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Summary RNA‐dependent RNA Polymerases (RdRPs) synthesize double‐stranded RNA (dsRNA) from a single‐stranded RNA (ssRNA) template. In plants, dsRNAs produced by RdRPs can be further processed into small interfering RNA (siRNAs) with different lengths, ranging from 21 to 24 nucleotides (nt). These siRNAs play a pivotal role in various biological processes, including antiviral responses, transposable elements silencing, DNA methylation, and the regulation of plant reproduction and development. Recent research has reported significant progress in uncovering the molecular mechanisms of plant RNA‐DEPENDENT RNA POLYMERASE 2 (RDR2), a representative RdRP involved in the RNA‐directed DNA methylation (RdDM) pathway. These discoveries provide a molecular basis underlying the principles of RdRP function and offer insights into potential advancements in crop breeding and antiviral defense strategies. [ABSTRACT FROM AUTHOR]

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 (<69%, <54%, and <50% identities with reptilian/amphibian AstVs, avastroviruses, and mamastroviruses, respectively). Phylogenetically, the group-I and group-II Mon-AstVs formed two distinct clusters, near the cluster of reptilian/amphibian AstVs, and were distantly related to avastroviruses and mamastroviruses. Since the mongooses were apparently healthy during sampling, we could not establish if the Mon-AstVs infected the animal or were of dietary origin. Although we could not ascertain the true host of the Mon-AstVs, phylogenetic analysis indicated that these viruses might have originated from lower vertebrates. To our knowledge, this is the first report on the detection and molecular characterization of AstVs in mongooses, highlighting the wide host range and significant genetic diversity within the family Astroviridae. [ABSTRACT FROM AUTHOR]

The global prevalence of RNA virus infections has presented significant challenges to public health in recent years, necessitating the expansion of its alternative therapeutic library. Due to its evolutional conservation, RNA‐dependent RNA polymerase (RdRp) has emerged as a potential target for broad‐spectrum antiviral nucleoside analogues. However, after over half a century of structural modification, exploring unclaimed chemical space using frequently‐used structural substitution methods to design new nucleoside analogues is challenging. In this study, we explore the use of the "ring‐opening" strategy to design new base mimics, thereby using these base mimics to design new nucleoside analogues with broad‐spectrum antiviral activities. A total of 29 compounds were synthesized. Their activity against viral RdRp was initially screened using an influenza A virus RdRp high‐throughput screening model. Then, the antiviral activity of 38a was verified against influenza virus strain A/PR/8/34 (H1N1), demonstrating a 50% inhibitory concentration (IC50) value of 9.95 μM, which was superior to that of ribavirin (the positive control, IC50 = 11.43 μM). Moreover, 38a also has inhibitory activity against coronavirus 229E with an IC50 of 30.82 μM. In addition, compounds 42 and 46f exhibit an 82% inhibition rate against vesicular stomatitis virus at a concentration of 20 μM and hardly induce cytotoxicity in host cells. This work demonstrates the feasibility of designing nucleoside analogues with "ring‐opening" bases and suggests the "ring‐opening" nucleosides may have greater polarity, and designing prodrugs is an important aspect of optimizing their antiviral activity. Future research should focus on enhancing the conformational restriction of open‐loop bases to mimic Watson‐Crick base pairing better and improve antiviral activity. [ABSTRACT FROM AUTHOR]

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. IMPORTANCE Japanese 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In a recent characterization of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variability present in 30 diagnostic samples from patients of the first COVID-19 pandemic wave, 41 amino acid substitutions were documented in the RNA-dependent RNA polymerase (RdRp) nsp12. Eight substitutions were selected in this work to determine whether they had an impact on the RdRp activity of the SARS-CoV-2 nsp12-nsp8-nsp7 replication complex. Three of these substitutions were found around the polymerase central cavity, in the template entry channel (D499G and M668V), and within the motif B (V560A), and they showed polymerization rates similar to the wild type RdRp. The remaining five mutations (P323L, L372F, L372P, V373A, and L527H) were placed near the nsp12-nsp8F contact surface; residues L372, V373, and L527 participated in a large hydrophobic cluster involving contacts between two helices in the nsp12 fingers and the long a-helix of nsp8F. The presence of any of these five amino acid substitutions resulted in important alterations in the RNA polymerization activity. Comparative primer elongation assays showed different behavior depending on the hydrophobicity of their side chains. The substitution of L by the bulkier F side chain at position 372 slightly promoted RdRp activity. However, this activity was dramatically reduced with the L372P, and L527H mutations, and to a lesser extent with V373A, all of which weaken the hydrophobic interactions within the cluster. Additional mutations, specifically designed to disrupt the nsp12-nsp8F interactions (nsp12-V330S, nsp12-V341S, and nsp8-R111A/D112A), also resulted in an impaired RdRp activity, further illustrating the importance of this contact interface in the regulation of RNA synthesis. [ABSTRACT FROM AUTHOR]

Arenaviridae is a family for ambisense RNA viruses with genomes of about 10.5 kb that infect mammals, snakes, and fish. The arenavirid genome consists of two or three single-stranded RNA segments and encodes a nucleoprotein (NP), a glycoprotein (GP) and a large (L) protein containing RNA-directed RNA polymerase (RdRP) domains; some arenavirids encode a zinc-binding protein (Z). This is a summary of the International Committee on Taxonomy of Viruses (ICTV) report on the family Arenaviridae, which is available at www.ictv.global/report/arenaviridae.

In April 2023, following the annual International Committee on Taxonomy of Viruses (ICTV) ratification vote on newly proposed taxa, the phylum Negarnaviricota was amended and emended. The phylum was expanded by one new family, 14 new genera, and 140 new species. Two genera and 538 species were renamed. One species was moved, and four were abolished. This article presents the updated taxonomy of Negarnaviricota as now accepted by the ICTV.

The RNA-dependent RNA polymerase (RdRp) represents a prominent target in the discovery and development of new antivirotics against RNA viruses, inhibiting the replication process. One of the most targeted RNA viruses of the last years is, without doubt, SARS-CoV-2, the cause of the recent COVID-19 pandemic. HeE1-2Tyr, a known inhibitor of flaviviral RdRp, has been discovered to also have antiviral potency against this coronavirus. In this study, we report three distinct modifications of HeE1-2Tyr: conversion of the core from a benzothiazole to a benzoxazole moiety and two different scaffold simplifications, respectively. We provide a novel synthetic approach and, in addition, evaluate the final molecules in an in vitro polymerase assay for biological activity.

Background: The RNA-dependent RNA polymerase (RdRp) inhibitors, molnupiravir and VV116, have the potential to maximize clinical benefits in the oral treatment of COVID-19. Subjects who consume these drugs may experience an increased incidence of adverse events. This study aimed to evaluate the safety profile of molnupiravir and VV116.Methods: A comprehensive search of scientific and medical databases, such as PubMed Central/Medline, Embase, Web of Science, and Cochrane Library, was conducted to find relevant articles in English from January 2020 to June 2023. Any kind of adverse events reported in the study were pooled and analyzed in the drug group versus the control group. Estimates of risk effects were summarized through the random effects model using Review Manager version 5.2, and sensitivity analysis was performed by Stata 17.0 software.Results: Fifteen studies involving 32,796 subjects were included. Eleven studies were placebo-controlled, and four were Paxlovid-controlled. Twelve studies reported adverse events for molnupiravir, and three studies described adverse events for VV116. The total odds ratio (OR) for adverse events in the RdRp inhibitor versus the placebo-controlled group was 1.01 (95% CI=0.84-1.22; I2=26%), P=0.88. The total OR for adverse events in the RdRp inhibitor versus the Paxlovid-controlled group was 0.32 (95% CI=0.16-0.65; I2=87%), P=0.002. Individual drug subgroup analysis in the placebo-controlled study showed that compared with the placebo group, a total OR for adverse events was 0.97 (95% CI, 0.85-1.10; I2=0%) in the molnupiravir group and 3.77 (95% CI=0.08-175.77; I2=85%) in the VV116 group.Conclusion: The RdRp inhibitors molnupiravir and VV116 are safe for oral treatment of COVID-19. Further evidence is necessary that RdRp inhibitors have a higher safety profile than Paxlovid.

I. Watanabe et al. isolated approximately 30 strains of RNA phages from various parts of Japan. To isolate RNA phages, they assessed the infection specificity of male Escherichia coli and RNase sensitivity. They found that the isolated strains of RNA phages could be serologically separated into three groups. Furthermore, most of them were serologically related, and the antiphage rabbit serum prepared by one of these phages neutralized most of the other phages. The only serologically unrelated phage was the RNA phage Qβ, which was isolated at the Institute for Virus Research, Kyoto University, in 1961.

Tomato Yellow Leaf Curl Virus (TYLCV) is one of the most devastating pathogens of tomato, worldwide. It is vectored by the globally prevalent whitefly, Bemisia tabaci, and is asymptomatic in a wide range of plant species that act as a virus reservoir. The most successful crop protection for tomato in the field has been from resistance genes, of which five loci have been introgressed fromwild relatives. Of these, the Ty-1/Ty-3 locus, which encodes an RNA-dependent RNA polymerase 3 (RDR3), has been the most effective. Nevertheless, several TYLCV strains that break this resistance are beginning to emerge, increasing the need for new sources of resistance. Here we use segregation analysis and CRISPR-mediated gene dysfunctionalisation to dissect the differential response of two isolates of Nicotiana benthamiana to TYLCV infection. Our study indicates the presence of a novel non-RDR3, but yet to be identified, TYLCV resistance gene in a wild accession of N. benthamiana. This gene has the potential to be incorporated into tomatoes. [ABSTRACT FROM AUTHOR]

Yellow leaf disease (YLD) poses a significant challenge to areca palm cultivation, yet its etiology remains uncertain. During our investigation of YLD-affected areca palm plants, transcriptome sequencing revealed an RNA contig exhibiting striking similarities to the RNA-dependent RNA polymerase (RdRp) of ormycoviruses. Subsequent gene cloning techniques yielded the full-length sequence of this RNA, potentially representing either the complete or partial genome of a hitherto unidentified ormycovirus, tentatively named areca palm yellow leaf-associated ormycovirus (APYLaOMV). RT-PCR detection found that APYLaOMV is present in over 30% of YLD-affected areca palm samples but is absent in healthy ones, suggesting a potential link between APYLaOMV and YLD. In summary, these data could be valuable in understanding the etiology of YLD in areca palms. [ABSTRACT FROM AUTHOR]

Themosquito-borne Rift Valley fever virus (RVFV) fromthe Phenuiviridae family is a single-stranded RNA virus that causes the re-emerging zoonotic disease Rift Valley fever (RVF). Classified as a Category A agent by the NIH, RVFV infection can cause debilitating disease or death in humans and lead to devastating economic impacts by causing abortion storms in pregnant cattle. In a previous study, we showed that the host chaperone protein HSP90 is an RVFV-associated host factor that plays a critical role post viral entry, during the active phase of viral genome replication/transcription. In this study, we have elucidated themolecular mechanisms behind the regulatory effect of HSP90 during infection with RVFV. Our results demonstrate that during the early infection phase, host HSP90 associates with the viral RNA-dependent RNA polymerase (L protein) and prevents its degradation through the proteasome, resulting in increased viral replication. [ABSTRACT FROM AUTHOR]

RNA-dependent RNA polymerase (RdRp) is a marker gene for RNA viruses; thus, it is widely used to identify RNA viruses from metatranscriptome data. However, because of the high diversity of RdRp domains, it remains difficult to identify RNA viruses using RdRp sequences. To overcome this problem, we created a NeoRdRp database containing 1,182 hidden Markov model (HMM) profiles utilizing 12,502 RdRp domain sequences. Since the development of this database, more RNA viruses have been discovered, mainly through metatranscriptome sequencing analyses. To identify RNA viruses comprehensively and specifically, we updated the NeoRdRp by incorporating recently reported RNA viruses. To this end, 557,197 RdRp-containing sequences were used as seed RdRp datasets. These sequences were processed through deduplication, clustering, alignment, and splitting, thereby generating 19,394 HMM profiles. We validated the updated NeoRdRp database, using the UniProtKB dataset and found that the recall and specificity rates were improved to 99.4% and 81.6%, from 97.2% and 76.8% in the previous version, respectively. Comparisons of eight different RdRp search tools showed that NeoRdRp2 exhibited balanced RdRp and nonspecific detection power. Expansion of the annotated RdRp datasets is expected to further accelerate the discovery of novel RNA viruses from various transcriptome datasets. The HMM profiles of NeoRdRp2 and their annotations are available at https://github.com/shoichisakaguchi/NeoRdRp. [ABSTRACT FROM AUTHOR]

Fusarium head blight is a destructive disease of small grains. The disease is predominantly caused by the haploid ascomycete fungus Fusarium graminearum in North America. To understand the genetics of quantitative traits for sensitivity to fungicides in this fungal pathogen, we conducted a genome-wide association study of sensitivity to two demethylation inhibition class fungicides, tebuconazole and prothioconazole, using an F. graminearum population of 183 isolates collected between 1981 and 2013 from North Dakota. Baseline sensitivity to tebuconazole and prothioconazole was established using 21 isolates collected between 1981 and 1994. Most fungal isolates were sensitive to both tebuconazole and prothioconazole; however, five isolates showed significantly reduced sensitivity to prothioconazole. The genome-wide association study identified one significant marker-trait association on chromosome 3 for tebuconazole resistance, whereas six significant marker-trait associations, one on chromosome 1, three on chromosome 2, and two on chromosome 4, were detected for prothioconazole resistance. Functional annotation of the marker-trait association for tebuconazole revealed a candidate gene encoding a basic helix-loop-helix domain-containing protein that reinforces sterol in the fungal membrane. Putative genes for prothioconazole resistance were also identified, which are involved in RNA interference, the detoxification by ubiquitin-proteasome pathway, and membrane integrity reinforcement. Considering the potential of the pathogen toward overcoming chemical control, continued monitoring of fungal sensitivities to commercially applied fungicides, especially those containing prothioconazole, is warranted to reduce risks of fungicide resistance in the pathogen populations. [ABSTRACT FROM AUTHOR]

Improved RNA virus understanding is critical to studying animal and plant health, and environmental processes. However, the continuous and rapid RNA virus evolution makes their identification and characterization challenging. While recent sequence-based advances have led to extensive RNA virus discovery, there is growing variation in how RNA viruses are identified, analyzed, characterized, and reported. To this end, an RdRp Summit was organized and a hybrid meeting took place in Valencia, Spain in May 2023 to convene leading experts with emphasis on early career researchers (ECRs) across diverse scientific communities. Here we synthesize key insights and recommendations and offer these as a first effort to establish a consensus framework for advancing RNA virus discovery. First, we need interoperability through standardized methodologies, data-sharing protocols, metadata provision and interdisciplinary collaborations and offer specific examples as starting points. Second, as an emergent field, we recognize the need to incorporate cutting-edge technologies and knowledge early and often to improve omic-based viral detection and annotation as novel capabilities reveal new biology. Third, we underscore the significance of ECRs in fostering international partnerships to promote inclusivity and equity in virus discovery efforts. The proposed consensus framework serves as a roadmap for the scientific community to collectively contribute to the tremendous challenge of unveiling the RNA virosphere. [ABSTRACT FROM AUTHOR]

RNA viruses are ubiquitous components of the global virosphere, yet relatively little is known about their genetic diversity or the cellular mechanisms by which they exploit the biology of their diverse eukaryotic hosts. A hallmark of (+)ssRNA (positive single-stranded RNA) viruses is the ability to remodel host endomembranes for their own replication. However, the subcellular interplay between RNA viruses and host organelles that harbor gene expression systems, such as mitochondria, is complex and poorly understood. Here we report the discovery of 763 new virus sequences belonging to the family Mitoviridae by metatranscriptomic analysis, the identification of previously uncharacterized mitovirus clades, and a putative new viral class. With this expanded understanding of the diversity of mitovirus and encoded RNA-dependent RNA polymerases (RdRps), we annotate mitovirus-specific protein motifs and identify hallmarks of mitochondrial translation, including mitochondrion-specific codons. This study expands the known diversity of mitochondrial viruses and provides additional evidence that they co-opt mitochondrial biology for their survival. IMPORTANCE Metatranscriptomic studies have rapidly expanded the cadre of known RNA viruses, yet our understanding of how these viruses navigate the cytoplasmic milieu of their hosts to survive remains poorly characterized. In this study, we identify and assemble 763 new viral sequences belonging to the Mitoviridae, a family of (+)ssRNA viruses thought to interact with and remodel host mitochondria. We exploit this genetic diversity to identify new clades of Mitoviridae, annotate clade-specific sequence motifs that distinguish the mitoviral RdRp, and reveal patterns of RdRp codon usage consistent with translation on host cell mitoribosomes. These results serve as a foundation for understanding how mitoviruses co-opt mitochondrial biology for their proliferation.

Influenza is one of the leading causes of disease-related mortalities worldwide. Several strategies have been implemented during the past decades to hinder the replication cycle of influenza viruses, all of which have resulted in the emergence of resistant virus strains. The most recent example is baloxavir marboxil, where a single mutation in the active site of the target endonuclease domain of the RNA-dependent-RNA polymerase renders the recent FDA approved compound ∼1000-fold less effective. Raltegravir is a first-in-class HIV inhibitor that shows modest activity to the endonuclease. Here, we have used structure-guided approaches to create rationally designed derivative molecules that efficiently engage the endonuclease active site. The design strategy was driven by our previously published structures of endonuclease-substrate complexes, which allowed us to target functionally conserved residues and reduce the likelihood of resistance mutations. We succeeded in developing low nanomolar equipotent inhibitors of both wild-type and baloxavir-resistant endonuclease. We also developed macrocyclic versions of these inhibitors that engage the active site in the same manner as their 'open' counterparts but with reduced affinity. Structural analyses provide clear avenues for how to increase the affinity of these cyclic compounds.

Abstract The interaction between viral components and cellular proteins plays a crucial role in viral replication. In a previous study, we showed that the 3′—untranslated region (3′—UTR) is an essential element for the replication of duck hepatitis A virus type 1 (DHAV-1). However, the underlying mechanism is still unclear. To gain a deeper understanding of this mechanism, we used an RNA pull-down and a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry assay to identify new host factors that interact with the 3′—UTR. We selected interleukin-2 enhancer binding factor 2 (ILF2) for further analysis. We showed that ILF2 interacts specifically with both the 3′—UTR and the 3D polymerase (3Dpol) of DHAV-1 through in vitro RNA pull-down and co-immunoprecipitation assays, respectively. We showed that ILF2 negatively regulates viral replication in duck embryo fibroblasts (DEFs), and that its overexpression in DEFs markedly suppresses DHAV-1 replication. Conversely, ILF2 silencing resulted in a significant increase in viral replication. In addition, the RNA-dependent RNA polymerase (RdRP) activity of 3Dpol facilitated viral replication by enhancing viral RNA translation efficiency, whereas ILF2 disrupted the role of RdRP in viral RNA translation efficiency to suppress DHAV-1 replication. At last, DHAV-1 replication markedly suppressed the expression of ILF2 in DEFs, duck embryo hepatocytes, and different tissues of 1 day-old ducklings. A negative correlation was observed between ILF2 expression and the viral load in primary cells and different organs of young ducklings, suggesting that ILF2 may affect the viral load both in vitro and in vivo.

Objective: Recent evidence reported that some dietary compounds like quercetin and apigenin as the most well-known flavonoids with anti-inflammatory effects may inhibit SARS-CoV-2 main protease. The hypothesis of the promising effects and possible mechanisms of action of quercetin against COVID-19 were assessed in this article.Materials and Methods: Related papers on the inhibitory effects of quercetin against COVID-19 were collected using the following search strategy: “corona or coronavirus or COVID or COVID-19 or viral or virus” AND “nutrient or flavonoid or Quercetin”.Results: The findings indicated that quercetin can be considered an effective agent against COVID-19 because of its SARS-CoV-2 main protease and RNA-dependent RNA polymerase inhibitory effects. In addition, quercetin may attenuate angiotensin-converting enzyme-2 (ACE-2) receptors leading to a reduction of SARS-CoV-2 ability to enter host cells. Moreover, the antiviral, anti-inflammatory, and immunomodulatory activities of quercetin have been frequently reported.Conclusion: Quercetin may be an effective agent for managing the complications of COVID-19. Further longitudinal human studies are warranted.

ABSTRACTThe Picornaviridae are a large group of positive-sense, single-stranded RNA viruses, and most research has focused on the Enterovirus genus, given they present a severe health risk to humans. Other picornaviruses, such as foot-and-mouth disease virus (FMDV) and senecavirus A (SVA), affect agricultural production with high animal mortality to cause huge economic losses. The 3Dpol protein of picornaviruses is widely known to be used for genome replication; however, a growing number of studies have demonstrated its non-polymerase roles, including modulation of host cell biological processes, viral replication complex assembly and localization, autophagy, and innate immune responses. Currently, there is no effective vaccine to control picornavirus diseases widely, and clinical therapeutic strategies have limited efficiency in combating infections. Many efforts have been made to develop different types of drugs to prohibit virus survival; the most important target for drug development is the virus polymerase, a necessary element for virus replication. For picornaviruses, there are also active efforts in targeted 3Dpol drug development. This paper reviews the interaction of 3Dpol proteins with the host and the progress of drug development targeting 3Dpol.

High-throughput RNA sequencing offers broad opportunities to explore the Earth RNA virome. Mining 5,150 diverse metatranscriptomes uncovered >2.5 million RNA virus contigs. Analysis of >330,000 RNA-dependent RNA polymerases (RdRPs) shows that this expansion corresponds to a 5-fold increase of the known RNA virus diversity. Gene content analysis revealed multiple protein domains previously not found in RNA viruses and implicated in virus-host interactions. Extended RdRP phylogeny supports the monophyly of the five established phyla and reveals two putative additional bacteriophage phyla and numerous putative additional classes and orders. The dramatically expanded phylum Lenarviricota, consisting of bacterial and related eukaryotic viruses, now accounts for a third of the RNA virome. Identification of CRISPR spacer matches and bacteriolytic proteins suggests that subsets of picobirnaviruses and partitiviruses, previously associated with eukaryotes, infect prokaryotic hosts.

The interaction between viral components and cellular proteins plays a crucial role in viral replication. In a previous study, we showed that the 3′—untranslated region (3′—UTR) is an essential element for the replication of duck hepatitis A virus type 1 (DHAV-1). However, the underlying mechanism is still unclear. To gain a deeper understanding of this mechanism, we used an RNA pull-down and a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry assay to identify new host factors that interact with the 3′—UTR. We selected interleukin-2 enhancer binding factor 2 (ILF2) for further analysis. We showed that ILF2 interacts specifically with both the 3′—UTR and the 3D polymerase (3Dpol) of DHAV-1 through in vitro RNA pull-down and co-immunoprecipitation assays, respectively. We showed that ILF2 negatively regulates viral replication in duck embryo fibroblasts (DEFs), and that its overexpression in DEFs markedly suppresses DHAV-1 replication. Conversely, ILF2 silencing resulted in a significant increase in viral replication. In addition, the RNA-dependent RNA polymerase (RdRP) activity of 3Dpol facilitated viral replication by enhancing viral RNA translation efficiency, whereas ILF2 disrupted the role of RdRP in viral RNA translation efficiency to suppress DHAV-1 replication. At last, DHAV-1 replication markedly suppressed the expression of ILF2 in DEFs, duck embryo hepatocytes, and different tissues of 1 day-old ducklings. A negative correlation was observed between ILF2 expression and the viral load in primary cells and different organs of young ducklings, suggesting that ILF2 may affect the viral load both in vitro and in vivo. [ABSTRACT FROM AUTHOR]

Viruses provide vital insights into gene expression control. Viral transactivators, with other viral and cellular proteins, regulate expression of self, other viruses, and host genes with profound effects on infected cells, underlying inflammation, control of immune responses, and pathogenesis. The multifunctional Tat proteins of lentiviruses (HIV-1, HIV-2, and SIV) transactivate gene expression by recruiting host proteins and binding to transacting responsive regions (TARs) in viral and host RNAs. SARS-CoV-2 nucleocapsid participates in early viral transcription, recruits similar cellular proteins, and shares intracellular, surface, and extracellular distribution with Tat. SARS-CoV-2 nucleocapsid interacting with the replication–transcription complex might, therefore, transactivate viral and cellular RNAs in the transcription and reactivation of self and other viruses, acute and chronic pathogenesis, immune evasion, and viral evolution. Here, we show, by using primary and secondary structural comparisons, that the leaders of SARS-CoV-2 and other coronaviruses contain TAR-like sequences in stem-loops 2 and 3. The coronaviral nucleocapsid C-terminal domains harbor a region of similarity to TAR-binding regions of lentiviral Tat proteins, and coronaviral nonstructural protein 12 has a cysteine-rich metal binding, dimerization domain, as do lentiviral Tat proteins. Although SARS-CoV-1 nucleocapsid transactivated gene expression in a replicon-based study, further experimental evidence for coronaviral transactivation and its possible implications is warranted. [ABSTRACT FROM AUTHOR]

A novel partitivirus, Fusarium commune partitivirus 1 (FcoPV1), was identified in Fusarium commune strain CP-SX-3 isolated from diseased roots of strawberry with symptoms of root rot. The complete genome of FcoPV1 comprises three double-stranded RNAs (dsRNAs): dsRNA1 (1,825 nt), dsRNA2 (1,592 nt), and dsRNA3 (1,421 nt). dsRNA1 contains a single open reading frame (ORF1) encoding an RNA-dependent RNA polymerase (RdRp), and dsRNA2 contains a single ORF (ORF2) encoding a coat protein (CP). dsRNA3 is a possible satellite RNA that does not appear to encode a known protein. BLASTp analysis revealed that RdRp (86.59%) and CP (74.13%) encoded by the two ORFs (ORF1 and ORF2) had the highest sequence similarity to their counterparts in Fusarium equiseti partitivirus 1 (FePV1). Phylogenetic analysis based on the complete amino acid sequence of RdRp suggested that FcoPV1 should be considered a member of a new species in the proposed genus “Zetapartitivirus” within the family Partitiviridae. To the best of our knowledge, this is the first report of a zetapartitivirus infecting phytopathogenic F. commune. [ABSTRACT FROM AUTHOR]

Dengue virus (DENV) causes dengue fever and dengue hemorrhagic fever, and DENV infection kills 20,000 people annually worldwide. Therefore, the development of anti-DENV drugs is urgently needed. Sofosbuvir (SOF) is an effective drug for HCV-related diseases, and its triphosphorylated metabolite inhibits viral RNA synthesis by the RNA-dependent RNA polymerase (RdRp) of HCV. (2′R)-2′-Deoxy-2′-fluoro-2′-methyluridine (FMeU) is the dephosphorylated metabolite produced from SOF. The effects of SOF and FMeU on DENV1 replication were analyzed using two DENV1 replicon-based methods that we previously established. First, a replicon-harboring cell assay showed that DENV1 replicon replication in human hepatic Huh7 cells was decreased by SOF but not by FMeU. Second, a transient replicon assay showed that DENV1 replicon replication in Huh7 cells was decreased by SOF; however, in hamster kidney BHK-21 cells, it was not suppressed by SOF. Additionally, the replicon replication in Huh7 and BHK-21 cells was not affected by FMeU. Moreover, we assessed the effects of SOF on infectious DENV1 production. SOF suppressed infectious DENV1 production in Huh7 cells but not in monkey kidney Vero cells. To examine the substrate recognition of the HCV and DENV1 RdRps, the complex conformation of SOF-containing DENV1 RdRp or HCV RdRp was predicted using AlphaFold 2. These results indicate that SOF may be used as a treatment for DENV1 infection. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Aims Despite metatranscriptomics becoming an emerging tool for pathogen surveillance, very little is known about the feasibility of this approach for understanding the fate of human-derived pathogens in drinking water sources. Methods and results We conducted multiplexed microfluidic cards and metatranscriptomic sequencing of the drinking water source in a border city of North Korea in four seasons. Microfluidic card detected norovirus, hepatitis B virus (HBV), enterovirus, and Vibrio cholerae in the water. Phylogenetic analyses showed that environmental-derived sequences from norovirus GII.17, genotype C of HBV, and coxsackievirus A6 (CA6) were genetically related to the local clinical isolates. Meanwhile, metatranscriptomic assembly suggested that several bacterial pathogens, including Acinetobacter johnsonii and V. cholerae might be prevalent in the studied region. Metatranscriptomic analysis recovered 349 species-level groups with substantial viral diversity without detection of norovirus, HBV, and CA6. Seasonally distinct virus communities were also found. Specifically, 126, 73, 126, and 457 types of viruses were identified in spring, summer, autumn, and winter, respectively. The viromes were dominated by the Pisuviricota phylum, including members from Marnaviridae, Dicistroviridae, Luteoviridae, Potyviridae, Picornaviridae, Astroviridae , and Picobirnaviridae families. Further phylogenetic analyses of RNA (Ribonucleic Acid)-dependent RNA polymerase (RdRp) sequences showed a diverse set of picorna-like viruses associated with shellfish, of which several novel picorna-like viruses were also identified. Additionally, potential animal pathogens, including infectious bronchitis virus, Bat dicibavirus, Bat nodavirus, Bat picornavirus 2, infectious bursal disease virus, and Macrobrachium rosenbergii nodavirus were also identified. Conclusions Our data illustrate the divergence between microfluidic cards and metatranscriptomics, highlighting that the combination of both methods facilitates the source tracking of human viruses in challenging settings without sufficient clinical surveillance. [ABSTRACT FROM AUTHOR]

The RNA‐dependent RNA polymerase (RdRp) is a crucial element in the replication and transcription of RNA viruses. Although the RdRps of lethal human coronaviruses severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), SARS‐CoV, and Middle East respiratory syndrome coronavirus (MERS‐CoV) have been extensively studied, the molecular mechanism of the catalytic subunit NSP12, which is involved in pathogenesis, remains unclear. In this study, the biochemical and cell biological results demonstrate the interactions between SARS‐CoV‐2 NSP12 and seven host proteins, including three splicing factors (SLU7, PPIL3, and AKAP8). The entry efficacy of SARS‐CoV‐2 considerably decreased when SLU7 or PPIL3 was knocked out, indicating that abnormal splicing of the host genome was responsible for this occurrence. Furthermore, the polymerase activity and stability of SARS‐CoV‐2 RdRp were affected by the three splicing factors to varying degrees. In addition, NSP12 and its homologues from SARS‐CoV and MERS‐CoV suppressed the alternative splicing of cellular genes, which were influenced by the three splicing factors. Overall, our research illustrates that SARS‐CoV‐2 NSP12 can engage with various splicing factors, thereby impacting virus entry, replication, and gene splicing. This not only improves our understanding of how viruses cause diseases but also lays the foundation for the development of antiviral therapies. [ABSTRACT FROM AUTHOR]

ABSTRACT Ascospores, forcibly released into the air from perithecia, are the primary inoculum for Fusarium head blight. In Fusarium graminearum, the biological functions of four RNA-dependent RNA polymerases (RdRPs) (Fgrdrp1–4) have been reported, but their regulatory mechanisms are poorly understood and the function of Fgrdrp5 is still unknown. In this study, we found that in addition to Fgrdrp1 and Fgrdrp2, Fgrdrp5 also plays an important role in ascospore discharge, and they all participate in the generation of turgor pressure in a polyol-dependent manner. Moreover, these three genes all affect the maturation of ascospores. Deep sequencing and co-analysis of small RNA and mRNA certified that Fgrdrp1, Fgrdrp2, and Fgrdrp5 partly share their functions in the biogenesis and accumulation of exonic small interference RNA (ex-siRNA), and these three RdRPs negatively regulate the expression levels of ex-siRNA corresponding genes, including certain genes associated with ascospore development or discharge. Furthermore, the differentially expressed genes of deletion mutants, those involved in lipid and sugar metabolism or transport as well as sexual development-related transcription factors, may also contribute to the defects in ascospore maturation or ascospore discharge. In conclusion, our study suggested that the components of the dicer-dependent ex-siRNA-mediated RNA interference pathway include at least Fgrdrp1, Fgrdrp2, and Fgrdrp5.IMPORTANCEWe found that in addition to Fgrdrp1 and Fgrdrp2, Fgrdrp5 also plays important roles in ascospore maturation and ascospore discharge of Fusarium graminearum. These three RNA-dependent RNA polymerases participate in the biogenesis and accumulation of exonic small interference RNA and then regulate ascospore discharge.

Coronaviruses contain one of the largest genomes among the RNA viruses, coding for 14–16 non-structural proteins (nsp) that are involved in proteolytic processing, genome replication and transcription, and four structural proteins that build the core of the mature virion. Due to conservation across coronaviruses, nsps form a group of promising drug targets as their inhibition directly affects viral replication and, therefore, progression of infection. A minimal but fully functional replication and transcription complex was shown to be formed by one RNA-dependent RNA polymerase (nsp12), one nsp7, two nsp8 accessory subunits, and two helicase (nsp13) enzymes. Our approach involved, targeting nsp12 and nsp13 to allow multiple starting point to interfere with virus infection progression. Here we report a combined in-vitro repurposing screening approach, identifying new and confirming reported SARS-CoV-2 nsp12 and nsp13 inhibitors.

The mosquito-borne Rift Valley fever virus (RVFV) from the Phenuiviridae family is a single-stranded RNA virus that causes the re-emerging zoonotic disease Rift Valley fever (RVF). Classified as a Category A agent by the NIH, RVFV infection can cause debilitating disease or death in humans and lead to devastating economic impacts by causing abortion storms in pregnant cattle. In a previous study, we showed that the host chaperone protein HSP90 is an RVFV-associated host factor that plays a critical role post viral entry, during the active phase of viral genome replication/transcription. In this study, we have elucidated the molecular mechanisms behind the regulatory effect of HSP90 during infection with RVFV. Our results demonstrate that during the early infection phase, host HSP90 associates with the viral RNA-dependent RNA polymerase (L protein) and prevents its degradation through the proteasome, resulting in increased viral replication.

Improved RNA virus understanding is critical to studying animal and plant health, and environmental processes. However, the continuous and rapid RNA virus evolution makes their identification and characterization challenging. While recent sequence-based advances have led to extensive RNA virus discovery, there is growing variation in how RNA viruses are identified, analyzed, characterized, and reported. To this end, an RdRp Summit was organized and a hybrid meeting took place in Valencia, Spain in May 2023 to convene leading experts with emphasis on early career researchers (ECRs) across diverse scientific communities. Here we synthesize key insights and recommendations and offer these as a first effort to establish a consensus framework for advancing RNA virus discovery. First, we need interoperability through standardized methodologies, data-sharing protocols, metadata provision and interdisciplinary collaborations and offer specific examples as starting points. Second, as an emergent field, we recognize the need to incorporate cutting-edge technologies and knowledge early and often to improve omic-based viral detection and annotation as novel capabilities reveal new biology. Third, we underscore the significance of ECRs in fostering international partnerships to promote inclusivity and equity in virus discovery efforts. The proposed consensus framework serves as a roadmap for the scientific community to collectively contribute to the tremendous challenge of unveiling the RNA virosphere.

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.

The polymerase complex of nonsegmented negative-strand RNA viruses primarily consists of a large (L) protein and a phosphoprotein (P). L is a multifunctional enzyme carrying out RNA-dependent RNA polymerization and all other steps associated with transcription and replication, while P is the nonenzymatic cofactor, regulating the function and conformation of L. The structure of a purified vesicular stomatitis virus (VSV) polymerase complex containing L and associated P segments has been determined; however, the location and manner of the attachments of L and P within each virion are unknown, limiting our mechanistic understanding of VSV RNA replication and transcription and hindering engineering efforts of this widely used anticancer and vaccine vector. Here, we have used cryo-electron tomography to visualize the VSV virion, revealing the attachment of the ring-shaped L molecules to VSV nucleocapsid proteins (N) throughout the cavity of the bullet-shaped nucleocapsid. Subtomogram averaging and three-dimensional classification of regions containing N and the matrix protein (M) have yielded the in situ structure of the polymerase complex. On average, ∼55 polymerase complexes are packaged in each virion. The capping domain of L interacts with two neighboring N molecules through flexible attachments. P, which exists as a dimer, bridges separate N molecules and the connector and C-terminal domains of L. Our data provide the structural basis for recruitment of L to N by P in virus assembly and for flexible attachments between L and N, which allow a quick response of L in primary transcription upon cell entry.

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. [ABSTRACT FROM AUTHOR]

The deformed wing virus (DWV) belongs to the genus Iflavirus and the family Iflaviridae within the order Picornavirales. It is an important pathogen of the Western honey bee, Apis mellifera, causing major losses among honey bee colonies in association with the ectoparasitic mite Varroa destructor. Although DWV is one of the best-studied insect viruses, the mechanisms of viral replication and polyprotein processing have been poorly studied in the past. We investigated the processing of the protease-polymerase region at the C-terminus of the polyprotein in more detail using recombinant expression, novel serological reagents, and virus clone mutagenesis. Edman degradation of purified maturated polypeptides uncovered the C- and N-termini of the mature 3C-like (3CL) protease and RNA-dependent RNA polymerase (3DL, RdRp), respectively. Autocatalytic processing of the recombinant DWV 3CL protease occurred at P1 Q2118 and P1′ G2119 (KPQ/GST) as well as P1 Q2393 and P1′ S2394 (HAQ/SPS) cleavage sites. New monoclonal antibodies (Mab) detected the mature 3CL protease with an apparent molecular mass of 32 kDa, mature 3DL with an apparent molecular mass of 55 kDa as well as a dominant 3CDL precursor of 90 kDa in DWV infected honey bee pupae. The observed pattern corresponds well to data obtained via recombinant expression and N-terminal sequencing. Finally, we were able to show that 3CL protease activity and availability of the specific protease cleavage sites are essential for viral replication, protein synthesis, and establishment of infection using our molecular clone of DWV-A. [ABSTRACT FROM AUTHOR]

RNA interference (RNAi) is one of the important defense responses against viral infection, but its mechanism and impact remain unclear in mycovirus infections. In our study, reverse genetics and virus-derived small RNA sequencing were used to show the antiviral responses of RNAi components in Aspergillus flavus infected with Aspergillus flavus partitivirus 1 (AfPV1). qRT-PCR revealed that AfPV1 infection induced the expression of the RNAi components in A. flavus compared with noninfected A. flavus. Knock mutants of each RNAi component were generated, but the mutants did not exhibit any obvious phenotypic changes compared with the A. flavus parental strain. However, after AfPV1 inoculation, production of AfPV1 was significantly less than in the parental strain. Furthermore, sporulation was greater in each AfPV1-infected mutant compared with the AfPV1-infected parental A. flavus. We also investigated the sensitivity of virus-free and AfPV1- infected RNAi mutants and the parental strain to cell wall stress, osmotic stress, genotoxic stress, and oxidative stress. The mutants of DCLs and AGOs infected by AfPV1 displayed more changes than RDRP mutants in response to the first three stresses. Small RNA sequencing analysis suggested that AfPV1 infection reduced the number of unique reads of sRNA in A. flavus, although there were many vsiRNA derived from the AfPV1 genome. GO term and KEGG pathway analyses revealed that the functions of sRNA affected by AfPV1 infection were closely related to vacuole production. These results provide a better understanding of the functional role of RNAi in the impact of AfPV1 on the hypovirulence of A. flavus. [ABSTRACT FROM AUTHOR]

Background Remdesivir is approved for treatment of coronavirus disease 2019 (COVID-19) in nonhospitalized and hospitalized adult and pediatric patients. Here we present severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) resistance analyses from the phase 3 ACTT-1 randomized placebo-controlled trial conducted in adult participants hospitalized with COVID-19. Methods Swab samples were collected at baseline and longitudinally through day 29. SARS-CoV-2 genomes were sequenced using next-generation sequencing. Phenotypic analysis was conducted directly on participant virus isolates and/or using SARS-CoV-2 subgenomic replicons expressing mutations identified in the Nsp12 target gene. Results Among participants with both baseline and postbaseline sequencing data, emergent Nsp12 substitutions were observed in 12 of 31 (38.7%) and 12 of 30 (40.0%) participants in the remdesivir and placebo arms, respectively. No emergent Nsp12 substitutions in the remdesivir arm were observed in more than 1 participant. Phenotyping showed low to no change in susceptibility to remdesivir relative to wild-type Nsp12 reference for the substitutions tested: A16V (0.8-fold change in EC50), P323L + V792I (2.2-fold), C799F (2.5-fold), K59N (1.0-fold), and K59N + V792I (3.4-fold). Conclusions The similar rate of emerging Nsp12 substitutions in the remdesivir and placebo arms and the minimal change in remdesivir susceptibility among tested substitutions support a high barrier to remdesivir resistance development in COVID-19 patients. Clinical Trials Registration.  NCT04280705. [ABSTRACT FROM AUTHOR]

A novel mycovirus, Ceratobasidium bipartite virus 1 (CBV1), was identified in Ceratobasidium sp. AG-A strain SHX-YJLC-1 isolated from diseased potato stems. The complete genome of CBV1 consists of two double-stranded RNA (dsRNA) segments: dsRNA1 (2311 bp) and dsRNA2 (1761 bp). dsRNA1 contains a single open reading frame (ORF1) encoding an RNA-dependent RNA polymerase (RdRp), while dsRNA2 contains a single ORF (ORF2) encoding a hypothetical protein (HP) with unknown function. BLASTp analysis revealed that RdRp (75.04%) and HP (61.86%) encoded by the two ORFs have the highest sequence similarity to their counterparts in Rhizoctonia solani dsRNA virus 11 (RsRV11). The genome organization and phylogenetic analysis indicated that the closest relatives to CBV1 are members of the proposed family "Bipartitiviridae". Based on the collective results, CBV1 is inferred to be a new member of the proposed family "Bipartitiviridae". This is the first report on the complete genome sequence of the novel bipartitivirus CBV1, which infects Ceratobasidium sp. AG-A strain SHX-YJLC-1. [ABSTRACT FROM AUTHOR]

The emergence of new coronaviruses poses a significant threat to animal husbandry and human health. Porcine epidemic diarrhea virus (PEDV) is considered a re-emerging porcine enteric coronavirus, which causes fatal watery diarrhea in piglets. Currently, there are no effective drugs to combat PEDV. Drug repurposing screens have emerged as an attractive strategy to accelerate antiviral drug discovery and development. Here, we screened 206 natural products for antiviral activity using live PEDV infection in Vero cells and identified ten candidate antiviral agents. Among them, Tubercidin, a nucleoside analog derived from Streptomyces tubercidicus, showed promising antiviral activity against PEDV infection. Furthermore, we demonstrated that Tubercidin exhibited significant antiviral activity against both classical and variant PEDV. Time of addition assay showed that Tubercidin displayed a significant inhibitory effect on viral post-entry events but not during other periods. Molecular docking analysis indicated that Tubercidin had better docking efficiency and formed hydrophobic interactions with the active pocket of RNA-dependent RNA polymerase (RdRp) of PEDV and other nidoviruses. Additionally, Tubercidin can effectively suppress other porcine nidoviruses, such as SADS-CoV and PRRSV, demonstrating its broad-spectrum antiviral properties. In summary, our findings provide valuable evidence for the antiviral activity of Tubercidin and offer insights into the development of new strategies for the prevention and treatment of coronavirus infections.

Seasonal influenza A and B viruses represent a global concern. Antiviral drugs are crucial to treat severe influenza in high-risk patients and prevent virus spread in case of a pandemic. The emergence of viruses showing drug resistance, in particular for the recently licensed polymerase inhibitor baloxavir marboxil, drives the need for developing alternative antivirals. The endonuclease activity residing in the N-terminal domain of the polymerase acidic protein (PAN) is crucial for viral RNA synthesis and a validated target for drug design. Its function can be impaired by molecules bearing a metal-binding pharmacophore (MBP) able to coordinate the two divalent metal ions in the active site. In the present work, the 2,3-dihydro-6,7-dihydroxy-1H-isoindol-1-one scaffold is explored for the inhibition of influenza virus PA endonuclease. The structure-activity relationship was analysed by modifying the substituents on the lipophilic moiety linked to the MBP. The new compounds exhibited nanomolar inhibitory activity in a FRET-based enzymatic assay, and a few compounds (15-17, 21) offered inhibition in the micromolar range, in a cell-based influenza virus polymerase assay. When investigated against a panel of PA-mutant forms, compound 17 was shown to retain full activity against the baloxavir-resistant I38T mutant. This was corroborated by docking studies providing insight into the binding mode of this novel class of PA inhibitors.

RNA-dependent RNA polymerases (RDRs) regulate important aspects of plant development and resistance to pathogens. The role of RDRs in virus resistance has been demonstrated using siRNA signal amplification and through the methylation of viral genomes. Cucumber (Cucumis sativus) has four RDR1 genes that are differentially induced during virus infection: CsRDR1a, CsRDR1b, and duplicated CsRDR1c1/c2. The mode of action of CsRDR1s during viral infection is unknown. Transient expression of the cucumber mosaic virus (CMV)-2b protein (the viral suppressor of RNA silencing) in cucumber protoplasts induced the expression of CsRDR1c, but not of CsRDR1a/1b. Results from the yeast two-hybrid system showed that CsRDR1 proteins interacted with CMV-2b and this was confirmed by bimolecular fluorescence complementation assays. In protoplasts, CsRDR1s localized in the cytoplasm as punctate spots. Colocalization experiments revealed that CsRDR1s and CMV-2b were uniformly dispersed throughout the cytoplasm, suggesting that CsRDR1s are redistributed as a result of interactions. Transient overexpression of individual CsRDR1a/1b genes in protoplasts reduced CMV accumulation, indicating their antiviral role. However, overexpression of CsRDR1c in protoplasts resulted in relatively higher accumulation of CMV and CMVΔ2b. In single cells, CsRDR1c enhances viral replication, leading to CMV accumulation and blocking secondary siRNA amplification of CsRDR1c by CMV-2b protein. This suggests that CMV-2b acts as both a transcription factor that induces CsRDR1c (controlling virus accumulation) and a suppressor of CsRDR1c activity.

Yellow leaf disease (YLD) poses a significant challenge to areca palm cultivation, yet its etiology remains uncertain. During our investigation of YLD-affected areca palm plants, transcriptome sequencing revealed an RNA contig exhibiting striking similarities to the RNA-dependent RNA polymerase (RdRp) of ormycoviruses. Subsequent gene cloning techniques yielded the full-length sequence of this RNA, potentially representing either the complete or partial genome of a hitherto unidentified ormycovirus, tentatively named areca palm yellow leaf-associated ormycovirus (APYLaOMV). RT-PCR detection found that APYLaOMV is present in over 30% of YLD-affected areca palm samples but is absent in healthy ones, suggesting a potential link between APYLaOMV and YLD. In summary, these data could be valuable in understanding the etiology of YLD in areca palms.

Laboratory evolution is a powerful approach to search for genetic adaptations to new or improved phenotypes, yet either relies on labour-intensive human-guided iterative rounds of mutagenesis and selection, or prolonged adaptation regimes based on naturally evolving cell populations. Here we present CRISPR- and RNA-assisted in vivo directed evolution (CRAIDE) of genomic loci using evolving chimeric donor gRNAs continuously delivered from an error-prone T7 RNA polymerase, and directly introduced as RNA repair donors into genomic targets under either Cas9 or dCas9 guidance. We validate CRAIDE by evolving novel functional variants of an auxotrophic marker gene, and by conferring resistance to a toxic amino acid analogue in baker's yeast Saccharomyces cerevisiae with a mutation rate >3,000-fold higher compared to spontaneous native rate, thus enabling the first demonstrations of in vivo delivery and information transfer from long evolving RNA donor templates into genomic context without the use of in vitro supplied and pre-programmed repair donors.

Mammalian reovirus (MRV) is the prototypical member of genus Orthoreovirus of family Reoviridae. However, lacking high-resolution structures of its RNA polymerase cofactor μ2 and infectious particle, limits understanding of molecular interactions among proteins and RNA, and their contributions to virion assembly and RNA transcription. Here, we report the 3.3 Å-resolution asymmetric reconstruction of transcribing MRV and in situ atomic models of its capsid proteins, the asymmetrically attached RNA-dependent RNA polymerase (RdRp) λ3, and RdRp-bound nucleoside triphosphatase μ2 with a unique RNA-binding domain. We reveal molecular interactions among virion proteins and genomic and messenger RNA. Polymerase complexes in three Spinoreovirinae subfamily members are organized with different pseudo-D3d symmetries to engage their highly diversified genomes. The above interactions and those between symmetry-mismatched receptor-binding σ1 trimers and RNA-capping λ2 pentamers balance competing needs of capsid assembly, external protein removal, and allosteric triggering of endogenous RNA transcription, before, during and after infection, respectively.

With advances in sequencing technology, metatranscriptome sequencing from a variety of environmental and biological sources has revealed the existence of various previously unknown RNA viruses. This review presents recent major RNA virome studies sampled from invertebrate and vertebrate species as well as aquatic environments. In particular, we focus on severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) and related RNA virus identification through metatranscriptome sequencing analyses. Recently developed bioinformatics software and databases for RNA virus identification are introduced. A relationship between newly identified RNA viruses and endogenous viral elements in host genomes is also discussed.

Abstract Background Giardia duodenalis (referred to as Giardia) is a flagellated binucleate protozoan parasite, which causes one of the most common diarrheal diseases, giardiasis, worldwide. Giardia can be infected by Giardiavirus (GLV), a small endosymbiotic dsRNA virus belongs to the Totiviridae family. However, the regulation of GLV and a positive correlation between GLV and Giardia virulence is yet to be elucidated. Methods To identify potential regulators of GLV, we performed a yeast two-hybrid (Y2H) screen to search for interacting proteins of RdRp. GST pull-down, co-immunoprecipitation and bimolecular fluorescence complementation (BiFC) assay were used to verify the direct physical interaction between GLV RdRp and its new binding partner. In addition, their in vivo interaction and colocalization in Giardia trophozoites were examined by using Duolink proximal ligation assay (Duolink PLA). Results From Y2H screen, the Giardia chaperone protein, Giardia DnaJ (GdDnaJ), was identified as a new binding partner for GLV RdRp. The direct interaction between GdDnaJ and GLV RdRp was verified via GST pull-down, co-immunoprecipitation and BiFC. In addition, colocalization and in vivo interaction between GdDnaJ and RdRp in Giardia trophozoites were confirmed by Duolink PLA. Further analysis revealed that KNK437, the inhibitor of GdDnaJ, can significantly reduce the replication of GLVs and the proliferation of Giardia. Conclusion Taken together, our results suggested a potential role of GdDnaJ in regulating Giardia proliferation and GLV replication through interaction with GLV RdRp. Graphical Abstract

Hand, foot, and mouth disease (HFMD), primarily instigated by Coxsackievirus A16 (CVA16), poses a serious health concern, necessitating effective therapeutic interventions. The RNA-dependent RNA polymerase (RdRp) of CVA16 emerges as a promising drug target for HFMD treatment. This study presents an in-silico pipeline for the identification of potential RdRp inhibitors against CVA16. A library of 91 natural compounds derived from Bacopa monnieri (brahmi) was virtually screened against the CVA16 RdRp. Here, Bacobitacin D emerged as a promising hit molecule, forming 8 hydrogen bonds including key catalytic site residues (Asp238 and Asp329) within the RdRp active site. Further, molecular dynamics (MD) simulations and MM/GBSA binding free energy calculations was applied on the top three hits that were selected based on exhaustive docking scores (≤−9.55 ​kcal/mol). Bacobitacin D exhibited sustainable stability, as evidenced by minimal deviation (RMSD ​= ​0.75 ​± ​0.02 ​nm) during a 100 ns MD simulation. Importantly, Bacopaside IV exhibited the lowest ΔGTOTAL binding free energy (−23.70 ​kcal/mol), while Bacobitacin D displayed a comparable ΔGTOTAL of −19.14 ​kcal/mol. Structural interpretation of the most populated cluster derived from MD simulations showed direct interactions of Bacobitacin D with pivotal catalytic residues, including Asp238 and Ser289. This comprehensive study confirmed Bacobitacin D as a potent inhibitor of CVA16 RdRp, offering a potential avenue for therapeutic intervention against HFMD. Experimental validation is required to confirm the inhibitory action of Bacobitacin D against HFMD.