RNA-dependent RNA Polymerase Research Articles

The Forgotten Tobamovirus Genes Encoding the 54 kDa Protein and the 4–6 kDa Proteins

This article reviews the literature concerning the largely forgotten tobamovirus gene products for which no functions have been ascribed. One of these gene products is the 54 kDa protein, representing the RNA-dependent RNA polymerase segment of the 183 kDa protein translated from the I1-subgenomic mRNA, but which has been found only by in vitro translation and not in plants. The other is a collection of small proteins, expressed from alternative reading frames (likely from internal ribosome entry sites) in either or both the movement protein gene or the capsid protein gene. Previously, two small proteins were referred to as the 4–6 kDa proteins, since only single proteins of such size had been characterized from tobacco mosaic virus and tomato mosaic virus genomes. Such putative proteins will be referred to here as P6 proteins, since many new proposed P6 open reading frames could be discerned, from an analysis of 45 of 47 tobamovirus genomes, with a coding capacity of >15 amino acids up to 94 amino acids, whereas other peptides with ≤15 amino acids were not considered here. The distribution of the putative P6 proteins among these tobamoviruses is described, as well as the various classes they fall into, based on their distribution with regard to the organization of other genes in the viral genomes. Models also are presented for possible functions of the 54 kDa protein and the P6 proteins, based on data in the literature.

Coxsackievirus B3-Induced m6A Modification of RNA Enhances Viral Replication via Suppression of YTHDF-Mediated Stress Granule Formation

N6-methyladenosine (m6A) is the most prevalent internal RNA modification. Here, we demonstrate that coxsackievirus B3 (CVB3), a common causative agent of viral myocarditis, induces m6A modification primarily at the stop codon and 3′ untranslated regions of its genome. As a positive-sense single-stranded RNA virus, CVB3 replicates exclusively in the cytoplasm through a cap-independent translation initiation mechanism. Our study shows that CVB3 modulates the expression and nucleo-cytoplasmic transport of the m6A machinery components—METTL3, ALKBH5 and YTHDFs—resulting in increased m6A modifications that enhance viral replication. Mechanistically, this enhancement is mediated through YTHDF-driven stress granule (SG) formation. We observed that YTHDF proteins co-localize with human antigen R (HuR), a protein facilitating cap-independent translation, in SGs during early infection. Later in infection, YTHDFs are cleaved, suppressing SG formation. Notably, for the first time, we identified that during early infection CVB3’s RNA-dependent RNA polymerase (3D) and double-stranded RNA (dsRNA) are stored in SGs, co-localizing with HuR. This early-stage sequestration likely protects viral components for use in late-phase replication, when SGs are disrupted due to YTHDF cleavage. In summary, our findings reveal that CVB3-induced m6A modifications enhance viral replication by regulating YTHDF-mediated SG dynamics. This study provides a potential therapeutic strategy for CVB3-induced myocarditis.

The first report of cotton leafroll dwarf virus infecting upland cotton plants in Arizona.

Cotton leafroll dwarf virus (CLRDV) represents a persistent threat to cotton production in the United States (U.S.) (Edula et al. 2023). Initially detected in Alabama (Avelar et al. 2019), CLRDV occurs in almost all the states in the U.S. cotton belt, extending from Virginia through Texas. The symptoms associated with CLRDV includes interveinal chlorosis, leaf rolling, stunting, and reduced boll sets. However, asymptomatic CLRDV-infected plants have also been reported (Edula et al. 2023). In the 2023 growing season, upland cotton plants (Gossypium hirsutum) presenting terminal splitting symptoms in the blooming stage were observed in a commercial field in Pinal County, Arizona, at 60% incidence. To evaluate if CLRDV may be associated with these symptoms, young fully expanded terminal leaves were collected from 20 symptomatic and ten asymptomatic plants. Moreover, ten samples were collected from a second asymptomatic block in the same field. Samples were shipped on dry ice to Cornell University in hermetic bags under APHIS PPQ permit 526-23-256-14384. Midribs and petioles were used to extract total nucleic acids from each sample using OPS Synergy 2.0 Plant DNA Extraction Kit (OPS Diagnostics) per the manufacturer's instructions. Complementary DNA (cDNA) was synthesized using an iScript Reverse transcriptase kit (BioRad) and used for the detection of a partial sequence of the coat protein (CP) of CLRDV using PCR assays as described previously (Mahas et al. 2022). The expected 309-bp product was obtained from only one symptomatic sample. CLRDV presence in the samples was further evaluated using CLRDV-specific primers targeting the RNA-dependent RNA polymerase (RdRp) gene. Primers CLRDV-Pol_innerF1 (5'- ACCCTCCAAGGAACAGAG -3') / R1 (5'- CGAATAATCTGATYGGGTCAC -3') and CLRDV-Pol_outerF1 (5'- AACGCGCCCAGTCCGCACAAATACC-3') / R1 (5'-ACCGGGTTTACTGGGGATTGCACGC-3'), designed based on virus isolates available in GenBank as of September 2022, were used to implement a single-tube nested RT-PCR as detailed by Dey et al. (2012) and to index the presence of CLRDV in all the samples. Two additional symptomatic samples and three asymptomatic samples (two from field one and one from field two) were positive for the virus. Direct Sanger sequencing of the one CP and two RdRp amplicons from symptomatic and asymptomatic samples (PP482918-20) demonstrated they shared >99% nucleotide identity to an isolate from South Carolina (OQ300129). To further evaluate the presence of CLRDV in Arizona, we performed double antibody sandwich (DAS)-ELISA on midrib and petiole samples using camelid single-chain antibodies against the CP as the capture antibody (Filed Patent 18/436,287) and the commercially available Anti-PLRV conjugate (Agdia, Elkhart, IN) as the secondary antibody. DAS-ELISA detected CLRDV in eight symptomatic and three asymptomatic samples, with the positive and negative controls testing positive and negative, respectively. Considering all the diagnostic approaches, ten symptomatic and five asymptomatic samples were positive for CLRDV (Table S1). To the best of our knowledge, this is the first report of CLRDV in Arizona. Future studies are required to evaluate the possible incidence and impact on the crop yield in the state and develop a reliable diagnostic assay to detect the virus in all infected samples. Since our study did not associate CLRDV with the terminal abortion or splitting symptoms, ongoing efforts are underway to elucidate if other viruses may be associated with the symptoms.

First report of citrus yellow vein clearing virus (CYVCV) on citrus in Italy.

In March 2024, symptoms of vein clearing (Fig. S1) were observed on leaves of six lemon (Citrus limon (L.) Burm. f.) trees grown in a private garden in Palma Campania (Campania region, Italy). Symptoms strongly resembled those of yellow vein clearing disease, caused by Potexvirus citriflavivenae (citrus yellow vein clearing virus, CYVCV), genus Potexvirus, sub-genus Mandarivirus (Loconsole et al., 2012; Cui et al., 2018). CYVCV was reported in Pakistan, India, China, Iran, Turkey, Korea and USA (Abrahamian et al., 2024; Catara et al., 1993; EPPO, 2024; Sun and Yokomi, 2024; Jin et al., 2024). Since 2022, considering the potential impact of this emerging virus, CYVCV has been included in the EPPO alert list and it has not been reported in any European Union (EU) Member State where the virus is not regulated. To ascertain possible infection with CYVCV, leaf samples were collected and tested by reverse transcription polymerase chain reaction (RT-PCR). Total nucleic acids (TNAs) from leaves of the symptomatic lemon and non-symptomatic lemon and sweet orange (Citrus sinensis (L.) Osbeck) trees grown in the same garden, were extracted using the silica-capture extraction method (Foissac et al. 2001). TNAs were tested by RT-PCR using CYVCV specific primers (Table S1) designed by Chen et al., (2014) targeting the coat protein gene. All symptomatic trees and an asymptomatic sweet orange from the same garden tested positive generating an amplicon of the expected size (612 bp). Direct Sanger sequencing of amplicons obtained from two symptomatic lemons and one non-symptomatic sweet orange (An PP842725-PP842727) followed by BLAST search showed 97.9-98.08% sequence identity (query coverage 100%) with CYVCV isolates previously reported from a mandarin (An OQ418501) and a lemon (An OQ418493) in California. In contrast, no amplicons were detected in samples from non-symptomatic lemon trees. Virus identity was confirmed by RT-PCR with different primers (Table S1) targeting the RNA-dependent RNA polymerase gene of CYVCV. Amplicons of the expected size (832 bp) were obtained only from samples previously tested positive to the virus and their sequences determined by Sanger sequence (An PQ284953-PQ284955) showed 97.98-98.11% identity (query coverage 100%) with CYVCV sequence previously reported from a mandarin (An OR251443) in New Delhi. Since CYVCV is transmitted by insects (Önelge et al., 2011a, 2011b, Zhang et al., 2018, 2019), TNAs (Foissac et al., 2001) from Aphis aurantii infesting symptomatic lemons, and of A. spiraecola and Dialeurodes citri infesting symptomatic lemon and non-symptomatic sweet orange trees were assayed by RT-PCR with primer pairs designed by Chen et al., (2014). Three samples of 15 specimens from three different trees were tested for each insect. In contrast to D. citri, which tested negative, an amplicon of 612 bp was detected from A. aurantii and A. spiraecola and Direct sequencing of amplicons (An PP842721-PP842724) confirmed the nature of the virus and showed that insect-derived viral sequences shared nucleotide identity of 99.3-100% with those from the infected trees. Altogether, these data support that CYVCV is present in Southern Italy. Likely, it has been introduced illegally through infected citrus propagation material. Given the large A. aurantii and A. spiraecola populations in the areas where CYVCV has been detected, a fast spread of the virus to other areas of Campania region can be expected. Even if the impact of CYVCV on citrus production in Italy remains to be assessed, prompt and extensive surveys may be helpful to establish the actual distribution of the virus. This would allow for a better evaluation of appropriate measures to be adopted to achieve sustainable citrus production. At the best of our knowledge, this is the first report of CYVCV in Italy and in the EU territory.

Revolutionizing Influenza Treatment: A Deep Dive into Targeted Drug Delivery Systems.

Influenza, a highly transmissible respiratory infection caused by influenza viruses A and B, poses a persistent threat to global public health due to its high mutation rate, ability to develop resistance to existing antiviral drugs, and capacity for rapid spread. Current treatment options, including four main classes of antiviral agents-adamantanes, neuraminidase inhibitors, RNA-dependent RNA polymerase inhibitors, and polymerase acidic endonuclease inhibitors- are limited by the emergence of drug-resistant viral strains, non-specific drug distribution, and adverse side effects. Moreover, the effectiveness of traditional vaccines is often compromised by antigenic drift and shift, necessitating the development of alternative therapeutic strategies. This review comprehensively explores the potential of novel targeted drug delivery systems to address these limitations and improve influenza management. Nanotechnology-based platforms, including lipid-based, polymer-based, inorganic, and hybrid nanoparticles, offer enhanced drug delivery through improved bioavailability, targeted action, and controlled release, thus minimizing systemic toxicity and optimizing therapeutic outcomes. Inhalation delivery systems such as dry powder inhalers (DPIs), nebulizers, and nanotechnology-based inhalation formulations provide direct delivery of antiviral agents to the respiratory tract, ensuring rapid onset of action with reduced systemic side effects. Transdermal delivery methods, including microneedle patches and hydrogel-based systems, offer non-invasive alternatives that enhance patient compliance and allow for sustained drug release. Furthermore, this review discusses recent innovations, such as responsive drug delivery systems and multifunctional nanoparticles capable of simultaneous delivery of multiple therapeutic agents, representing a significant advancement in the fight against influenza. These novel approaches promise improved targeting and efficacy and enable personalized treatment strategies, enhancing patient outcomes in both seasonal flu and pandemic scenarios. Integrating these advanced drug delivery systems into clinical practice could revolutionize the management of influenza, offering a promising pathway toward more effective and safer therapies.

Molecular characterization of a novel mycotombus‑like virus isolated from the phytopathogenic fungus Nigrospora oryzae.

In this study, we identified a new mycotombus-like mycovirus from the phytopathogenic fungus Nigrospora oryzae, which was tentatively designated as "Nigrospora oryzae umbra-like virus 1" (NoULV1). The complete genome of NoULV1 is 3,381 nt long, containing two open reading frames (ORF1 and ORF2). ORF1 encodes a hypothetical protein with an unknown function, while ORF2 encodes an RNA-dependent RNA polymerase (RdRp) with a conserved RdRp domain containing a metal-binding 'GDN' triplet in motif C, which is distinct from the 'GDD' motif found in most + ssRNA mycoviruses. A homology search revealed that the RdRp encoded by ORF2 was similar to the RdRp of umbra-like mycoviruses. Phylogenetic analysis based on the RdRp indicated that NoULV1 was grouped into a clade together with umbra-like mycoviruses belonging to the proposed family "Mycotombusviridae".

Molecular characterization of a novel fusarivirus infecting the plant-pathogenic fungus Nigrospora sphaerica.

Here, we describe a novel mycovirus, tentatively designated as "Nigrospora sphaerica fusarivirus 2" (NsFV2), which was isolated from the phytopathogenic fungus Nigrospora sphaerica strain HNXX-Ns20. NsFV2 has a single-stranded positive-sense RNA (+ ssRNA) genome of 6,156 nucleotides, excluding the poly(A) tail, and contains two putative open reading frames (ORFs). ORF1 encodes a large polypeptide of 1,509 amino acids (aa) containing a conserved RNA-dependent RNA polymerase (RdRp) domain and a viral helicase domain. The ORF1-encoded polypeptide shares 29.40-68.48% sequence identity with other fusariviruses and shares the highest sequence identity (68.48%) with Nigrospora sphaerica fusarivirus 1 (NsFV1). The small ORF2 encodes a polypeptide of 483 aa that contains a conserved chromosome segregation ATPase (Smc) domain. Multiple sequence alignments and phylogenetic analysis based on the ORF1-encoded polypeptide indicated that NsFV2 should be considered a new member of the genus Alphafusarivirus of the family Fusariviridae.

Resistance gene Ty-1 restricts TYLCV infection in tomato by increasing RNA silencing

A major antiviral mechanism in plants is mediated by RNA silencing through the action of DICER-like (DCL) proteins, which cleave dsRNA into discrete small RNA fragments, and ARGONAUTE (AGO) proteins, which use the small RNAs to target single-stranded RNA. RNA silencing can also be amplified through the action of RNA-dependent RNA polymerases (RDRs), which use single stranded RNA to generate dsRNA that in turn is targeted by DCL proteins. As a counter-defense, plant viruses encode viral suppressors of RNA silencing (VSRs) that target different components in the RNA silencing pathway. The tomato Ty-1 gene confers resistance to the DNA virus tomato yellow leaf curl virus (TYLCV) and has been reported to encode an RDRγ protein. However, the molecular mechanisms by which Ty-1 controls TYLCV infection, including whether Ty-1 is involved in RNA silencing, are unknown. Here, by using a transient expression assay, we have confirmed that Ty-1 shows antiviral activity against TYLCV in Nicotiana benthamiana. Also, in transient expression-based silencing assays, Ty-1 augmented systemic transgene silencing in GFP transgenic N. benthamiana plants. Furthermore, co-expression of Ty-1 or other RDRγ proteins from N. benthamiana or Arabidopsis with various proteins resulted in lower protein expression. These results are consistent with a model wherein Ty-1-mediated resistance to TYLCV is due, at least in part, to an increase in RNA silencing activity.

3'-Dehydroxypurpurogallin-4-carboxamides as Influenza A Endonuclease Inhibitors: Synthesis, SARAnalysis, and Structural Characterization of Protein Complex.

The influenza RNA-dependent RNA polymerase harbours an endonuclease subunit characterized by a catalytic site housing two divalent metal ions. By effectively chelating both Mg2+ and Mn2+ ions, a low-molecular-weight inhibitor with a metal-binding pharmacophore can halt endonuclease activity. Herein, two 3'-dehydroxypurpurogallin-4-carboxamide series, namely twelve C-4' unsubstituted and twelve C-4' phenyl substituted congeners were designed and prepared to be tested as inhibitors of the metal-dependent viral enzyme. These inhibitors were accessed through the chemoenzymatic reaction of gallic acid with either pyrocatechol or phenylpyrocatechol moderated by laccase, followed by amidation. Experimental IC50 values were determined using AlphaScreen technology, with the most potent inhibitors exhibiting IC50 values around 0.35 μM. Using X-ray crystallography, we analyzed structure of the endonuclease in complex with one potent 3'-dehydroxypurpurogallin-carboxamide at 2.0 Å resolution, revealing the coordination of the compound's triad of oxygen atoms with the two metal ions in the influenza A endonuclease active site.

Distinct chikungunya virus polymerase palm subdomains contribute to viral protein accumulation and virion production.

Alphaviruses encode an error-prone RNA-dependent RNA polymerase (RdRp), nsP4, required for genome synthesis, yet how the RdRp functions in the complete alphavirus life cycle is not well-defined. Previous work using chikungunya virus has established the importance of the nsP4 residue cysteine 483 in replication. Given the location of residue C483 in the nsP4 palm domain, we hypothesized that other residues within this domain and surrounding subdomains would also contribute to polymerase function. To test this hypothesis, we designed a panel of nsP4 variants via homology modeling based on the coxsackievirus B3 3D polymerase. We rescued each variant in mammalian and mosquito cells and discovered that the palm domain and ring finger subdomain contribute to host-specific replication. In C6/36 cells, we found that while the nsP4 variants had replicase function similar to that of wild-type CHIKV, many variants presented changes in protein accumulation and virion production even when viral nonstructural and structural proteins were produced. Finally, we found that WT CHIKV and nsP4 variant replication and protein production could be enhanced in mammalian cells at 28°C, yet growing virus under these conditions led to changes in virus infectivity. Taken together, these studies highlight that distinct nsP4 subdomains are required for proper RNA transcription and translation, having major effects on virion production.

Complete genome sequence of a novel mitovirus identified in the phytopathogenic fungus Alternaria tenuissima.

In this study, a novel positive-sense single-stranded RNA (+ ssRNA) mycovirus, Alternaria tenuissima mitovirus 1 (AtMV1), was identified in Alternaria tenuissima strain YQ-2-1, a phytopathogenic fungus causing leaf blight on muskmelon. The genome of AtMV1 is a single RNA molecule that is 3013 nt in length with an A + U content of 66.58% and contains a single open reading frame (ORF) using the fungal mitochondrial genetic code. The ORF was predicted to encode a 313-amino-acid RNA-dependent RNA polymerase (RdRp) with a molecular mass of 35.48 kDa, which contains six conserved motifs with the highly conserved GDD tripeptide in motif IV. The 5' and 3' untranslated regions were predicted to fold into stem-loop and panhandle secondary structures. The results of a BLASTp search revealed that the amino acid (aa) sequence of RdRp of AtMV1 shared the highest sequence similarity (51.04% identity) with that of Sichuan mito-like virus 30, a member of the genus Duamitovirus within the family Mitoviridae. Phylogenetic analysis based on the aa sequence of the RdRp suggested that AtMV1 is a novel member of the genus Duamitovirus. To our knowledge, this is the first report of the complete genome sequence of a new mitovirus infecting A. tenuissima.

Structure of the RNA-dependent RNA polymerase P2 from the cystovirus φ8

The replication of RNA viruses relies on the activity of RNA-dependent RNA polymerases (RdRps). Despite large variations in their genomic sequences, viral RdRps share a common architecture generally known as a closed right hand. The P2 polymerase of cystovirus φ6 is currently among the best characterized viral RdRps. This polymerase is responsible for carrying out both replication and transcription of the viral double-stranded RNA genome using de novo initiation. Despite the extensive biochemical and structural studies conducted on φ6 P2, further structural information on other cystoviral RdRps is crucial to elucidate the structural and functional diversity of viral RdRps. Here, we have determined the atomic X-ray structure of the RdRp P2 from the φ6-related cystovirus φ8 at 3Å resolution. This structure completes the existing set of structural information on the φ8 polymerase complex and sheds light on the difference and similarities with related cystoviral RdRps.

Comparison of Nucleocapsid Antigen, ORIF 1ab and RdRp (RNA-Dependent RNA Polymerase) in the Diagnosis of COVID-19

Comparison of Nucleocapsid Antigen, ORIF 1ab and RdRp (RNA-Dependent RNA Polymerase) in the Diagnosis of COVID-19

Structure of the Nipah virus polymerase phosphoprotein complex

The Nipah virus (NiV), a member of the Paramyxoviridae family, is notorious for its high fatality rate in humans. The RNA polymerase machinery of NiV, comprising the large protein L and the phosphoprotein P, is essential for viral replication. This study presents the 2.9-Å cryo-electron microscopy structure of the NiV L-P complex, shedding light on its assembly and functionality. The structure not only demonstrates the molecular details of the conserved N-terminal domain, RNA-dependent RNA polymerase (RdRp), and GDP polyribonucleotidyltransferase of the L protein, but also the intact central oligomerization domain and the C-terminal X domain of the P protein. The P protein interacts extensively with the L protein, forming an antiparallel β-sheet among the P protomers and with the fingers subdomain of RdRp. The flexible linker domain of one P promoter extends its contact with the fingers subdomain to reach near the nascent RNA exit, highlighting the distinct characteristic of the NiV L-P interface. This distinctive tetrameric organization of the P protein and its interaction with the L protein provide crucial molecular insights into the replication and transcription mechanisms of NiV polymerase, ultimately contributing to the development of effective treatments and preventive measures against this Paramyxoviridae family deadly pathogen.

Remdesivir triphosphate is a valid substrate to initiate synthesis of DNA primers by human PrimPol

Remdesivir is a broad-spectrum antiviral drug which has been approved to treat COVID-19. Remdesivir is in fact a prodrug, which is metabolized in vivo into the active form remdesivir triphosphate (RTP), an analogue of adenosine triphosphate (ATP) with a cyano group substitution in the carbon 1’ of the ribose (1’-CN). RTP is a substrate for RNA synthesis and can be easily incorporated by viral RNA-dependent RNA polymerases (RdRp). Importantly, once remdesivir is incorporated (now monophosphate), it will act as a delayed chain terminator, thus blocking viral RNA synthesis. It has been reported that mitochondrial Polγ is also blocked in vitro by RTP, but the low impact in vivo on mitochondrial DNA replication stalling is likely due to repriming by the human DNA-directed DNA Primase/Polymerase (HsPrimPol), which also operates in mitochondria. In this work, we have tested if RTP is a valid substrate for both DNA primase and DNA polymerase activities of HsPrimPol, and its impact in the production of mature DNA primers. RTP resulted to be an invalid substrate for elongation, but it can be used to initiate primers at the 5´site, competing with ATP. Nevertheless, RTP-initiated primers are abortive, ocassionally reaching a maximal length of 4–5 nucleotides, and do not support elongation mediated by primer/template distortions. However, considering that the concentration of ATP, the natural substrate, is much higher than the intracellular concentration of RTP, it is unlikely that HsPrimPol would use RTP for primer synthesis during a remdesivir treatment in real patients.

Identification and Characterization of Four Novel Viruses in Balclutha incisa.

Balclutha incisa (Cicadellidae: Deltocephalinae), a leafhopper prevalent in tropical and temperate regions, is notably abundant in grasses and rice. The virome of B. incisa was investigated using deep transcriptome sequencing, leading to the first identification of four viruses belonging to the families Aliusviridae, Iflaviridae, and Totiviridae in B. incisa. These viruses have been provisionally named B. incisa ollusvirus 1 (BiOV1), B. incisa ollusvirus 2 (BiOV2), B. incisa iflavirus 1 (BiIV1), and B. incisa totivirus 1 (BiTV1). The complete genome sequences of these viruses were obtained through rapid amplification of cDNA ends (RACE). BiOV1 has a linear genome of 15,125 nucleotides (nt), while BiOV2 possesses a circular genome of 14,853 nt. The BiIV1 genome, excluding the poly(A) tail, is 10,903 nt in length and encodes a single open reading frame (ORF) for a polyprotein consisting of 3194 amino acids (aa). The BiTV1 genome is 4357 nt long and contains two overlapping ORFs, with the viral RNA-dependent RNA polymerase (RdRp) translated via a -1 ribosomal frameshift. Phylogenetic and sequence identity analyses suggest that all these viruses are novel members of their respective families. This study significantly expands our understanding of the virome associated with B. incisa by reporting and characterizing these novel viruses.

VirID: Beyond Virus Discovery-An Integrated Platform for Comprehensive RNA Virus Characterization.

RNA viruses exhibit vast phylogenetic diversity and can significantly impact public health and agriculture. However, current bioinformatics tools for viral discovery from metagenomic data frequently generate false positive virus results, overestimate viral diversity, and misclassify virus sequences. Additionally, current tools often fail to determine virus-host associations, which hampers investigation of the potential threat posed by a newly detected virus. To address these issues we developed VirID, a software tool specifically designed for the discovery and characterization of RNA viruses from metagenomic data. The basis of VirID is a comprehensive RNA-dependent RNA polymerase database to enhance a workflow that includes RNA virus discovery, phylogenetic analysis, and phylogeny-based virus characterization. Benchmark tests on a simulated data set demonstrated that VirID had high accuracy in profiling viruses and estimating viral richness. In evaluations with real-world samples, VirID was able to identify RNA viruses of all types, but also provided accurate estimations of viral genetic diversity and virus classification, as well as comprehensive insights into virus associations with humans, animals, and plants. VirID therefore offers a robust tool for virus discovery and serves as a valuable resource in basic virological studies, pathogen surveillance, and early warning systems for infectious disease outbreaks.

SMILES-based QSAR virtual screening to identify potential therapeutics for COVID-19 by targeting 3CLpro and RdRp viral proteins

The COVID-19 pandemic has prompted the medical systems of many countries to develop effective treatments to combat the high rate of infection and death caused by the disease. Within the array of proteins found in SARS-CoV-2, the 3 chymotrypsin-like protease (3CLpro) holds significance as it plays a crucial role in cleaving polyprotein peptides into distinct functional nonstructural proteins. Meanwhile, RNA-dependent RNA polymerase (RdRp) takes center stage as the key enzyme tasked with replicating the viral genomic RNA within host cells. These proteins, 3CLpro and RdRp, are deemed optimal subjects for QSAR modeling due to their pivotal functions in the viral lifecycle. In this study, SMILES-based QSAR classification models were developed for a dataset of 2377 compounds that were defined as either active or inactive against 3CLpro and RdRp. Pharmacophore (PH4) and QSAR modeling were used for the virtual screening on 60.2 million compounds including ZINC, ChEMBL, Molport, and MCULE databases to identify new potent inhibitors against 3CLpro and RdRp. Then, a filter was established based on typical molecular characteristics to identify drug-like molecules. The molecular docking was also performed to evaluate the binding affinity of 156 AND 51 potential inhibitors to 3CLpro and RdRp, respectively. Among the 15 hits identified based on molecular docking scores, M3, N2, and N4 were identified as promising inhibitors due to their good synthetic accessibility scores (3.07, 3.11, and 3.29 out of 10 for M3, N2, and N4 respectively). These compounds contain amine functional groups, which are known for their crucial role in the binding interactions between drugs and their targets. Consequently, these hits have been chosen for further biological assay studies to validate their activity. They may represent novel 3CLpro and RdRp inhibitors possessing drug-like properties suitable for COVID-19 therapy.

Characterization of the Conformational Hotspots of the RNA-Dependent RNA Polymerase Complex Identifies a Unique Structural Malleability of nsp8.

Several antiviral therapeutic approaches have been targeted toward the RNA-dependent RNA polymerase (RdRp) complex that is involved in viral genome replication. In SARS-CoV-2, although the RdRp is a multiprotein complex, the focus has been on the ligand binding catalytic core (nonstructural protein nsp12), and not the multiprotein functional dynamics. In this study, we focus on the conformational ensembles of the RdRp complex and their modulation by the presence of RNA, performing comprehensive microsecond-scale atomistic simulations of the apo- and RNA-bound complex. We delineate the differential impact of RNA on the constituent proteins, such as conformational polymorphisms, dominant segment-specific fluctuations, and the switch in dynamical crosstalk within the complex. We distinguish dynamical signatures of nsp7, nsp8, and nsp12 in the apo-state that are reduced in the presence of the RNA and appear to "prime" the complex for activity. Importantly, we identify a unique structural malleability of the nsp8 protein with high conformational heterogeneity in the apo state, especially at three sites (Y71 for nsp8A, and D52 and A66 for nsp8B). Our work highlights the functional implications of the polymorphism of nsp8 structures and reveals possibilities for the development of allosteric inhibitors.

Cy-1, a major QTL for tomato leaf curl New Delhi virus resistance, harbors a gene encoding a DFDGD-Class RNA-dependent RNA polymerase in cucumber (Cucumis sativus)

BackgroundTomato leaf curl New Delhi virus (ToLCNDV) (family Geminiviridae, genus Begomovirus) is a significant threat to cucumber (Cucumis sativus) production in many regions. Previous studies have reported the genetic mapping of loci related to ToLCNDV resistance, but no resistance genes have been identified.ResultsWe conducted map-based cloning of the ToLCNDV resistance gene in cucumber accession No.44. Agroinfiltration and graft-inoculation analyses confirmed the resistance of No.44 to ToLCNDV isolates from the Mediterranean and Asian countries. Initial mapping involving two rounds of phenotyping with two independent F2 populations generated by crossing the begomovirus-susceptible cultivar SHF and No.44 consistently detected major quantitative trait loci (QTLs) on chromosomes 1 and 2 that confer resistance to ToLCNDV. Fine-mapping of Cy-1, the dominant QTL on chromosome 1, using F3 populations narrowed the candidate region to a 209-kb genomic segment harboring 24 predicted genes. Among these genes, DFDGD-class RNA-dependent RNA polymerase (CsRDR3), an ortholog of Ty-1/Ty-3 of tomato and Pepy-2 of capsicum, was found to be a strong candidate conferring ToLCNDV resistance. The CsRDR3 sequence of No.44 contained multiple amino acid substitutions; the promoter region of CsRDR3 in No.44 had a large deletion; and the CsRDR3 transcript levels were greater in No.44 than in SHF. Virus-induced gene silencing (VIGS) of CsRDR3 using two chromosome segment substitution lines harboring chromosome 1 segments derived from No.44 compromised resistance to ToLCNDV.ConclusionsForward and reverse genetic approaches identified CsRDR3, which encodes a DFDGD-class RNA-dependent RNA polymerase, as the gene responsible for ToLCNDV resistance at the major QTL Cy-1 on chromosome 1 in cucumber. Marker-assisted breeding of ToLCNDV resistance in cucumber will be expedited by using No.44 and the DNA markers developed in this study.