Here, we characterized a novel mitovirus from the fungus Nigrospora oryzae, which was named \"Nigrospora oryzae mitovirus 3\" (NoMV3). The NoMV3 genome is 2,492 nt in length with a G + C content of 33%, containing a single large open reading frame (ORF) using the fungal mitochondrial genetic code. The ORF encodes an RNA-dependent RNA polymerase (RdRp) of 775 amino acids with a molecular mass of 88.75 kDa. BLASTp analysis revealed that the RdRp of NoMV3 had 68.6%, 50.6%, and 48.6% sequence identity to those of Nigrospora oryzae mitovirus 2, Suillus luteus mitovirus 6, and Fusarium proliferatum mitovirus 3, respectively, which belong to the genus Unuamitovirus within the family Mitoviridae. Phylogenetic analysis based on amino acid sequences supported the classification of NoMV3 as a member of a new species in the genus Unuamitovirus within the family Mitoviridae.

Since the emergence of SARS-CoV-2, mutations in all subunits of the RNA-dependent RNA polymerase (RdRp) of the virus have been repeatedly reported. Although RdRp represents a primary target for antiviral drugs, experimental studies exploring the phenotypic effect of these mutations have been limited. This study focuses on the phenotypic effects of substitutions in the three RdRp subunits: nsp7, nsp8, and nsp12, selected based on their occurrence rate and potential impact. We employed nano-differential scanning fluorimetry and microscale thermophoresis to examine the impact of these mutations on protein stability and RdRp complex assembly. We observed diverse impacts; notably, a single mutation in nsp8 significantly increased its stability as evidenced by a 13°C increase in melting temperature, whereas certain mutations in nsp7 and nsp8 reduced their binding affinity to nsp12 during RdRp complex formation. Using a fluorometric enzymatic assay, we assessed the overall effect on RNA polymerase activity. We found that most of the examined mutations altered the polymerase activity, often as a direct result of changes in stability or affinity to the other components of the RdRp complex. Intriguingly, a combination of nsp8 A21V and nsp12 P323L mutations resulted in a 50% increase in polymerase activity. To our knowledge, this is the first biochemical study to demonstrate the impact of amino acid mutations across all components constituting the RdRp complex in emerging SARS-CoV-2 subvariants.

Avian influenza viruses (AIVs) are the origin of multiple mammal influenza viruses. The genetic determinants of AIVs adapted to humans have been widely elucidated, however, the molecular mechanism of cross-species transmission and adaptation of AIVs to canines are still poorly understood. In this study, two H3N2 influenza viruses isolated from a live poultry market (A/environment/Guangxi/13431/2018, GX13431) and a swab sample from a canine (A/canine/Guangdong/0601/2019, GD0601) were used to investigate the possible molecular basis that determined H3N2 AIV adapting to canine. We found that GD0601 exhibited more robust polymerase activity in cells and higher pathogenicity in mice compared with its evolution ancestor H3N2 AIV GX13431. A series of reassortments of the ribonucleoprotein (RNP) complex showed that the PB2 subunit was the crucial factor that conferred high polymerase activity of GD0601, and the substitution of I714S in the PB2 subunit of GD0601 attenuated the replication and pathogenicity in mammal cells and the mouse model. Mechanistically, the reverse mutation of I714S in the PB2 polymerase subunit which was identified in AIV GX13431 reduced the nuclear import efficiency of PB2 protein and interfered with the interactions of PB2-PA/NP that affected the assembly of the viral RNP complex. Our study reveals amino acid mutation at the position of 714 in the nuclear localization signal (NLS) area in PB2 plays an important role in overcoming the barrier from poultry to mammals of the H3N2 canine influenza virus and provides clues for further study of mammalian adaptation mechanism of AIVs.

The benzofuran core inhibitors HCV-796, BMS-929075, MK-8876, compound 2, and compound 9B exhibit good pan-genotypic activity against various genotypes of NS5B polymerase. To elucidate their mechanism of action, multiple molecular simulation methods were used to investigate the complex systems of these inhibitors binding to GT1a, 1b, 2a, and 2b NS5B polymerases. The calculation results indicated that these five inhibitors can not only interact with the residues in the palm II subdomain of NS5B polymerase, but also with the residues in the palm I subdomain or the palm I/III overlap region. Interestingly, the binding of inhibitors with longer substituents at the C5 position (BMS-929075, MK-8876, compound 2, and compound 9B) to the GT1a and 2b NS5B polymerases exhibits different binding patterns compared to the binding to the GT1b and 2a NS5B polymerases. The interactions between the para-fluorophenyl groups at the C2 positions of the inhibitors and the residues at the binding pockets, together with the interactions between the substituents at the C5 positions and the residues at the reverse β-fold (residues 441-456), play a key role in recognition and the induction of the binding. The relevant studies could provide valuable information for further research and development of novel anti-HCV benzofuran core pan-genotypic inhibitors.

In this study, a novel mitovirus, tentatively designated as \"Alternaria alternata mitovirus 2\" (AaMV2), was isolated from the fungus Alternaria alternata f. sp. mali causing apple leaf blotch disease. The complete genome of AaMV2 is 3,157 nucleotides in length, with an A+U content of 68.10%. The genome has a single large open reading frame (ORF) encoding an RNA-dependent RNA polymerase (RdRp) protein with a molecular mass of 98.10 kDa. BLAST analysis revealed that AaMV2 has the highest sequence identity to Leptosphaeria biglobosa mitovirus 6, with 79.76% and 82.86% identity at the amino acid and nucleotide level, respectively. Phylogenetic analysis suggested that AaMV2 is a new member of the genus Duamitovirus within the family Mitoviridae. This is the first report of the complete genome sequence analysis of a mitovirus in A. alternata.

Viral diseases pose a serious global health threat due to their rapid transmission and widespread impact. The RNA-dependent RNA polymerase (RdRp) participates in the synthesis, transcription, and replication of viral RNA in host. The current study investigates the antiviral potential of secondary metabolites particularly those derived from bacteria, fungi, and plants to develop novel medicines. Using a virtual screening approach that combines molecular docking and molecular dynamics (MD) simulations, we aimed to discover compounds with strong interactions with RdRp of five different retroviruses. The top five compounds were selected for each viral RdRp based on their docking scores, binding patterns, molecular interactions, and drug-likeness properties. The molecular docking study uncovered several metabolites with antiviral activity against RdRp. For instance, cytochalasin Z8 had the lowest docking score of -8.9 (kcal/mol) against RdRp of SARS-CoV-2, aspulvinone D (-9.2 kcal/mol) against HIV-1, talaromyolide D (-9.9 kcal/mol) for hepatitis C, aspulvinone D (-9.9 kcal/mol) against Ebola and talaromyolide D also maintained the lowest docking score of -9.2 kcal/mol against RdRp enzyme of dengue virus. These compounds showed remarkable antiviral potential comparable to standard drug (remdesivir -7.4 kcal/mol) approved to target RdRp and possess no significant toxicity. The molecular dynamics simulation confirmed that the best selected ligands were firmly bound to their respective target proteins for a simulation time of 200 ns. The identified lead compounds possess distinctive pharmacological characteristics, making them potential candidates for repurposing as antiviral drugs against SARS-CoV-2. Further experimental evaluation and investigation are recommended to ascertain their efficacy and potential.

Bovine viral diarrhea virus (BVDV) is a widespread pathogen of cattle and other mammals that causes major economic losses in the livestock industry. N4-TSC and 6NO2-TSC are two thiosemicarbazones derived from 1-indanone that exhibit anti-BVDV activity in vitro. These compounds selectively inhibit BVDV and are effective against both cytopathic and non-cytopathic BVDV-1 and BVDV-2 strains. We confirmed that N4-TSC acts at the onset of viral RNA synthesis, as previously reported for 6NO2-TSC. Moreover, resistance selection and characterization showed that N4-TSCR mutants were highly resistant to N4-TSC but remained susceptible to 6NO2-TSC. In contrast, 6NO2-TSCR mutants were resistant to both compounds. Additionally, mutations N264D and A392E were found in the viral RNA-dependent RNA polymerase (RdRp) of N4-TSCR mutants, whereas I261 M was found in 6NO2-TSCR mutants. These mutations lay in a hydrophobic pocket within the fingertips region of BVDV RdRp that has been described as a \"hot spot\" for BVDV non-nucleoside inhibitors.

Self-amplifying RNAs (saRNAs) are versatile vaccine platforms that take advantage of a viral RNA-dependent RNA polymerase (RdRp) to amplify the messenger RNA (mRNA) of an antigen of interest encoded within the backbone of the viral genome once inside the target cell. In recent years, more saRNA vaccines have been clinically tested with the hope of reducing the vaccination dose compared to the conventional mRNA approach. The use of N1-methyl-pseudouridine (1mΨ), which enhances RNA stability and reduces the innate immune response triggered by RNAs, is among the improvements included in the current mRNA vaccines. In the present study, we evaluated the effects of this modified nucleoside on various saRNA platforms based on different viruses. The results showed that different stages of the replication process were affected depending on the backbone virus. For TNCL, an insect virus of the Alphanodavirus genus, replication was impaired by poor recognition of viral RNA by RdRp. In contrast, the translation step was severely abrogated in coxsackievirus B3 (CVB3), a member of the Picornaviridae family. Finally, the effects of 1mΨ on Semliki forest virus (SFV), were not detrimental in in vitro studies, but no advantages were observed when immunogenicity was tested in vivo.

Japanese encephalitis virus (JEV) is an arthropod-borne, plus-strand flavivirus causing viral encephalitis in humans with a high case fatality rate. The JEV non-structural protein 5 (NS5) with the RNA-dependent RNA polymerase activity interacts with the viral and host proteins to constitute the replication complex. We have identified the multifunctional protein Nucleolin (NCL) as one of the several NS5-interacting host proteins. We demonstrate the interaction and colocalization of JEV NS5 with NCL in the virus-infected HeLa cells. The siRNA-mediated knockdown of NCL indicated that it was required for efficient viral replication. Importantly, JEV grew to higher titers in cells over-expressing exogenous NCL, demonstrating its pro-viral role. We demonstrated that NS5 interacted with the RRM and GAR domains of NCL. We show that the NCL-binding aptamer AS1411 containing the G-quadruplex (GQ) structure and the GQ ligand BRACO-19 caused significant inhibition of JEV replication. The antiviral effect of AS1411 and BRACO-19 could be overcome in HeLa cells by the overexpression of exogenous NCL. We demonstrated that the synthetic RNAs derived from the 3\'-NCR of JEV genomic RNA containing the GQ sequence could bind NCL in vitro. The replication complex binding to the 3\'-NCR is required for the viral RNA synthesis. It is likely that NCL present in the replication complex destabilizes the GQ structures in the genomic RNA, thus facilitating the movement of the replication complex resulting in efficient virus replication.IMPORTANCEJapanese encephalitis virus (JEV) is endemic in most parts of South-East Asia and the Western Pacific region, causing epidemics of encephalitis with a high case fatality rate. While a tissue culture-derived JEV vaccine is available, no antiviral therapy exists. The JEV NS5 protein has RNA-dependent RNA polymerase activity. Together with several host and viral proteins, it constitutes the replication complex necessary for virus replication. Understanding the interaction of NS5 with the host proteins could help design novel antivirals. We identified Nucleolin (NCL) as a crucial host protein interactor of JEV NS5 having a pro-viral role in virus replication. The NS5-interacting NCL binds to the G-quadruplex (GQ) structure sequence in the 3\'-NCR of JEV RNA. This may smoothen the movement of the replication complex along the genomic RNA, thereby facilitating the virus replication. This study is the first report on how NCL, a host protein, helps in JEV replication through GQ-binding.

The Mononegavirales, or non-segmented negative-sense RNA viruses (nsNSVs), includes significant human pathogens, such as respiratory syncytial virus, parainfluenza virus, measles virus, Ebola virus, and rabies virus. Although these viruses differ widely in their pathogenic properties, they are united by each having a genome consisting of a single strand of negative-sense RNA. Consistent with their shared genome structure, the nsNSVs have evolved similar ways to transcribe their genome into mRNAs and replicate it to produce new genomes. Importantly, both mRNA transcription and genome replication are performed by a single virus-encoded polymerase. A fundamental and intriguing question is: how does the nsNSV polymerase commit to being either an mRNA transcriptase or a replicase? The polymerase must become committed to one process or the other either before it interacts with the genome template or in its initial interactions with the promoter sequence at the 3´ end of the genomic RNA. This review examines the biochemical, molecular biology, and structural biology data regarding the first steps of transcription and RNA replication that have been gathered over several decades for different families of nsNSVs. These findings are discussed in relation to possible models that could explain how an nsNSV polymerase initiates and commits to either transcription or genome replication.