subsequent hybridization of complementary strands. However, the yield of properly annealed biologically active dsRNA molecules is low. As an alternative approach, we have developed methods based on components derived from bacteriophage phi6, a dsRNA virus encoding RNA-dependent RNA polymerase (RdRp). Phi6 RdRp can be harnessed for the enzymatic production of high-quality dsRNA molecules. The isolated RdRp efficiently synthesizes dsRNA in vitro on a heterologous ssRNA template of any length and sequence. To scale up dsRNA production, we have developed an in vivo system where phi6 polymerase complexes produce target dsRNA molecules inside Pseudomonas cells.

In-silico Investigations for the Identification of Novel Inhibitors Targeting Hepatitis C Virus RNA-dependent RNA Polymerase.

BACKGROUND: Hepatitis C is an inflammatory condition of the liver caused by the hepatitis C virus, exhibiting acute and chronic manifestations with severity ranging from mild to severe and lifelong illnesses leading to liver cirrhosis and cancer. According to the World Health Organization's global estimates, a population of about 58 million have chronic hepatitis C virus infection, with around 1.5 million new infections occurring every year. OBJECTIVE: The present study aimed to identify novel molecules targeting the Hepatitis C viral RNA Dependent RNA polymerases, which play a crucial role in genome replication, mRNA synthesis, etc. Methods: Structure-based virtual screening of chemical libraries of small molecules was done using AutoDock/Vina. The top-ranking pose for every ligand was complexed with the protein and used for further protein-ligand interaction analysis using the Protein-ligand interaction Profiler. Molecules from virtual screening were further assessed using the pkCSM web server. The proteinligand interactions were further subjected to molecular dynamics simulation studies to establish dynamic stability. RESULTS: Molecular docking-based virtual screening of the database of small molecules, followed by screening based on pharmacokinetic and toxicity parameters, yielded eight probable RNA Dependent RNA polymerase inhibitors. The docking scores for the proposed candidates ranged from - 8.04 to -9.10 kcal/mol. The potential stability of the ligands bound to the target protein was demonstrated by molecular dynamics simulation studies. CONCLUSION: Data from exhaustive computational studies proposed eight molecules as potential anti-viral candidates, targeting Hepatitis C viral RNA Dependent RNA polymerases, which can be further evaluated for their biological potential.

Hepatitis C virus NS5B triggers an MDA5-mediated innate immune response by producing dsRNA without the replication of viral genomes.

During the replication of viral genomes, RNA viruses produce double-stranded RNA (dsRNA), through the activity of their RNA-dependent RNA polymerases (RdRps) as viral replication intermediates. Recognition of viral dsRNA by host pattern recognition receptors - such as retinoic acid-induced gene-I (RIG-I)-like receptors and Toll-like receptor 3 - triggers the production of interferon (IFN)-ß via the activation of IFN regulatory factor (IRF)-3. It has been proposed that, during the replication of viral genomes, each of RIG-I and melanoma differentiation-associated gene 5 (MDA5) form homodimers for the efficient activation of a downstream signalling pathway in host cells. We previously reported that, in the non-neoplastic human hepatocyte line PH5CH8, the RdRp NS5B derived from hepatitis C virus (HCV) could induce IFN-ß expression by its RdRp activity without the actual replication of viral genomes. However, the exact mechanism by which HCV NS5B produced IFN-ß remained unknown. In the present study, we first showed that NS5B derived from another Flaviviridae family member, GB virus B (GBV-B), also possessed the ability to induce IFN-ß in PH5CH8 cells. Similarly, HCV NS5B, but not its G317V mutant, which lacks RdRp activity, induced the dimerization of MDA5 and subsequently the activation of IRF-3. Interestingly, immunofluorescence analysis showed that HCV NS5B produced dsRNA. Like HCV NS5B, GBV-B NS5B also triggered the production of dsRNA and subsequently the dimerization of MDA5. Taken together, our results show that HCV NS5B triggers an MDA5-mediated innate immune response by producing dsRNA without the replication of viral genomes in human hepatocytes.

Diversity and connectedness of brine shrimp viruses in global hypersaline ecosystems.

Brine shrimp (Artemia) has existed on Earth for 400 million years and has major ecological importance in hypersaline ecosystems. As a crucial live food in aquaculture, brine shrimp cysts have become one of the most important aquatic products traded worldwide. However, our understanding of the biodiversity, prevalence and global connectedness of viruses in brine shrimp is still very limited. A total of 143 batches of brine shrimp (belonging to seven species) cysts were collected from six continents including 21 countries and more than 100 geographic locations worldwide during 1977-2019. In total, 55 novel RNA viruses were identified, which could be assigned to 18 different viral families and related clades. Eleven viruses were dsRNA viruses, 16 were +ssRNA viruses, and 28 were-ssRNA viruses. Phylogenetic analyses of the RNA-directed RNA polymerase (RdRp) showed that brine shrimp viruses were often grouped with viruses isolated from other invertebrates and fungi. Remarkably, most brine shrimp viruses were related to those from different hosts that might feed on brine shrimp or share the same ecological niche. A notable case was the novel brine shrimp noda-like virus 3, which shared 79.25% (RdRp) and 63.88% (capsid proteins) amino acid identity with covert mortality nodavirus (CMNV) that may cause losses in aquaculture. In addition, both virome composition and phylogenetic analyses revealed global connectedness in certain brine shrimp viruses, particularly among Asia and Northern America. This highlights the incredible species diversity of viruses in these ancient species and provides essential data for the prevalence of RNA viruses in the global aquaculture industry. More broadly, these findings provide novel insights into the previously unrecognized RNA virosphere in hypersaline ecosystems worldwide and demonstrate that human activity might have driven the global connectedness of brine shrimp viruses.

Atypical activation of the RNA sensor MDA5 by hepatitis C virus.

Hepatitis C virus (HCV) is a significant human pathogen that can cause a number of serious diseases including chronic inflammation of the liver, cirrhosis, and hepatocellular carcinoma. A key enzyme in the HCV life cycle is the nonstructural protein 5B (NS5B), which functions as an RNA-dependent RNA polymerase (RdRp) responsible for replicating the viral RNA genome. In their recent study, Dansako and colleagues showed that HCV NS5B induces type I interferon via activation of the RNA receptor MDA5, an activity that was dependent on the RdRp enzymatic activity but independent of viral RNA replication. Their data further indicated that the NS5B enzymes of HCV and the related GB virus-B produce cellular double-stranded RNA (dsRNA) species with potential immunostimulatory activity. These findings unveil an unconventional mechanism of activation of MDA5-mediated host immunity by viral RdRp enzymes, which is expected to spur new research directions in viral immunology.

Synthesis and biological evaluation of Ribo 7-N/O/S pyrimidine 9-deaza C-nucleoside analogs as new antiviral agents for inhibiting HCV RNA-dependent RNA polymerases.

Hepatitis C infection is caused by the bloodborne pathogen hepatitis C virus (HCV) and can lead to serious liver diseases and, ultimately, death if the treatment is ineffective. This work reports the synthesis and preclinical evaluation of 7 novel 9-O/N/S pyrimidine nucleosides, including compound 12, the triphosphate of known compound 7b. The nucleosides are 9-deaza modifications of adenosine and guanosine with ß-2'-C-methyl substituent on the ribose. Within this series of compounds, a 9-deaza furopyrimidine analog of adenosine, compound 7b, showed high anti-HCV activity in vitro, good stability, low toxicity, and low genotoxicity when administrated in low doses, and an adequate pharmacokinetics profile. An improved synthesis of compound 7b compared to a previous study is also reported. Compound 12 was synthesized as a control to verify phosphorylation of 7b occurred in vivo.

Tiratricol inhibits yellow fever virus replication through targeting viral RNA-dependent RNA polymerase of NS5.

Yellow fever virus (YFV) infection is a major public concern that threatens a large population in South America and Africa. No specific antiviral drugs are available for treating yellow fever. Here, we report that tiratricol (triiodothyroacetic acid, TRIAC), a clinically approved drug used to treat thyroid hormone resistance syndrome (THRS), is a potent YFV inhibitor both in host cells and in animal models.An in vitro study demonstrates that TRIAC remarkably suppresses viral RNA synthesis and protein expression in a dose-dependent manner in human hepatoma cell lines (Huh-7) with an EC50 value of 2.07 µM and a CC50 value of 385.77 µM respectively. The surface plasmon resonance assay and molecular docking analysis indicate that TRIAC hinders viral replication by binding to the RNA-dependent RNA polymerase (RdRp) domain of viral nonstructural protein NS5, probably through interacting with the active sites of RdRp.The inhibitory effect of TRIAC in vivo is also confirmed in 3-week old C57BL/6 mice challenged with YFV infection, from which the survival of the mice as well as lesions and infection in their tissues and serum issignificantly promoted following oral administration of TRIAC (0.2 mg/kg/day). Additionally, TRIAC shows a broad-spectrum antiviral activity against multiple flaviviruses such as TBEV, WNV,ZIKV, andJEV in vitro. Our data demonstrate that the TH analogue TRIAC is an effective anti-YFV compound and may act as a potential therapeutic candidate for the treatment of YFV infection if its clinical importance is determined in patients in future.

Sangivamycin is preferentially incorporated into viral RNA by the SARS-CoV-2 polymerase.

Sangivamycin (S) is an adenosine (A) nucleoside analog with low nanomolar antiviral activity against SARS-CoV-2 in vitro. Previously, low nanomolar antiviral efficacy was revealed when tested against multiple viral variants in several cell types. SARS-CoV-2 RNA isolated from live virus infected cells and the virions released from these cells was analyzed by mass spectrometry (MS) for S incorporation. Dose-dependent incorporation occurred up to 1.8 S per 1,000 nucleotides (49 S per genome) throughout the viral genomes isolated from both infected cells and viral particles, but this incorporation did not change the viral mutation rate. In contrast, host mRNA, affinity purified from the same infected and treated cells, contained little or no S. Sangivamycin triphosphate (STP) was synthesized to evaluate its incorporation into RNA by recombinant SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) under defined in vitro conditions. SARS-CoV-2 RdRp showed that S was not a chain terminator and S containing oligonucleotides templated as A. Though the antiviral mechanism remains to be determined, the data suggests that SARS-CoV-2 RdRp incorporates STP into SARS-CoV-2 RNA, which does not significantly impair viral RNA synthesis or the mutation rate.

Replication-competent influenza virus with a protein-responsive multiplication ability.

Applications of influenza A viruses (IAV) for virotherapy and biotechnology have accelerated substantially with the development of reverse genetic technology and advances in the understanding of packaging signals. While the use of a replication-competent IAV is particularly promising, owing to its efficient transmission to organ depths with high infectivity, there is also a risk that its multiplication cannot be controlled in a cell-type-specific manner, causing an infectious disease. Therefore, here a simple and effective replication-competent IAV-based cell-targeting system has been developed. It was demonstrated that the activity of the ribonucleoprotein complex (RNP) of IAV could be regulated by the interaction between the endogenous protein and a nanobody fused to the subunit of RNA-dependent RNA polymerase (RdRp). To validate the feasibility of the method, it was demonstrated that RNP containing RdRp fused with Nb139, a nanobody against p53, is inactive in HEK293T cells expressing endogenous p53, but active in p53-defective Saos-2 cells. Finally, a replication-competent IAV was successfully generated that multiplies only in p53-defective tumor cells and an IAV vector was developed that can deliver a foreign gene in cell type-specific manner. The method is flexible because the nanobody can be easily altered to target a different cell type, offering a valuable platform for virotherapy and biotechnology.

Hepatitis C virus RNA is 5'-capped with flavin adenine dinucleotide.

RNA viruses have evolved elaborate strategies to protect their genomes, including 5' capping. However, until now no RNA 5' cap has been identified for hepatitis C virus1,2 (HCV), which causes chronic infection, liver cirrhosis and cancer3. Here we demonstrate that the cellular metabolite flavin adenine dinucleotide (FAD) is used as a non-canonical initiating nucleotide by the viral RNA-dependent RNA polymerase, resulting in a 5'-FAD cap on the HCV RNA. The HCV FAD-capping frequency is around 75%, which is the highest observed for any RNA metabolite cap across all kingdoms of life4-8. FAD capping is conserved among HCV isolates for the replication-intermediate negative strand and partially for the positive strand. It is also observed in vivo on HCV RNA isolated from patient samples and from the liver and serum of a human liver chimeric mouse model. Furthermore, we show that 5'-FAD capping protects RNA from RIG-I mediated innate immune recognition but does not stabilize the HCV RNA. These results establish capping with cellular metabolites as a novel viral RNA-capping strategy, which could be used by other viruses and affect anti-viral treatment outcomes and persistence of infection.

An efficient computational protocol for template-based design of peptides that inhibit interactions involving SARS-CoV-2 proteins.

The RNA-dependent RNA polymerase (RdRp) complex of SARS-CoV-2 lies at the core of its replication and transcription processes. The interfaces between holo-RdRp subunits are highly conserved, facilitating the design of inhibitors with high affinity for the interaction interface hotspots. We, therefore, take this as a model protein complex for the application of a structural bioinformatics protocol to design peptides that inhibit RdRp complexation by preferential binding at the interface of its core subunit nonstructural protein, nsp12, with accessory factor nsp7. Here, the interaction hotspots of the nsp7-nsp12 subunit of RdRp, determined from a long molecular dynamics trajectory, are used as a template. A large library of peptide sequences constructed from multiple hotspot motifs of nsp12 is screened in-silico to determine sequences with high geometric complementarity and interaction specificity for the binding interface of nsp7 (target) in the complex. Two lead designed peptides are extensively characterized using orthogonal bioanalytical methods to determine their suitability for inhibition of RdRp complexation. Binding affinity of these peptides to accessory factor nsp7, determined using a surface plasmon resonance (SPR) assay, is slightly better than that of nsp12: dissociation constant of 133nM and 167nM, respectively, compared to 473nM for nsp12. A competitive ELISA is used to quantify inhibition of nsp7-nsp12 complexation, with one of the lead peptides giving an IC50 of 25µM . Cell penetrability and cytotoxicity are characterized using a cargo delivery assay and MTT cytotoxicity assay, respectively. Overall, this work presents a proof-of-concept of an approach for rational discovery of peptide inhibitors of SARS-CoV-2 protein-protein interactions.

An in silico prediction of interaction models of influenza A virus PA and human C14orf166 protein from yeast-two-hybrid screening data.

The human C14orf166 protein, also known as RNA transcription, translation, and transport factor, shows positive modulatory activity on the cellular RNA polymerase II enzyme. This protein is a component of the tRNA-splicing ligase complex and is involved in RNA metabolism. It also functions in the nucleo-cytoplasmic transport of RNA molecules. The C14orf166 protein has been reported to be associated with some types of cancer. It has been shown that the C14orf166 protein binds to the influenza A virus RNA polymerase PA subunit and has a stimulating effect on viral replication. In this study, candidate interactor proteins for influenza A virus PA protein were screened with a Y2H assay using HEK293 Matchmaker cDNA. The C14orf166 protein fragments in different sizes were found to interact with the PA. The three-dimensional structures of the viral PA and C14orf166 proteins interacting with the PA were generated using the I-TASSER algorithm. The interaction models between these proteins were predicted with the ClusPro protein docking algorithm and analyzed with PyMol software. The results revealed that the carboxy-terminal end of the C14orf166 protein is involved in this interaction, and it is highly possible that it binds to the carboxy-terminal of the PA protein. Although amino acid residues in the interaction area of the PA protein with the C14orf166 showed distribution from 450th to 700th position, the intense interaction region was revealed to be at amino acid positions 610-630.

Molecular characterization of a novel fungal alphaflexivirus reveals potential inter-species horizontal gene transfer.

Sclerotinia sclerotiorum is a notorious phytopathogenic fungus that harbors diverse mycoviruses. A novel positive-sense single-stranded RNA virus, Sclerotinia sclerotiorum alphaflexivirus 2 (SsAFV2), was isolated from the hypovirulent strain 32-9 of S. sclerotiorum, and its complete genome was determined. The SsAFV2 genome contains 7,162 nucleotides (nt), excluding the poly (A) structure, and is composed of four open reading frames (ORF1-4). ORF1 encodes a polyprotein that contains three conserved domains: methyltransferase, helicase, and RNA-dependent RNA polymerase (RdRp). The ORF3 putative encodes coat proteins (CP), with ORF2 and ORF4 encoding hypothetical proteins of unknown functions. Phylogenetic analysis revealed that SsAFV2 clustered with Botrytis virus X (BVX) based on multiple alignments of helicase, RdRp, and CP, but the methyltransferase of SsAFV2 was most closely related to Sclerotinia sclerotiorum alphaflexivirus 1, suggesting that SsAFV2 is a new member of the Botrexvirus genus within the Alphaflexiviridae family, and also revealed the occurrence of potential inter-species horizontal gene transfer events within the Botrexvirus genus during the evolutionary process. Our results contribute to the current knowledge regarding the evolution and divergence of Botrexviruses.

A mutation in the coronavirus nsp13-helicase impairs enzymatic activity and confers partial remdesivir resistance.

Coronaviruses (CoVs) encode nonstructural proteins 1-16 (nsps 1-16) which form replicase complexes that mediate viral RNA synthesis. Remdesivir (RDV) is an adenosine nucleoside analog antiviral that inhibits CoV RNA synthesis. RDV resistance mutations have been reported only in the nonstructural protein 12 RNA-dependent RNA polymerase (nsp12-RdRp). We here show that a substitution mutation in the nsp13-helicase (nsp13-HEL A335V) of the betacoronavirus murine hepatitis virus (MHV) that was selected during passage with the RDV parent compound confers partial RDV resistance independently and additively when expressed with co-selected RDV resistance mutations in the nsp12-RdRp. The MHV A335V substitution did not enhance replication or competitive fitness compared to WT MHV and remained sensitive to the active form of the cytidine nucleoside analog antiviral molnupiravir (MOV). Biochemical analysis of the SARS-CoV-2 helicase encoding the homologous substitution (A336V) demonstrates that the mutant protein retained the ability to associate with the core replication proteins nsps 7, 8, and 12 but had impaired helicase unwinding and ATPase activity. Together, these data identify a novel determinant of nsp13-HEL enzymatic activity, define a new genetic pathway for RDV resistance, and demonstrate the importance of surveillance for and testing of helicase mutations that arise in SARS-CoV-2 genomes. IMPORTANCE Despite the development of effective vaccines against COVID-19, the continued circulation and emergence of new variants support the need for antivirals such as RDV. Understanding pathways of antiviral resistance is essential for surveillance of emerging variants, development of combination therapies, and for identifying potential new targets for viral inhibition. We here show a novel RDV resistance mutation in the CoV helicase also impairs helicase functions, supporting the importance of studying the individual and cooperative functions of the replicase nonstructural proteins 7-16 during CoV RNA synthesis. The homologous nsp13-HEL mutation (A336V) has been reported in the GISAID database of SARS-CoV-2 genomes, highlighting the importance of surveillance of and genetic testing for nucleoside analog resistance in the helicase.