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The trans-replication system explores the concept of separating the viral RNA involved in the translation of the replicase protein from the replication of the viral genome and has been successfully used to study the replication mechanisms of alphaviruses. We tested the feasibility of this system with potato virus X (PVX), an alpha-like virus, in planta. A viral RNA template was designed which does not produce the replicase and prevents virion formation but remains recognizable by the replicase. The replicase construct encodes for the replicase protein, while lacking other virus-specific recognition sequences. Both the constructs were delivered into Nicotiana benthamiana leaves via Agrobacterium-mediated infiltration. Templates of various lengths were tested, with the longer templates not replicating at 4 and 6 days post inoculation, when the replicase protein was provided in trans. Co-expression of helper component proteinase with the short template led to its trans-replication. The cells where replication had been initiated were observed to be scattered across the leaf lamina. This study established that PVX is capable of trans-replicating and can likely be further optimized, and that the experimental freedom offered by the system can be utilized to delve deeper into understanding the replication mechanism of the virus.

Applying a pan-astrovirus (AstV) RT-hemi-nested PCR assay, we report here high detection rates (28.3%, 15/53) of AstVs in the small Indian mongoose (Urva auropunctata) on the Caribbean Island of St. Kitts. Based on deduced amino acid (aa) identities and phylogenetic analysis of long RNA-dependent RNA polymerase (RdRp) sequences (~315 aa, partial RdRp), the AstVs detected in the mongooses (designated as Mon-AstVs) were classified into two distinct groups (deduced aa identities of 66.45–67.30% between the groups). The putative RdRps of the Mon-AstVs shared low deduced aa identities with those of AstVs from other host species (Astroviridae.

RNA virus polymerases carry out multiple functions necessary for successful genome replication and transcription. A key tool for molecular studies of viral RNA-dependent RNA polymerases (RdRps) is a ‘minigenome’ or ‘minireplicon’ assay, in which viral RdRps are reconstituted in cells in the absence of full virus infection. Typically, plasmids expressing the viral polymerase protein(s) and other co-factors are co-transfected, along with a plasmid expressing an RNA encoding a fluorescent or luminescent reporter gene flanked by viral untranslated regions containing cis-acting elements required for viral RdRp recognition. This reconstitutes the viral transcription/replication machinery and allows the viral RdRp activity to be measured as a correlate of the reporter protein signal. Here, we report on the development of a ‘first-generation’ plasmid-based minigenome assay for species A rotavirus using a firefly luciferase reporter gene.

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.

From the first isolation of the cystovirus bacteriophage Φ6 from Pseudomonas syringae 50 years ago, we have progressed to a better understanding of the structure and transformations of many parts of the virion. The three-layered virion, encapsulating the tripartite double-stranded RNA (dsRNA) genome, breaches the cell envelope upon infection, generates its own transcripts, and coopts the bacterial machinery to produce its proteins. The generation of a new virion starts with a procapsid with a contracted shape, followed by the packaging of single-stranded RNA segments with concurrent expansion of the capsid, and finally replication to reconstitute the dsRNA genome. The outer two layers are then added, and the fully formed virion released by cell lysis. Most of the procapsid structure, composed of the proteins P1, P2, P4, and P7 is now known, as well as its transformations to the mature, packaged nucleocapsid. The outer two layers are less well-studied. One additional study investigated the binding of the host protein YajQ to the infecting nucleocapsid, where it enhances the transcription of the large RNA segment that codes for the capsid proteins. Finally, I relate the structural aspects of bacteriophage Φ6 to those of other dsRNA viruses, noting the similarities and differences.

Chronic HCV infection is a risk factor for end-stage liver disease, leading to a major burden on public health. Mitophagy is a specific form of selective autophagy that eliminates mitochondria to maintain mitochondrial integrity. HCV NS5A is a multifunctional protein that regulates the HCV life cycle and may induce host mitophagy. However, the molecular mechanism by which HCV NS5A activates mitophagy remains largely unknown. Here, for the first time, we delineate the dynamic process of HCV NS5A-activated PINK1/Parkin-dependent mitophagy. By performing live-cell imaging and CLEM analyses of HCV NS5A-expressing cells, we demonstrate the degradation of mitochondria within autophagic vacuoles, a process that is dependent on Parkin and ubiquitin translocation onto mitochondria and PINK1 stabilization. In addition, the cargo receptors of mitophagy, NDP52 and OPTN, are recruited to the mitochondria and required for HCV NS5A-induced mitophagy. Moreover, ATG5 and DFCP1, which function in autophagosome closure and phagophore formation, are translocated near mitochondria for HCV NS5A-induced mitophagy. Furthermore, autophagy-initiating proteins, including ATG14 and ULK1, are recruited near the mitochondria for HCV NS5A-triggered mitophagy. Together, these findings demonstrate that HCV NS5A may induce PINK1/Parkin-dependent mitophagy through the recognition of mitochondria by cargo receptors and the nascent formation of phagophores close to mitochondria.

(1) Background: SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) is a promising therapeutic target to fight COVID-19, and many RdRp inhibitors nucleotide/nucleoside analogs, such as remdesivir, have been identified or are in clinical studies. However, the appearance of resistant mutations could reduce their efficacy. In the present work, we structurally evaluated the impact of RdRp mutations found at baseline in 39 patients treated with remdesivir and associated with a different degree of antiviral response in vivo. (2) Methods: A refined bioinformatics approach was applied to assign SARS-CoV-2 clade and lineage, and to define RdRp mutational profiles. In line with such a method, the same mutations were built and analyzed by combining docking and thermodynamics evaluations with both molecular dynamics and representative pharmacophore models. (3) Results: Clinical studies revealed that patients bearing the most prevalent triple mutant P323L+671S+M899I, which was present in 41% of patients, or the more complex mutational profile P323L+G671S+L838I+D738Y+K91E, which was found with a prevalence of 2.6%, showed a delayed reduced response to remdesivir, as confirmed by the increase in SARS-CoV-2 viral load and by a reduced theoretical binding affinity versus RdRp (ΔGbindWT = −122.70 kcal/mol; ΔGbindP323L+671S+M899I = −84.78 kcal/mol; ΔGbindP323L+G671S+L838I+D738Y+K91E = −96.74 kcal/mol). Combined computational approaches helped to rationalize such clinical observations, offering a mechanistic understanding of the allosteric effects of mutants on the global motions of the viral RNA synthesis machine and in the changes of the interactions patterns of remdesivir during its binding.

The World Health Organization (WHO) has designated the Zika virus (ZIKV) as a significant risk to the general public’s health. Currently, there are no vaccinations or medications available to treat or prevent infection with the Zika virus. Thus, it is urgently required to develop a highly efficient therapeutic molecule. In the presented study, a computationally intensive search was carried out to identify potent compounds that have the potential to bind and block the activity of ZIKV NS5 RNA-dependent RNA polymerase (RdRp). The anti-dengue chemical library was subjected to high-throughput virtual screening and MM/GBSA analysis in order to rate the potential candidates. The top three compounds were then chosen. According to the MM/GBSA analysis, compound 127042987 from the database had the highest binding affinity to the protein with a minimum binding free energy of −77.16 kcal/mole. Compound 127042987 had the most stable RMSD trend and the greatest number of hydrogen bond interactions when these chemical complexes were evaluated further under a 100 ns molecular dynamics simulation. Compound 127042987 displayed the best binding free energy (GBind) of −96.50 kcal/mol, surpassing the native ligand binding energy (−66.17 kcal/mole). Thereafter, an MM/GBSA binding free energy study was conducted to validate the stability of selected chemical complexes. Overall, this study illustrated that compound 127042987 showed preferred binding free energies, suggesting a possible inhibitory mechanism against ZIKV-RdRp. As per this study, it was proposed that compound 127042987 could be used as a therapeutic option to prevent Zika virus infection. These compounds need to be tested in experiments for further validation.

Early detection and characterization of new variants and their impacts enable improved genomic surveillance. This study aims to evaluate the subvariant distribution of Omicron strains isolated from Turkish cases to determine the rate of antiviral resistance of RdRp and 3CLpro inhibitors. The Stanford University Coronavirus Antiviral & Resistance Database online tool was used for variant analyses of the strains uploaded to GISAID as Omicron (n = 20.959) between January 2021 and February,2023. Out of 288 different Omicron subvariants, B.1, BA.1, BA.2, BA.4, BE.1, BF.1, BM.1, BN.1, BQ.1, CK.1, CL.1, and XBB.1 were the main determined subvariants, and BA.1 (34.7%), BA.2 (30.8%), and BA.5 (23.6%) were reported most frequently. RdRp and 3CLPro-related resistance mutations were determined in n = 150, 0.72% sequences, while the rates of resistance against RdRp and 3CLpro inhibitors were reported at 0.1% and 0.6%, respectively. Mutations that were previously associated with a reduced susceptibility to remdesivir, nirmatrelvir/r, and ensitrelvir were most frequently detected in BA.2 (51.3%). The mutations detected at the highest rate were A449A/D/G/V (10.5%), T21I (10%), and L50L/F/I/V (6%). Our findings suggest that continuous monitoring of variants, due to the diversity of Omicron lineages, is necessary for global risk assessment. Although drug-resistant mutations do not pose a threat, the tracking of drug mutations will be necessary due to variant heterogenicity.

The unusual and interesting architecture of the catalytic chamber of the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) was recently explored using Cryogenic Electron Microscopy (Cryo-EM), which revealed the presence of two distinctive binding cavities within the catalytic chamber. In this report, first, we mapped out and fully characterized the variations between the two binding sites, BS1 and BS2, for significant differences in their amino acid architecture, size, volume, and hydrophobicity. This was followed by investigating the preferential binding of eight antiviral agents to each of the two binding sites, BS1 and BS2, to understand the fundamental factors that govern the preferential binding of each drug to each binding site. Results showed that, in general, hydrophobic drugs, such as remdesivir and sofosbuvir, bind better to both binding sites than relatively less hydrophobic drugs, such as alovudine, molnupiravir, zidovudine, favilavir, and ribavirin. However, suramin, which is a highly hydrophobic drug, unexpectedly showed overall weaker binding affinities in both binding sites when compared to other drugs. This unexpected observation may be attributed to its high binding solvation energy, which disfavors overall binding of suramin in both binding sites. On the other hand, hydrophobic drugs displayed higher binding affinities towards BS1 due to its higher hydrophobic architecture when compared to BS2, while less hydrophobic drugs did not show a significant difference in binding affinities in both binding sites. Analysis of binding energy contributions revealed that the most favorable components are the ΔEele, ΔEvdw, and ΔGgas, whereas ΔGsol was unfavorable. The ΔEele and ΔGgas for hydrophobic drugs were enough to balance the unfavorable ΔGsol, leaving the ΔEvdw to be the most determining factor of the total binding energy. The information presented in this report will provide guidelines for tailoring SARS-CoV-2 inhibitors with enhanced binding profiles.

PB1, acting as the catalytic subunit of the influenza polymerase, has numerous sequentially and structurally conserved regions. It has been observed that the slight modification of residues in PB1 would greatly affect the polymerase activity and even host adaptation ability. Here, we identified a critical residue, 362M, on the polymerase activity and virus replication. By means of the minireplicon assay, we assured the importance of the hydrophobicity of PB1 362, and the possibility that the size and charge of the side chain might directly interfere with the polymerase function. We also proposed a hydrophobic core between the PA-arch and the PB1 β-hairpin motifs and showed the importance of the core to the polymerase function.

Norovirus is the leading viral agent of gastroenteritis in humans. RNA-dependent RNA polymerase (RdRp) is essential in the replication of norovirus RNA. Here, we present a comprehensive evolutionary analysis of the norovirus GI RdRp gene. Our results show that the norovirus GI RdRp gene can be divided into three groups, and that the most recent common ancestor was 1484. The overall evolutionary rate of GI RdRp is 1.821 × 10−3 substitutions/site/year. Most of the amino acids of the GI RdRp gene were under negative selection, and only a few positively selected sites were recognized. Amino acid substitutions in the GI RdRp gene accumulated slowly over time. GI.P1, GI.P3 and GI.P6 owned the higher evolutionary rates. GI.P11 and GI.P13 had the faster accumulation rate of amino acid substitutions. GI.P2, GI.P3, GI.P4, GI.P6 and GI.P13 presented a strong linear evolution. These results reveal that the norovirus GI RdRp gene evolves conservatively, and that the molecular evolutionary characteristics of each P-genotype are diverse. Sequencing in RdRp and VP1 of norovirus should be advocated in the surveillance system to explore the effect of RdRp on norovirus activity.

Dengue fever (DF) continues to be one of the tropical and subtropical health concerns. Its prevalence tends to increase in some places in these regions. This disease is caused by the dengue virus (DENV), which is transmitted through the mosquitoes Aedes aegypti and A. albopictus. The treatment of DF to date is only supportive and there is no definitive vaccine to prevent this disease. The non-structural DENV protein, RNA-dependent RNA Polymerase (RdRp), is involved in viral replication. The RdRp-derived peptides can be used in the construction of a universal dengue vaccine. These peptides can be utilized as epitopes to induce immunity. This study was an in silico evaluation of the affinity of the potential epitope for the universal dengue vaccine to dendritic cells and the bonds between the epitope and the dendritic cell receptor. The peptide sequence MGKREKKLGEFGKAKG generated from dengue virus subtype 2 (DENV-2) RdRp was antigenic, did not produce allergies, was non-toxic, and had no homology with the human genome. The potential epitope-based vaccine MGKREKKLGEFGKAKG binds stably to dendritic cell receptors with a binding free energy of −474,4 kcal/mol. This epitope is anticipated to induce an immunological response and has the potential to serve as a universal dengue virus vaccine candidate.

Positive-sense single-stranded RNA viruses replicate in virus-induced membranous organelles for maximum efficiency and immune escaping. The replication of potato virus X (PVX) takes place on the endoplasmic reticulum (ER); however, how PVX-encoded RNA-dependent RNA polymerase (RdRp) is associated with the ER is still unknown. A proline-kinked amphipathic α-helix was recently found in the MET domain of RdRp. In this study, we further illustrate that the first α-helix of the MET domain is also required for ER association. Moreover, we found that the MET domain forms multimers on ER and the first α-helix is essential for multimerization. These results suggest that the RdRp of PVX adopts more than one hydrophobic motif for membrane association and for multimerization.

Cucumber mosaic virus (CMV), a major tomato pathogen, is aphid-vectored in the non-persistent manner. We investigated if CMV-induced volatile organic compounds (VOCs) or other virus-induced cues alter aphid–tomato interactions. Y-tube olfactometry showed that VOCs emitted by plants infected with CMV (strain Fny) attracted generalist (Myzus persicae) and Solanaceae specialist (Macrosiphum euphorbiae) aphids. Myzus persicae preferred settling on infected plants (3 days post-inoculation: dpi) at 1h post-release, but at 9 and 21 dpi, aphids preferentially settled on mock-inoculated plants. Macrosiphum euphorbiae showed no strong preference for mock-inoculated versus infected plants at 3 dpi but settled preferentially on mock-inoculated plants at 9 and 21 dpi. In darkness aphids showed no settling or migration bias towards either mock-inoculated or infected plants. However, tomato VOC blends differed in light and darkness, suggesting aphids respond to a complex mix of olfactory, visual, and other cues influenced by infection. The LS-CMV strain induced no changes in aphid–plant interactions. Experiments using inter-strain recombinant and pseudorecombinant viruses showed that the Fny-CMV 2a and 2b proteins modified tomato interactions with Macrosiphum euphorbiae and Myzus persicae, respectively. The defence signal salicylic acid prevents excessive CMV-induced damage to tomato plants but is not involved in CMV-induced changes in aphid–plant interactions.

The on-going global pandemic of COVID-19 is caused by SARS-CoV-2, which features a proofreading mechanism to facilitate the replication of its large RNA genome. The 3′-to-5′ exoribonuclease (ExoN) activity of SARS-CoV-2 non-structural protein 14 (nsp14) removes nucleotides misincorporated during RNA synthesis by the low-fidelity viral RNA-dependent RNA polymerase (RdRp) and thereby compromises the efficacy of antiviral nucleoside/nucleotide analogues. Here we show biochemically that SARS-CoV-2 nsp14 can excise the natural antiviral chain-terminating nucleotide, 3′-deoxy-3′,4′-didehydro-cytidine 5′-monophosphate (ddhCMP), incorporated by RdRp at the 3′ end of an RNA strand. Nsp14 ExoN processes an RNA strand terminated with ddhCMP more efficiently than that with a non-physiological chain terminator 3′-deoxy-cytidine monophosphate (3′-dCMP), whereas RdRp is more susceptible to chain termination by 3′-dCTP than ddhCTP. These results suggest that nsp14 ExoN could play a role in protecting SARS-CoV-2 from ddhCTP, which is produced as part of the innate immune response against viral infections, and that the SARS-CoV-2 enzymes may have adapted to minimize the antiviral effect of ddhCTP.

Dengue virus (DENV) is the causative agent of DENV infection. To tackle DENV infection, the development of therapeutic molecules as direct-acting antivirals (DAAs) has been demonstrated as a truly effective approach. Among various DENV drug targets, non-structural protein 5 (NS5)—a highly conserved protein among the family Flaviviridae—carries the RNA-dependent RNA polymerase (DENVRdRp) domain at the C-terminal, and its “N-pocket” allosteric site is widely considered for anti-DENV drug development. Therefore, in this study, we developed a pharmacophore model by utilising 41 known inhibitors of the DENVRdRp domain, and performed model screening against the FDA’s approved drug database for drug repurposing against DENVRdRp. Herein, drugs complying with the pharmacophore hypothesis were further processed through standard-precision (SP) and extra-precision (XP) docking scores (DSs) and binding pose refinement based on MM/GBSA binding energy (BE) calculations. This resulted in the identification of four potential potent drugs: (i) desmopressin (DS: −10.52, BE: −69.77 kcal/mol), (ii) rutin (DS: −13.43, BE: −67.06 kcal/mol), (iii) lypressin (DS: −9.84, BE: −67.65 kcal/mol), and (iv) lanreotide (DS: −8.72, BE: −64.7 kcal/mol). The selected drugs exhibited relevant interactions with the allosteric N-pocket of DENVRdRp, including priming-loop and entry-point residues (i.e., R729, R737, K800, and E802). Furthermore, 100 ns explicit-solvent molecular dynamics simulations and end-point binding free energy assessments support the considerable stability and free energy of the selected drugs in the targeted allosteric pocket of DENVRdRp. Hence, these four drugs, repurposed as potent inhibitors of the allosteric site of DENVRdRp, are recommended for further validation using experimental assays.

For the last few years, the world has been going through a difficult time, and the reason behind this is severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), one of the significant members of the Coronaviridae family. The major research groups have shifted their focus towards finding a vaccine and drugs against SARS-CoV-2 to reduce the infection rate and save the life of human beings. Even the WHO has permitted using certain vaccines for an emergency attempt to cut the infection curve down. However, the virus has a great sense of mutation, and the vaccine’s effectiveness remains questionable. No natural medicine is available at the community level to cure the patients for now. In this study, we have screened the vast library of experimental drugs of Drug Bank with Schrodinger’s maestro by using three algorithms: high-throughput virtual screening (HTVS), standard precision, and extra precise docking followed by Molecular Mechanics/Generalized Born Surface Area (MMGBSA). We have identified 3-(7-diaminomethyl-naphthalen-2-YL)-propionic acid ethyl ester and Thymidine-5′-thiophosphate as potent inhibitors against the SARS-CoV-2, and both drugs performed impeccably and showed stability during the 100 ns molecular dynamics simulation. Both of the drugs are among the category of small molecules and have an acceptable range of ADME properties. They can be used after their validation in in-vitro and in-vivo conditions.

With the recent global spread of new SARS-CoV-2 variants, there remains an urgent need to develop effective and variant-resistant oral drugs. Recently, we reported in vitro results validating the use of combination drugs targeting both the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) and proofreading exonuclease (ExoN) as potential COVID-19 therapeutics. For the nucleotide analogues to be efficient SARS-CoV-2 inhibitors, two properties are required: efficient incorporation by RdRp and substantial resistance to excision by ExoN. Here, we have selected and evaluated nucleotide analogues with a variety of structural features for resistance to ExoN removal when they are attached at the 3′ RNA terminus. We found that dideoxynucleotides and other nucleotides lacking both 2′- and 3′-OH groups were most resistant to ExoN excision, whereas those possessing both 2′- and 3′-OH groups were efficiently removed. We also found that the 3′-OH group in the nucleotide analogues was more critical than the 2′-OH for excision by ExoN. Since the functionally important sequences in Nsp14/10 are highly conserved among all SARS-CoV-2 variants, these identified structural features of nucleotide analogues offer invaluable insights for designing effective RdRp inhibitors that can be simultaneously efficiently incorporated by the RdRp and substantially resist ExoN excision. Such newly developed RdRp terminators would be good candidates to evaluate their ability to inhibit SARS-CoV-2 in cell culture and animal models, perhaps combined with additional exonuclease inhibitors to increase their overall effectiveness.

A vast diversity of 16 influenza hemagglutinin (HA) subtypes are found in birds. Interestingly, viruses from only two subtypes, H5 and H7, have so far evolved into highly pathogenic avian influenza viruses (HPAIVs) following insertions or substitutions at the HA cleavage site by the viral polymerase. The mechanisms underlying this striking subtype specificity are still unknown. Here, we compiled a comprehensive dataset of 20,488 avian influenza virus HA sequences to investigate differences in nucleotide and amino acid usage at the HA cleavage site between subtypes and how these might impact the genesis of HPAIVs by polymerase stuttering and realignment. We found that sequences of the H5 and H7 subtypes stand out by their high purine content at the HA cleavage site. In addition, fewer substitutions were necessary in H5 and H7 HAs than in HAs from other subtypes to acquire an insertion-prone HA cleavage site sequence, as defined based on in vitro and in vivo data from the literature. Codon usage was more favorable for HPAIV genesis in sequences of viruses isolated from species or geographical regions in which HPAIV genesis is more frequently observed in nature. The results of the present analyses suggest that the subtype restriction of HPAIV genesis to H5 and H7 influenza viruses might be due to the particular codon usage at the HA cleavage site in these subtypes.

Six novel mycoviruses that collectively represent the mycovirome of Rhizoctonia solani anastomosis group (AG)-3 PT strain ZJ-2H, which causes potato black scurf, were identified through metatranscriptome sequencing and putatively designated as Rhizoctonia solani fusarivirus 4 [RsFV4, positive single-stranded RNA (+ssRNA)], Rhizoctonia solani fusarivirus 5 (RsFV5, +ssRNA), Rhizoctonia solani mitovirus 40 (RsMV40, +ssRNA), Rhizoctonia solani partitivirus 10 [RsPV10, double-stranded RNA (dsRNA)], Rhizoctonia solani partitivirus 11 (RsPV11, dsRNA), and Rhizoctonia solani RNA virus 11 (RsRV11, dsRNA). Whole genome sequences of RsFV4, RsMV40, RsPV10, RsPV11, and RsRV11, as well as a partial genome sequence of RsFV5, were obtained. The 3’- and 5’- untranslated regions of the five mycoviruses with complete genome sequences were folded into stable stem-loop or panhandle secondary structures. RsFV4 and RsFV5 are most closely related to Rhizoctonia solani fusarivirus 1 (RsFV1), however, the first open reading frame (ORF) of RsFV4 and RsFV5 encode a hypothetical protein that differs from the first ORF of RsFV1, which encodes a helicase. We confirmed that RsPV10 and RsPV11 assemble into the spherical virus particles (approximately 30 nm in diameter) that were extracted from strain ZJ-2H. This is the first report that +ssRNA and dsRNA viruses co-infect a single strain of R. solani AG-3 PT.

The hepatitis C virus (HCV) is a major global health challenge and a leading cause of morbidity and mortality. Many direct-acting antivirals (DAAs) target essential macromolecules involved in the virus’ life cycle. Although such DAAs achieve great success in reducing the viral load in genotype 1 infections, other genotypes demonstrate different levels of response. This study focused on mutation sites associated with patients with genotype 4a infections that failed to respond to treatment with sofosbuvir. The genotyping of HCV samples from patients with virological failure, and responder patients, was conducted using Geno2Pheno webserver-based full NS5B sequences. We constructed 3D structural models for all the samples and used structural analysis to investigate the effect of amino acid substitution on the observed resistance to SOF-based treatment, and the docking of sofosbuvir into the active sites of the 10 models was performed. Finally, 10 molecular dynamic (MD) simulation experiments were conducted to compare the stability of the 3D models of the resistant samples against the stability of the 3D models of the responder samples. The results highlighted the presence of HCV subtype 4a in all ten samples; in addition, an amino acid (aa) substitution in the palm region may hinder HCV polymerase activity. In this study, we provide evidence that a mutation in the NS5B gene that induces resistance to sofosbuvir in patients with the S282T/C/R mutant virus is present in the Egyptian population. Overall, the docking and MD results support our findings and highlight the significant impact of the identified mutations on the resistance of HCV NS5B RNA-dependent RNA polymerase to direct-acting antivirals (DAAs).

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2), the causative agent of Coronavirus Disease 2019 (COVID-19), has seriously threatened global health. Alongside the approved vaccines, the discovery of prospective anti-COVID-19 drugs has been progressively targeted. Essential oils (EOs) provide a rich source of compounds with valuable antiviral activities that may contribute as effective agents against COVID-19. In this study, the EO of Agathus robusta bark was investigated for its chemical composition and its antiviral activity against SARS-CoV2. Overall, 26 constituents were identified by gas chromatography-mass spectrometry (GC-MS) analysis. α-Pinene, tricyclene, α-terpineol, limonene, d-camphene, trans-pinocarveol, α-phellandren-8-ol, L-β-pinene and borneol were the major components. In silico docking of these constituents against viral key enzymes, spike receptor-binding domain (RBD), main protease (Mpro) and RNA-dependent RNA polymerase (RdRp), using Molecular Operating Environment (MOE) software revealed good binding affinities of the components to the active site of the selected targets, especially, the RBD. In Vitro antiviral MTT and cytopathic effect inhibition assays demonstrated a promising anti SARS-CoV2 for A. robusta bark EO, with a significant selectivity index of 17.5. The results suggested using this EO or its individual components for the protection against or treatment of COVID-19.

ss(+)RNA viruses represent the dominant group of plant viruses. They owe their evolutionary superiority to the large number of mutations that occur during replication, courtesy of RNA-dependent RNA polymerase. Natural selection rewards successful viral subtypes, whose effective tuning of the ecosystem regulates the interactions between its participants. Thus, ss(+)RNA viruses act as shuttles for the functionally important genes of the participants in symbiotic relationships within the ecosystem, of which the most common ecological triad is “plant–virus–insect”. Due to their short life cycle and large number of offspring, RNA viruses act as skillful tuners of the ecosystem, which benefits both viruses and the system as a whole. A fundamental understanding of this aspect of the role played by viruses in the ecosystem makes it possible to apply this knowledge to the creation of DNA insecticides. In fact, since the genes that viruses are involved in transferring are functionally important for both insects and plants, silencing these genes (for example, in insects) can be used to regulate the pest population. RNA viruses are increasingly treated not as micropathogens but as necessary regulators of ecosystem balance.

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), urgently needs effective prophylactic and therapeutic drugs. RNA-dependent RNA polymerase (RdRp), essential for replicating and transcribing a viral RNA genome, is highly conserved in coronaviruses; thus, it is a potential target for inhibiting coronavirus infection. In this study, we generated the cell-based SARS-CoV-2 RdRp activity assay system by modifying a previously reported cell-based MERS-CoV RdRp activity assay system to screen for SARS-CoV-2 RdRp inhibitors. The assay system consisted of an expression plasmid encoding SARS-CoV-2 RdRp and an RdRp activity reporter plasmid. RdRp activity in the cells could be conveniently detected by luminescence after transfection. We confirmed that SARS-CoV-2 RdRp replicated double-stranded RNA using immunofluorescence staining and the inhibition of RdRp activity by remdesivir and lycorine using this system. Moreover, the Z-factor of this system was calculated to be 0.798, suggesting the reproducibility and reliability of the high-throughput screening system. Finally, we screened nucleoside and nucleotide analogs and identified adefovir dipivoxil, emtricitabine, telbivudine, entecavir hydrate, moroxydine and rifampin as novel SARS-CoV-2 RdRp inhibitors and therapeutic candidates for COVID-19 This system provides an effective high-throughput screening system platform for developing potential prophylactic and therapeutic drugs for COVID-19 and emerging coronavirus infections.

The 3′untranslated region (3′UTR) and NS5B of classical swine fever virus (CSFV) play vital roles in viral genome replication. In this study, two chimeric viruses, vC/SM3′UTR and vC/b3′UTR, with 3′UTR substitution of CSFV Shimen strain or bovine viral diarrhea virus (BVDV) NADL strain, were constructed based on the infectious cDNA clone of CSFV vaccine C strain, respectively. After virus rescue, each recombinant chimeric virus was subjected to continuous passages in PK-15 cells. The representative passaged viruses were characterized and sequenced. Serial passages resulted in generation of mutations and the passaged viruses exhibited significantly increased genomic replication efficiency and infectious virus production compared to parent viruses. A proline to threonine mutation at position 162 of NS5B was identified in both passaged vC/SM3′UTR and vC/b3′UTR. We generated P162T mutants of two chimeras using the reverse genetics system, separately. The single P162T mutation in NS5B of vC/SM3′UTR or vC/b3′UTR played a key role in increased viral genome replication and infectious virus production. The P162T mutation increased vC/SM3′UTRP162T replication in rabbits. From RNA-dependent RNA polymerase (RdRp) assays in vitro, the NS5B containing P162T mutation (NS5BP162T) exhibited enhanced RdRp activity for different RNA templates. We further identified that the enhanced RdRp activity originated from increased initiation efficiency of RNA synthesis. These findings revealed a novel function for the NS5B residue 162 in modulating pestivirus replication.

SARS-CoV-2 has caused an extensive pandemic of COVID-19 all around the world. Key viral enzymes are suitable molecular targets for the development of new antivirals against SARS-CoV-2 which could represent potential treatments of the corresponding disease. With respect to its essential role in the replication of viral RNA, RNA-dependent RNA polymerase (RdRp) is one of the prime targets. HeE1-2Tyr and related derivatives were originally discovered as inhibitors of the RdRp of flaviviruses. Here, we present that these pyridobenzothiazole derivatives also significantly inhibit SARS-CoV-2 RdRp, as demonstrated using both polymerase- and cell-based antiviral assays.

The paramyxoviral phosphoprotein (P protein) is the non-catalytic subunit of the viral RNA polymerase, and coordinates many of the molecular interactions required for RNA synthesis. All paramyxoviral P proteins oligomerize via a centrally located coiled-coil that is connected to a downstream binding domain by a dynamic linker. The C-terminal region of the P protein coordinates interactions between the catalytic subunit of the polymerase, and the viral nucleocapsid housing the genomic RNA. The inherent flexibility of the linker is believed to facilitate polymerase translocation. Here we report biophysical and structural characterization of the C-terminal region of the P protein from Menangle virus (MenV), a bat-borne paramyxovirus with zoonotic potential. The MenV P protein is tetrameric but can dissociate into dimers at sub-micromolar protein concentrations. The linker is globally disordered and can be modeled effectively as a worm-like chain. However, NMR analysis suggests very weak local preferences for alpha-helical and extended beta conformation exist within the linker. At the interface between the disordered linker and the structured C-terminal binding domain, a gradual disorder-to-order transition occurs, with X-ray crystallographic analysis revealing a dynamic interfacial structure that wraps the surface of the binding domain.

Picobirnaviruses (PBVs) have been detected in several species of animals worldwide; however, data pertaining to their presence in Australian wild and domestic animals are limited. Although PBVs are mostly found in faecal samples, their detection in blood and respiratory tract samples raises questions concerning their tropism and pathogenicity. We report here PBV detection in wild deer and cattle from southeastern Australia. Through metagenomics, the presence of PBV genogroups I (GI) and II (GII) were detected in deer serum and plasma. Molecular epidemiology studies targeting the partial RNA-dependent RNA polymerase gene were performed in a wide range of specimens (serum, faeces, spleen, lung, nasal swabs, and trachea) collected from wild deer and cattle, with PCR amplification obtained in all specimen types except lung and spleen. Our results reveal the predominance of GI and concomitant detection of both genogroups in wild deer and cattle. In concordance with other studies, the detected GI sequences displayed high genetic diversity, however in contrast, GII sequences clustered into three distinct clades. Detection of both genogroups in the upper respiratory tract (trachea and nasal swab) of deer in the present study gives more evidence about the respiratory tract tropism of PBV. Although much remains unknown about the epidemiology and tropism of PBVs, our study suggests a wide distribution of these viruses in southeastern Australia.

The work is devoted to the study of the complementarity of the electronic structures of the ligands and SARS-CoV-2 RNA-dependent RNA polymerase. The research methodology was based on determining of 3D maps of electron densities of complexes using an original quantum free-orbital AlteQ approach. We observed a positive relationship between the parameters of the electronic structure of the enzyme and ligands. A complementarity factor of the enzyme-ligand complexes has been proposed. The console applications of the AlteQ complementarity assessment for Windows and Linux (alteq_map_enzyme_ligand_4_win.exe and alteq_map_enzyme_ligand_4_linux) are available for free at the ChemoSophia webpage.

Tembusu virus (TMUV), a highly infectious pathogenic flavivirus, causes severe egg-drop and encephalitis in domestic waterfowl, while the determinants responsible for viral pathogenicity are largely unknown. In our previous studies, virulent strain JXSP2-4 had been completely attenuated by successive passages in BHK-21 cells and the avirulent strain was designated as JXSP-310. Based on the backbone of JXSP2-4, a series of chimeric viruses were generated according to the amino acid substitutions in NS5 and their infectivities were also analyzed in cell cultures and ducklings. The results showed that the viral titers of RNA-dependent RNA polymerase (RdRp) domain-swapped cheimeric mutant (JXSP-310RdRp) in cells and ducklings were both markedly decreased compared with JXSP2-4, indicating that mutations in the RdRp domain affected viral replication. There are R543K and V711A two amino acid substitutions in the RdRp domain. Further site-directed mutagenesis showed that single-point R543K mutant (JXSP-R543K) exhibited similar replication efficacy compared with JXSP2-4 in cells, but the viral loads in JXSP-R543K-infected ducklings were significantly lower than that of JXSP2-4 and higher than JXSP-310RdRp. Surprisingly, the single-point V711A mutation we introduced rapidly reverted. In addition, qRT-PCR and Western blot confirmed that the mutations in the RdRp domain significantly affected the replication of the virus. Taken together, these results show that R543K substitution in the RdRp domain impairs the in vivo growth of TMUV, but sustaining its attenuated infectivity requires the concurrent presence of the V711A mutation.

Arenaviruses cause chronic and asymptomatic infections in their natural host, rodents, and several arenaviruses cause severe hemorrhagic fever that has a high mortality in infected humans, seriously threatening public health. There are currently no FDA-licensed drugs available against arenaviruses; therefore, it is important to develop novel antiviral strategies to combat them, which would be facilitated by a detailed understanding of the interactions between the viruses and their hosts. To this end, we performed a transcriptomic analysis on cells infected with arenavirus lymphocytic choriomeningitis virus (LCMV), a neglected human pathogen with clinical significance, and found that the signal transducer and activator of transcription 3 (STAT3) signaling pathway was activated. A further investigation indicated that STAT3 could be activated by the RNA-dependent RNA polymerase L protein (Lp) of LCMV. Our functional analysis found that STAT3 cannot affect LCMV multiplication in A549 cells. We also found that STAT3 was activated by the Lp of Mopeia virus and Junin virus, suggesting that this activation may be conserved across certain arenaviruses. Our study explored the interactions between arenaviruses and STAT3, which may help us to better understand the molecular and cell biology of arenaviruses.

RNA silencing pathways control eukaryotic gene expression transcriptionally or posttranscriptionally in a sequence-specific manner. In RNA silencing, the production of double-stranded RNA (dsRNA) gives rise to various classes of 20–24 nucleotide (nt) small RNAs (smRNAs). In Arabidopsis thaliana, smRNAs are often derived from long dsRNA molecules synthesized by one of the six genomically encoded RNA-dependent RNA Polymerase (RDR) proteins. However, the full complement of the RDR-dependent smRNAs and functions that these proteins and their RNA-binding cofactors play in plant RNA silencing has not been fully uncovered. To address this gap, we performed a global genomic analysis of all six RDRs and two of their cofactors to find new substrates for RDRs and targets of the resulting RDR-derived siRNAs to uncover new functions for these proteins in plants. Based on these analyses, we identified substrates for the three RDRγ clade proteins (RDR3–5), which had not been well-characterized previously. We also identified new substrates for the other three RDRs (RDR1, RDR2, and RDR6) as well as the RDR2 cofactor RNA-directed DNA methylation 12 (RDM12) and the RDR6 cofactor suppressor of gene silencing 3 (SGS3). These findings revealed that the target substrates of SGS3 are not limited to those solely utilized by RDR6, but that this protein seems to be a more general cofactor for the RDR family of proteins. Additionally, we found that RDR6 and SGS3 are involved in the production of smRNAs that target transcripts related to abiotic stresses, including water deprivation, salt stress, and ABA response, and as expected the levels of these mRNAs are increased in rdr6 and sgs3 mutant plants. Correspondingly, plants that lack these proteins (rdr6 and sgs3 mutants) are hypersensitive to ABA treatment, tolerant to high levels of PEG8000, and have a higher survival rate under salt treatment in comparison to wild-type plants. In total, our analyses have provided an extremely data-rich resource for uncovering new functions of RDR-dependent RNA silencing in plants, while also revealing a previously unexplored link between the RDR6/SGS3-dependent pathway and plant abiotic stress responses.

RNA viruses are the fastest evolving known biological entities. Consequently, the sequence similarity between homologous viral proteins disappears quickly, limiting the usability of traditional sequence-based phylogenetic methods in the reconstruction of relationships and evolutionary history among RNA viruses. Protein structures, however, typically evolve more slowly than sequences, and structural similarity can still be evident, when no sequence similarity can be detected. Here, we used an automated structural comparison method, homologous structure finder, for comprehensive comparisons of viral RNA-dependent RNA polymerases (RdRps). We identified a common structural core of 231 residues for all the structurally characterized viral RdRps, covering segmented and non-segmented negative-sense, positive-sense, and double-stranded RNA viruses infecting both prokaryotic and eukaryotic hosts. The grouping and branching of the viral RdRps in the structure-based phylogenetic tree follow their functional differentiation. The RdRps using protein primer, RNA primer, or self-priming mechanisms have evolved independently of each other, and the RdRps cluster into two large branches based on the used transcription mechanism. The structure-based distance tree presented here follows the recently established RdRp-based RNA virus classification at genus, subfamily, family, order, class and subphylum ranks. However, the topology of our phylogenetic tree suggests an alternative phylum level organization.

Our knowledge of the diversity and evolution of the virosphere will likely increase dramatically with the study of microbial eukaryotes, including the microalgae within which few RNA viruses have been documented. By combining total RNA sequencing with sequence and structural-based homology detection, we identified 18 novel RNA viruses in cultured samples from two major groups of microbial algae: the chlorophytes and the chlorarachniophytes. Most of the RNA viruses identified in the green algae class Ulvophyceae were related to the Tombusviridae and Amalgaviridae viral families commonly associated with land plants. This suggests that the evolutionary history of these viruses extends to divergence events between algae and land plants. Seven Ostreobium sp-associated viruses exhibited sequence similarity to the mitoviruses most commonly found in fungi, compatible with horizontal virus transfer between algae and fungi. We also document, for the first time, RNA viruses associated with chlorarachniophytes, including the first negative-sense (bunya-like) RNA virus in microalgae, as well as a distant homolog of the plant virus Virgaviridae, potentially signifying viral inheritance from the secondary chloroplast endosymbiosis that marked the origin of the chlorarachniophytes. More broadly, these data suggest that the scarcity of RNA viruses in algae results from limited investigation rather than their absence.

The coronavirus disease-2019 (COVID-19) pandemic continues to challenge health care systems around the world. Scientists and pharmaceutical companies have promptly responded by advancing potential therapeutics into clinical trials at an exponential rate. Initial encouraging results have been realized using remdesivir and dexamethasone. Yet, the research continues so as to identify better clinically relevant therapeutics that act either as prophylactics to prevent the infection or as treatments to limit the severity of COVID-19 and substantially decrease the mortality rate. Previously, we reviewed the potential therapeutics in clinical trials that block the early stage of the viral life cycle. In this review, we summarize potential anti-COVID-19 therapeutics that block/inhibit the post-entry stages of the viral life cycle. The review presents not only the chemical structures and mechanisms of the potential therapeutics under clinical investigation, i.e., listed in clinicaltrials.gov, but it also describes the relevant results of clinical trials. Their anti-inflammatory/immune-modulatory effects are also described. The reviewed therapeutics include small molecules, polypeptides, and monoclonal antibodies. At the molecular level, the therapeutics target viral proteins or processes that facilitate the post-entry stages of the viral infection. Frequent targets are the viral RNA-dependent RNA polymerase (RdRp) and the viral proteases such as papain-like protease (PLpro) and main protease (Mpro). Overall, we aim at presenting up-to-date details of anti-COVID-19 therapeutics so as to catalyze their potential effective use in fighting the pandemic.

At the beginning of 2020, the world was swept with a wave of a new coronavirus disease, named COVID-19 by the World Health Organization (WHO 2). The causative agent of this infection is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The data available on one of the promising therapeutic agents—nucleotide analog remdesivir (Gilead Sciences number GS-5734)—were evaluated. These data were concerned with remdesivir activation from the prodrug to the active molecule—triphosphate containing 1′-cyano group and modified nucleobase. This triphosphate competes with the natural substrate adenosine triphosphate. Additionally, its mechanisms of action based on RNA and proofreading exonuclease inhibition, leading to the delayed RNA chain termination of infected cells, and basic pharmacological data were assessed. Additionally, the analytical determination of remdesivir and its metabolites in cells and body liquids and also some data from remdesivir use in other RNA infections—such as Ebola, Nipah virus infection, and Middle East Respiratory Syndrome (MERS)—were summarized. More recent and more detailed data on the clinical use of remdesivir in COVID-19 were reported, showing the intensive efforts of clinicians and scientists to develop a cure for this new disease. Remdesivir as such represents one of the more promising alternatives for COVID-19 therapy, however the current understanding of this disease and the possible ways of dealing with it requires further investigation.

The Crimean–Congo hemorrhagic fever virus (CCHFV) is a segmented negative-sense RNA virus that can cause severe human disease. The World Health Organization (WHO) has listed CCHFV as a priority pathogen with an urgent need for enhanced research activities to develop effective countermeasures. We report on the expression, characterization, and inhibition of the CCHFV full-length L-protein that provides an important tool in this regard. The requirements for high biosafety measures hamper drug discovery and development efforts with infectious CCHFV. Hence, we decided to adopt a biochemical approach that targets the viral RNA-dependent RNA polymerase (RdRp). The CCHFV RdRp activity is part of a multifunctional L protein that is unusually large, with a molecular weight of ~450 kDa. The CCHFV L-protein also contains an ovarian tumor (OTU) domain that exhibits deubiquitinating (DUB) activity. Previous studies have shown that DUB activity interferes with innate immune responses and viral replication. Here, we utilized the baculovirus expression system and generated a full-length CCHFV L protein. RdRp activity was seen in the presence of divalent metal ions, and inhibition of RNA synthesis was demonstrated with nucleotide analogues. The ubiquitin analogue CC.4 inhibits the CCHFV-associated DUB activity of the full-length L protein and the isolated DUB domain to a similar extent. We have finally shown that RdRp and DUB activities are functionally independent. The full-length CCHFV L-protein provides an important tool for the discovery of antiviral agents. High-throughput screening (HTS) campaigns are now feasible. The same enzyme preparations can be utilized to identify polymerase and DUB inhibitors.

The bovine viral diarrhea virus (BVDV), a pestivirus from the family of Flaviviridae is ubiquitous and causes a range of clinical manifestations in livestock, mainly cattle. Two quinolinecarboxamide analogues were identified in a CPE-based screening effort, as selective inhibitors of the in vitro bovine viral diarrhea virus (BVDV) replication, i.e., TO505-6180/CSFCI (average EC50 = 0.07 µM, SD = 0.02 µM, CC50 > 100 µM) and TO502-2403/CSFCII (average EC50 = 0.2 µM, SD = 0.06 µM, CC50 > 100 µM). The initial antiviral activity observed for both hits against BVDV was corroborated by measuring the inhibitory effect on viral RNA synthesis and the production of infectious virus. Modification of the substituents on the quinolinecarboxamide scaffold resulted in analogues that proved about 7-fold more potent (average EC50 = 0.03 with a SD = 0.01 µM) and that were devoid of cellular toxicity, for the concentration range tested (SI = 3333). CSFCII resistant BVDV variants were selected and were found to carry the F224P mutation in the viral RNA-dependent RNA polymerase (RdRp), whereas CSFCI resistant BVDV carried two mutations in the same region of the RdRp, i.e., N264D and F224Y. Likewise, molecular modeling revealed that F224P/Y and N264D are located in a small cavity near the fingertip domain of the pestivirus polymerase. CSFC-resistant BVDV proved to be cross-resistant to earlier reported pestivirus inhibitors (BPIP, AG110, LZ37, and BBP) that are known to target the same region of the RdRp. CSFC analogues did not inhibit the in vitro activity of recombinant BVDV RdRp but inhibited the activity of BVDV replication complexes (RCs). CSFC analogues likely interact with the fingertip of the pestivirus RdRp at the same position as BPIP, AG110, LZ37, and BBP. This indicates that this region is a “hot spot” for the inhibition of pestivirus replication.

Influenza A viruses (IAVs) are a significant human pathogen that cause seasonal epidemics and occasional pandemics. Avian waterfowl are the natural reservoir of IAVs, but a wide range of species can serve as hosts. Most IAV strains are adapted to one host species and avian strains of IAV replicate poorly in most mammalian hosts. Importantly, IAV polymerases from avian strains function poorly in mammalian cells but host adaptive mutations can restore activity. The 2009 pandemic H1N1 (H1N1pdm09) virus acquired multiple mutations in the PA gene that activated polymerase activity in mammalian cells, even in the absence of previously identified host adaptive mutations in other polymerase genes. These mutations in PA localize within different regions of the protein suggesting multiple mechanisms exist to activate polymerase activity. Additionally, an immunomodulatory protein, PA-X, is expressed from the PA gene segment. PA-X expression is conserved amongst many IAV strains but activity varies between viruses specific for different hosts, suggesting that PA-X also plays a role in host adaptation. Here, we review the role of PA in the emergence of currently circulating H1N1pdm09 viruses and the most recent studies of host adaptive mutations in the PA gene that modulate polymerase activity and PA-X function.

The Sabin I poliovirus live, attenuated vaccine strain encodes for four amino acid changes (i.e., D53N, Y73H, K250E, and T362I) in the RNA-dependent RNA polymerase (RdRp). We have previously shown that the T362I substitution leads to a lower fidelity RdRp, and viruses encoding this variant are attenuated in a mouse model of poliovirus. Given these results, it was surprising that the nucleotide incorporation rate and nucleobase fidelity of the Sabin I RdRp is similar to that of wild-type enzyme, although the Sabin I RdRp is less selective against nucleotides with modified sugar groups. We suggest that the other Sabin amino acid changes (i.e., D53N, Y73H, K250E) help to re-establish nucleotide incorporation rates and nucleotide discrimination near wild-type levels, which may be a requirement for the propagation of the virus and its efficacy as a vaccine strain. These results also suggest that the nucleobase fidelity of the Sabin I RdRp likely does not contribute to viral attenuation.

Genome replication in flavivirus requires (—) strand RNA synthesis, (+) strand RNA synthesis, and 5’-RNA capping and methylation. To carry out viral genome replication, flavivirus assembles a replication complex, consisting of both viral and host proteins, on the cytoplasmic side of the endoplasmic reticulum (ER) membrane. Two major components of the replication complex are the viral non-structural (NS) proteins NS3 and NS5. Together they possess all the enzymatic activities required for genome replication, yet how these activities are coordinated during genome replication is not clear. We provide an overview of the flaviviral genome replication process, the membrane-bound replication complex, and recent crystal structures of full-length NS5. We propose a model of how NS3 and NS5 coordinate their activities in the individual steps of (—) RNA synthesis, (+) RNA synthesis, and 5’-RNA capping and methylation.

RNA viruses typically encode their own RNA-dependent RNA polymerase (RdRP) to ensure genome replication within the infected cells. RdRP function is critical not only for the virus life cycle but also for its adaptive potential. The combination of low fidelity of replication and the absence of proofreading and excision activities within the RdRPs result in high mutation frequencies that allow these viruses a rapid adaptation to changing environments. In this review, we summarize the current knowledge about structural and functional aspects on RdRP catalytic complexes, focused mainly in the Picornaviridae family. The structural data currently available from these viruses provided high-resolution snapshots for a range of conformational states associated to RNA template-primer binding, rNTP recognition, catalysis and chain translocation. As these enzymes are major targets for the development of antiviral compounds, such structural information is essential for the design of new therapies.

Potyviruses represent the largest group of known plant RNA viruses and include many agriculturally important viruses, such as Plum pox virus, Soybean mosaic virus, Turnip mosaic virus, and Potato virus Y. Potyviruses adopt polyprotein processing as their genome expression strategy. Among the 11 known viral proteins, the nuclear inclusion protein b (NIb) is the RNA-dependent RNA polymerase responsible for viral genome replication. Beyond its principal role as an RNA replicase, NIb has been shown to play key roles in diverse virus−host interactions. NIb recruits several host proteins into the viral replication complexes (VRCs), which are essential for the formation of functional VRCs for virus multiplication, and interacts with the sumoylation pathway proteins to suppress NPR1-mediated immunity response. On the other hand, NIb serves as a target of selective autophagy as well as an elicitor of effector-triggered immunity, resulting in attenuated virus infection. These contrasting roles of NIb provide an excellent example of the complex co-evolutionary arms race between plant hosts and potyviruses. This review highlights the current knowledge about the multifunctional roles of NIb in potyvirus infection, and discusses future research directions.

We report high rates of detection (35.36%, 29/82) of genogroup-I (GI) picobirnaviruses (PBVs) in non-diarrheic fecal samples from the small Indian mongoose (Urva auropunctata). In addition, we identified a novel PBV-like RNA-dependent RNA polymerase (RdRp) gene sequence that uses an alternative mitochondrial genetic code (that of mold or invertebrate) for translation. The complete/nearly complete gene segment-2/RdRp gene sequences of seven mongoose PBV GI strains and the novel PBV-like strain were obtained by combining a modified non-specific primer-based amplification method with conventional RT-PCRs, facilitated by the inclusion of a new primer targeting the 3′-untranslated region (UTR) of PBV gene segment-2. The mongoose PBV and PBV-like strains retained the various features that are conserved in gene segment-2/RdRps of other PBVs. However, high genetic diversity was observed among the mongoose PBVs within and between host species. This is the first report on detection of PBVs in the mongoose. Molecular characterization of the PBV and PBV-like strains from a new animal species provided important insights into the various features and complex diversity of PBV gene segment-2/putative RdRps. The presence of the prokaryotic ribosomal binding site in the mongoose PBV genomes, and analysis of the novel PBV-like RdRp gene sequence that uses an alternative mitochondrial genetic code (especially that of mold) for translation corroborated recent speculations that PBVs may actually infect prokaryotic or fungal host cells.

Flaviviruses, most of which are emerging and re-emerging human pathogens and significant public health concerns worldwide, are positive-sense RNA viruses. Flavivirus replication occurs on the ER and is regulated by many mechanisms and factors. NS5, which consists of a C-terminal RdRp domain and an N-terminal methyltransferase domain, plays a pivotal role in genome replication and capping. The C-terminal RdRp domain acts as the polymerase for RNA synthesis and cooperates with diverse viral proteins to facilitate productive RNA proliferation within the replication complex. Here, we provide an overview of the current knowledge of the functions and characteristics of the RdRp, including the subcellular localization of NS5, as well as the network of interactions formed between the RdRp and genome UTRs, NS3, and the methyltransferase domain. We posit that a detailed understanding of RdRp functions may provide a target for antiviral drug discovery and therapeutics.

RNA silencing has evolved as a widespread antiviral strategy in many eukaryotic organisms. Antiviral RNA silencing is mediated by virus-derived small RNAs (vsiRNAs), created by the cleavage of double-stranded viral RNA substrates by Dicer (Dcr) in animals or Dicer-like (DCL) proteins in plants. However, little is known about how the RNA silencing mechanisms of different hosts respond to the same virus infection. We performed high-throughput small RNA sequencing in Nephotettix cincticeps and Oryza sativa infected with Rice dwarf phytoreovirus and analyzed the distinct accumulation of vsiRNAs in these two hosts. The results suggested a potential branch in the evolution of antiviral RNA silencing of insect and plant hosts. The rice vsiRNAs were predominantly 21 and 22 nucleotides (nt) long, suggesting that OsDCL4 and OsDCL2 are involved in their production, whereas 21-nt vsiRNAs dominated in leafhopper, suggesting the involvement of a Dcr-2 homolog. Furthermore, we identified ~50-fold more vsiRNAs in rice than in leafhoppers, which might be partially attributable to the activity of RNA-dependent RNA polymerase 6 (RDR6) in rice and the lack of RDR genes in leafhoppers. Our data established a basis for further comparative studies on the evolution of RNA silencing-based interactions between a virus and its hosts, across kingdoms.

Vesicular stomatitis virus (VSV) represents a promising platform for developing oncolytic viruses, as well as vaccines against significant human pathogens. To safely control VSV infection in humans, small-molecule drugs that selectively inhibit VSV infection may be needed. Here, using a cell-based high-throughput screening assay followed by an in vitro transcription assay, compounds with a 7-hydroxy-6-methyl-3,4-dihydroquinolin-2(1H)-one structure and an aromatic group at position 4 (named vesiculopolins, VPIs) were identified as VSV RNA polymerase inhibitors. The most effective compound, VPI A, inhibited VSV-induced cytopathic effects and in vitro mRNA synthesis with micromolar to submicromolar 50% inhibitory concentrations. VPI A was found to inhibit terminal de novo initiation rather than elongation for leader RNA synthesis, but not mRNA capping, with the VSV L protein, suggesting that VPI A is targeted to the polymerase domain in the L protein. VPI A inhibited transcription of Chandipura virus, but not of human parainfluenza virus 3, suggesting that it specifically acts on vesiculoviral L proteins. These results suggest that VPIs may serve not only as molecular probes to elucidate the mechanisms of transcription of vesiculoviruses, but also as lead compounds to develop antiviral drugs against vesiculoviruses and other related rhabdoviruses.

Foot-and-mouth disease virus (FMDV) is an RNA virus belonging to the Picornaviridae family that contains three small viral proteins (VPgs), named VPg1, VPg2 and VPg3, linked to the 5′-end of the viral genome. These VPg proteins act as primers for RNA replication, which is initiated by the consecutive binding of two UMP molecules to the hydroxyl group of Tyr3 in VPg. This process, termed uridylylation, is catalyzed by the viral RNA-dependent RNA polymerase named 3Dpol. 5-Fluorouridine triphosphate (FUTP) is a potent competitive inhibitor of VPg uridylylation. Peptide analysis showed FUMP covalently linked to the Tyr3 of VPg. This fluorouridylylation prevents further incorporation of the second UMP residue. The molecular basis of how the incorporated FUMP blocks the incorporation of the second UMP is still unknown. To investigate the mechanism of inhibition of VPg uridylylation by FUMP, we have prepared a simplified 15-mer model of VPg1 containing FUMP and studied its x-ray crystal structure in complex with 3Dpol. Unfortunately, the fluorouridylylated VPg1 was disordered and not visible in the electron density maps; however, the structure of 3Dpol in the presence of VPg1-FUMP showed an 8 Å movement of the β9-α11 loop of the polymerase towards the active site cavity relative to the complex of 3Dpol with VPg1-UMP. The conformational rearrangement of this loop preceding the 3Dpol B motif seems to block the access of the template nucleotide to the catalytic cavity. This result may be useful in the design of new antivirals against not only FMDV but also other picornaviruses, since all members of this family require the uridylylation of their VPg proteins to initiate the viral RNA synthesis.

Zika virus (ZIKV), an emerging flavivirus that causes neurodevelopmental impairment to fetuses and has been linked to Guillain-Barré syndrome continues to threaten global health due to the absence of targeted prophylaxis or treatment. Nucleoside analogues are good examples of efficient anti-viral inhibitors, and prodrug strategies using phosphate masking groups (ProTides) have been employed to improve the bioavailability of ribonucleoside analogues. Here, we synthesized and tested a small library of 13 ProTides against ZIKV in human neural stem cells. Strong activity was observed for 2′-C-methyluridine and 2′-C-ethynyluridine ProTides with an aryloxyl phosphoramidate masking group. Substitution of a 2-(methylthio) ethyl phosphoramidate for the aryloxyl phosphoramidate ProTide group of 2′-C-methyluridine completely abolished antiviral activity of the compound. The aryloxyl phosphoramidate ProTide of 2′-C-methyluridine outperformed the hepatitis C virus (HCV) drug sofosbuvir in suppression of viral titers and protection from cytopathic effect, while the former compound’s triphosphate active metabolite was better incorporated by purified ZIKV NS5 polymerase over time. These findings suggest both a nucleobase and ProTide group bias for the anti-ZIKV activity of nucleoside analogue ProTides in a disease-relevant cell model.