RdRps Of Viruses Research Articles

Identification of RNA Virus-Derived RdRp Sequences in Publicly Available Transcriptomic Data Sets.

RNA viruses are abundant and highly diverse and infect all or most eukaryotic organisms. However, only a tiny fraction of the number and diversity of RNA virus species have been catalogued. To cost-effectively expand the diversity of known RNA virus sequences, we mined publicly available transcriptomic data sets. We developed 77 family-level Hidden Markov Model profiles for the viral RNA-dependent RNA polymerase (RdRp)-the only universal "hallmark" gene of RNA viruses. By using these to search the National Center for Biotechnology Information Transcriptome Shotgun Assembly database, we identified 5,867 contigs encoding RNA virus RdRps or fragments thereof and analyzed their diversity, taxonomic classification, phylogeny, and host associations. Our study expands the known diversity of RNA viruses, and the 77 curated RdRp Profile Hidden Markov Models provide a useful resource for the virus discovery community.

Recapitulating Trafficking of Nucleosides Into the Active Site of Polymerases of RNA Viruses: The Challenge and the Prize.

Nucleoside analogs are very effective antiviral agents with currently over 25 compounds approved for the therapy of viral infections. Still, their successful use against RNA viruses is very recent, despite RNA viruses comprising some of the most damaging human pathogens (e.g., Coronaviruses, Influenza viruses, or Flaviviridae such as dengue, Zika and hepatitis C viruses). The breakthrough came in 2013–2014, when the nucleoside analog Sofosbuvir became one of the cornerstones of current curative treatments for hepatitis C virus (HCV). An analog designed on the same principles, Remdesivir, has been the first approved compound against SARS-CoV-2, the coronavirus that causes the current COVID-19 pandemic. Both of these nucleoside analogs target the RNA-dependent RNA polymerase (RdRp) (NS5B for HCV, nsp12 for SARS-CoV-2). RdRps of RNA viruses display a peculiar elaboration of the classical polymerase architecture that leads to their active site being caged. Thus, triphosphate nucleosides and their analogs must access this active site in several steps along a narrow and dynamic tunnel. This makes straightforward computational approaches such as docking unsuitable for getting atomic-level details of this process. Here we give an account of ribose-modified nucleoside analogs as inhibitors of viral RdRps and of why taking into account the dynamics of these polymerases is necessary to understand nucleotide selection by RdRps. As a case study we use a computational protocol we recently described to examine the approach of the NTP tunnel of HCV NS5B by cellular metabolites of Sofosbuvir. We find major differences with natural nucleotides even at this early stage of nucleotide entry.

Mechanism of Inhibition of the Reproduction of SARS-CoV-2 and Ebola Viruses by Remdesivir.

Remdesivir is an antiviral drug initially designed against the Ebola virus. The results obtained with it both in biochemical studies in vitro and in cell line assays in vivo were very promising, but it proved to be ineffective in clinical trials. Remdesivir exhibited far better efficacy when repurposed against SARS-CoV-2. The chemistry that accounts for this difference is the subject of this study. Here, we examine the hypothesis that remdesivir monophosphate (RMP)-containing RNA functions as a template at the polymerase site for the second run of RNA synthesis, and as mRNA at the decoding center for protein synthesis. Our hypothesis is supported by the observation that RMP can be incorporated into RNA by the RNA-dependent RNA polymerases (RdRps) of both viruses, although some of the incorporated RMPs are subsequently removed by exoribonucleases. Furthermore, our hypothesis is consistent with the fact that RdRp of SARS-CoV-2 selects RMP for incorporation over AMP by 3-fold in vitro, and that RMP-added RNA can be rapidly extended, even though primer extension is often paused when the added RMP is translocated at the i + 3 position (with i the nascent base pair at an initial insertion site of RMP) or when the concentrations of the subsequent nucleoside triphosphates (NTPs) are below their physiological concentrations. These observations have led to the hypothesis that remdesivir might be a delayed chain terminator. However, that hypothesis is challenged under physiological concentrations of NTPs by the observation that approximately three-quarters of RNA products efficiently overrun the pause.

Four Novel Botourmiaviruses Co-Infecting an Isolate of the Rice Blast Fungus Magnaporthe oryzae

Via virome sequencing, six viruses were detected from Magnaporthe oryzae strains YC81-2, including one virus in the family Tombusviridae, one virus in the family Narnaviridae and four viruses in the family Botourmiaviridae. Since the RNA-dependent RNA polymerase (RdRp) of one botourmiavirus show the highest identity (79%) with Magnaporthe oryzae ourmia-like virus 1 (MOLV1), the virus that was grouped into the genus Magoulivirus was designated as Magnaporthe oryzae botourmiavirus 2 (MOBV2). The three other novel botourmiaviruses were selected for further study. The complete nucleotide sequences of the three botourmiaviruses were determined. Sequence analysis showed that virus 1, virus 2, and virus 3 were 2598, 2385, and 2326 nts in length, respectively. The variable 3′ untranslated region (3′-UTR) and 5′-UTR of each virus could be folded into a stable stem-loop secondary structure. Each virus consisted of a unique ORF encoding a putative RdRp. The putative proteins with a conserved GDD motif of RdRp showed the highest sequence similarity to RdRps of viruses in the family Botourmiaviridae. Phylogenetic analysis demonstrated that these viruses were three distinct novel botourmiaviruses, clustered into the Botourmiaviridae family but not belonging to any known genera of this family. Thus, virus 1, virus 2, and virus 3 were designated as Magnaporthe oryzae botourmiavirus 5, 6, and 7 (MOBV5, MOBV6, and MOBV7), respectively. Our results suggest that four distinct botourmiaviruses, MOBV2, MOBV5, MOBV6, and MOBV7, co-infect a single strain of Magnaporthe oryzae, and MOBV5, MOBV6, and MOBV7 are members of three unclassified genera in the family Botourmiaviridae.

Evidence for Internal Initiation of RNA Synthesis by the Hepatitis C Virus RNA-Dependent RNA Polymerase NS5B In Cellulo

Initiation of RNA synthesis by the hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) NS5B has been extensively studied in vitro and in cellulo Intracellular replication is thought to rely exclusively on terminal de novo initiation, as it conserves all genetic information of the genome. In vitro, however, additional modes of initiation have been observed. In this study, we aimed to clarify whether the intracellular environment allows for internal initiation of RNA replication by the HCV replicase. We used a dual luciferase replicon harboring a terminal and an internal copy of the viral genomic 5' untranslated region, which was anticipated to support noncanonical initiation. Indeed, a shorter RNA species was detected by Northern blotting with low frequency, depending on the length and sequence composition upstream of the internal initiation site. By introducing mutations at either site, we furthermore established that internal and terminal initiation shared identical sequence requirements. Importantly, lethal point mutations at the terminal site resulted exclusively in truncated replicons. In contrast, the same mutations at the internal site abrogated internal initiation, suggesting a competitive selection of initiation sites, rather than recombination or template-switching events. In conclusion, our data indicate that the HCV replicase is capable of internal initiation in its natural environment, although functional replication likely requires only terminal initiation. Since many other positive-strand RNA viruses generate subgenomic messenger RNAs during their replication cycle, we surmise that their capability for internal initiation is a common and conserved feature of viral RdRps.IMPORTANCE Many aspects of viral RNA replication of hepatitis C virus (HCV) are still poorly understood. The process of RNA synthesis is driven by the RNA-dependent RNA polymerase (RdRp) NS5B. Most mechanistic studies on NS5B so far were performed with in vitro systems using isolated recombinant polymerase. In this study, we present a replicon model, which allows the intracellular assessment of noncanonical modes of initiation by the full HCV replicase. Our results add to the understanding of the biochemical processes underlying initiation of RNA synthesis by NS5B by the discovery of internal initiation in cellulo Moreover, they validate observations made in vitro, showing that the viral polymerase acts very similarly in isolation and in complex with other viral and host proteins. Finally, these observations provide clues about the evolution of RdRps of positive-strand RNA viruses, which might contain the intrinsic ability to initiate internally.

The hepatitis C virus RNA-dependent RNA polymerase directs incoming nucleotides to its active site through magnesium-dependent dynamics within its F motif

RNA viruses synthesize new genomes in the infected host thanks to dedicated, virally-encoded RNA-dependent RNA polymerases (RdRps). As such, these enzymes are prime targets for antiviral therapy, as has recently been demonstrated for hepatitis C virus (HCV). However, peculiarities in the architecture and dynamics of RdRps raise fundamental questions about access to their active site during RNA polymerization. Here, we used molecular modeling and molecular dynamics simulations, starting from the available crystal structures of HCV NS5B in ternary complex with template-primer duplexes and nucleotides, to address the question of ribonucleotide entry into the active site of viral RdRp. Tracing the possible passage of incoming UTP or GTP through the RdRp-specific entry tunnel, we found two successive checkpoints that regulate nucleotide traffic to the active site. We observed that a magnesium-bound nucleotide first binds next to the tunnel entry, and interactions with the triphosphate moiety orient it such that its base moiety enters first. Dynamics of RdRp motifs F1 + F3 then allow the nucleotide to interrogate the RNA template base prior to nucleotide insertion into the active site. These dynamics are finely regulated by a second magnesium dication, thus coordinating the entry of a magnesium-bound nucleotide with shuttling of the second magnesium necessary for the two-metal ion catalysis. The findings of our work suggest that at least some of these features are general to viral RdRps and provide further details on the original nucleotide selection mechanism operating in RdRps of RNA viruses.

Mycoviruses of an endophytic fungus can replicate in plant cells: evolutionary implications

So far there is no record of a specific virus able to infect both fungal and plant hosts in nature. However, experimental evidence shows that some plant virus RdRPs are able to perform replication in trans of genomic or DI RNAs in the yeast Saccharomyces cerevisiae. Furthermore, tobacco mosaic virus was recently shown to replicate in a filamentous ascomycetous fungus. Thus, at least experimentally, some plant viruses can infect some fungi. Endophytic fungi have been reported from many plants and several of these fungi have been shown to contain viruses. Here we tested if mycoviruses derived from a marine plant endophyte can replicate in plant cells. For this purpose, we used partially purified viral particles from isolate MUT4330 of Penicillium aurantiogriseum var. viridicatum which harbors six virus species, some having dsRNA and some positive-strand ssRNA genomes. These were transfected into three distinct plant protoplast cell systems. Time-course analysis of absolute RNA accumulation provided for the first time evidence that viruses of two species belonging to the Partitiviridae and Totiviridae families, can replicate in plant cells without evidence of host adaptation, i.e, changes in their nucleotide sequence.

Substrate selectivity of Dengue and Zika virus NS5 polymerase towards 2′-modified nucleotide analogues

In targeting the essential viral RNA-dependent RNA-polymerase (RdRp), nucleotide analogues play a major role in antiviral therapies. In the Flaviviridae family, the hepatitis C virus (HCV) can be eradicated from chronically infected patients using a combination of drugs which generally include the 2′-modified uridine analogue Sofosbuvir, delivered as nucleotide prodrug. Dengue and Zika viruses are emerging flaviviruses whose RdRp is closely related to that of HCV, yet no nucleoside drug has been clinically approved for these acute infections. We have purified dengue and Zika virus full-length NS5, the viral RdRps, and used them to assemble a stable binary complex made of NS5 and virus-specific RNA primer/templates. The complex was used to assess the selectivity of NS5 towards nucleotide analogues bearing modifications at the 2′-position. We show that dengue and Zika virus RdRps exhibit the same discrimination pattern: 2′-O-Me > 2′-C-Me-2′-F > 2′-C-Me nucleoside analogues, unlike HCV RdRp for which the presence of the 2′-F is beneficial rendering the discrimination pattern 2′-O-Me > 2′-C-Me ≥ 2′-C-Me-2′-F. Both 2′-C-Me and 2′-C-Me-2′-F analogues act as non-obligate RNA chain terminators. The dengue and Zika NS5 nucleotide selectivity towards 2′-modified NTPs mirrors potency of the corresponding analogues in infected cell cultures.

Molecular characterization of Botrytis ourmia-like virus, a mycovirus close to the plant pathogenic genus Ourmiavirus

The molecular characterization of a novel single-stranded RNA virus, obtained by next generation sequencing using Illumina platform, in a field grapevine isolate of the plant pathogenic fungus Botrytis, is reported in this work. The sequence comparison of this virus against the NCBI database showed a strong identity with RNA dependent RNA polymerases (RdRps) of plant pathogenic viruses belonging to the genus Ourmiavirus, therefore, this novel virus was named Botrytis ourmia-like virus (BOLV). BOLV has one open reading frame of 2169 nucleotides, which encodes a protein of 722 amino acids showing conserved domains of plant RNA viruses RdRps such as the most conserved GDD active domain. Our analyses showed that BOLV is phylogenetically closer to the fungal Narnavirus and the plant Ourmiavirus than to Mitovirus of the family Narnaviridae. Hence, we proposed that BOLV might represent the link between fungal viruses of the family Narnaviridae and the plant ourmiaviruses.

Genome sequence of a novel mycovirus of Rhizoctonia solani, a plant pathogenic fungus.

Here we present the genome sequence of a novel dsRNA virus we designed as Rhizoctonia solani RNA virus HN008 (RsRV-HN008) from a filamentous fungus R. solani. Its genome (7596 nucleotides) contains two non-overlapping open reading frames (ORF1 and ORF2). ORF1 encoded a 128 kDa protein that showed no significant identity to any other virus sequence in the NCBI database. ORF2 encoded a protein with a molecular weight of 140 kDa and shared a low percentage of sequence identity to the RdRps of unclassified dsRNA viruses. Sequence analysis revealed that RsRV-HN008 may be a member of a novel unclassified family of mycoviruses.

Plant viruses of the Amalgaviridae family evolved via recombination between viruses with double-stranded and negative-strand RNA genomes

Plant viruses of the recently recognized family Amalgaviridae have monopartite double-stranded (ds) RNA genomes and encode two proteins: an RNA-dependent RNA polymerase (RdRp) and a putative capsid protein (CP). Whereas the RdRp of amalgaviruses has been found to be most closely related to the RdRps of dsRNA viruses of the family Partitiviridae, the provenance of their CP remained obscure. Here we show that the CP of amalgaviruses is homologous to the nucleocapsid proteins of negative-strand RNA viruses of the genera Phlebovirus (Bunyaviridae) and Tenuivirus. The chimeric genomes of amalgaviruses are a testament to the effectively limitless gene exchange between viruses that shaped the evolution of the virosphere.Reviewers: This article was reviewed by Lakshminarayan M. Iyer and Nick V. Grishin. For complete reviews, see the Reviewers’ Reports section.Electronic supplementary materialThe online version of this article (doi:10.1186/s13062-015-0047-8) contains supplementary material, which is available to authorized users.

A Closed-Handed Affair: Positive-Strand RNA Virus Polymerases

ABSTRACT RNA viruses are important emerging pathogens that cause human and animal infectious diseases. Antiviral therapies have to deal with the high mutational capacity of RNA viruses, which quickly adapt to new environments. A primary target for antiviral drug development is the viral RNA-dependent RNA polymerase (RdRp), which is the central enzyme of the viral RNA replication/transcription machinery. Here, we review the current mechanistic and structural knowledge on RdRps of positive-strand RNA viruses gained through crystallography and biochemistry. In addition, we review the growing body of information on RdRp-mediated strategies, such as proofreading and genome end repair, used by positive-strand RNA viruses to maintain their genome integrity.

Mutational Evidence for a Structural Model of the Lassa Virus RNA Polymerase Domain and Identification of Two Residues, Gly1394 and Asp1395, That Are Critical for Transcription but Not Replication of the Genome

The RNA-dependent RNA polymerase (RdRp) of arenaviruses is an integral part of the L protein, a 200-kDa multifunctional and multidomain protein. In view of the paucity of structural data, we recently proposed a model for the RdRp domain of arenaviruses based on the folding of RdRps of plus-strand viruses (S. Vieth et al., Virology 318:153-168, 2004). In the present study, we have chosen a large-scale mutagenesis approach to gain insight into the structure and function of the Lassa virus RdRp domain. A total of 180 different mutants of the domain were generated by using a novel PCR-based mutagenesis technique and tested in the context of the Lassa virus replicon system. Nearly all residues, which were essential for function, clustered in the center of the three-dimensional model including the catalytic site, while residues that were less important for function mapped to the periphery of the model. The combined bioinformatics and mutagenesis data allowed deducing candidate residues for ligand interaction. Mutation of two adjacent residues in the putative palm-thumb subdomain junction, G1394 and D1395 (strain AV), led to a defect in mRNA synthesis but did not affect antigenomic RNA synthesis. In conclusion, the data provide circumstantial evidence for the existence of an RdRp domain between residues 1040 and 1540 of the Lassa virus L protein and the folding model of the domain. A functional element within the RdRp was identified, which is important for transcription but not replication of the genome.

A second, non-canonical RNA-dependent RNA polymerase in SARS Coronavirus

In (+) RNA coronaviruses, replication and transcription of the giant ∼30 kb genome to produce genome- and subgenome-size RNAs of both polarities are mediated by a cognate membrane-bound enzymatic complex. Its RNA-dependent RNA polymerase (RdRp) activity appears to be supplied by non-structural protein 12 (nsp12) that includes an RdRp domain conserved in all RNA viruses. Using SARS coronavirus, we now show that coronaviruses uniquely encode a second RdRp residing in nsp8. This protein strongly prefers the internal 5′-(G/U)CC-3′ trinucleotides on RNA templates to initiate the synthesis of complementary oligonucleotides of <6 residues in a reaction whose fidelity is relatively low. Distant structural homology between the C-terminal domain of nsp8 and the catalytic palm subdomain of RdRps of RNA viruses suggests a common origin of the two coronavirus RdRps, which however may have evolved different sets of catalytic residues. A parallel between the nsp8 RdRp and cellular DNA-dependent RNA primases is drawn to propose that the nsp8 RdRp produces primers utilized by the primer-dependent nsp12 RdRp.

Intergeneric relationship between the Aspergillus ochraceous virus F and the Penicillium stoloniferum virus S

It was reported that the ‘slow’ component (PsV-S) of Penicillium stoloniferum virus complex also occurred in a second genus, Aspergillus ochraceous. The responsible virus for this intergeneric occurrence was considered to be the ‘fast’ component (AoV-F) of A. ochraceous virus complex. In this investigation, AoV dsRNA 1, that was previously shown to cross-hybridize with PsV-S dsRNA, has been cloned. It was 1754 bp in length and contained one open reading frame of 539 amino acids (p63), had the same genome organization as PsV-S dsRNA S1 and also had the conserved sequence motif of the PsV-S dsRNAs (5′-GCGCAAAA-3′) at the 5′ terminus. A BLAST search indicated that p63 was a putative dsRNA-dependent RNA polymerase (RdRp), had 81% of sequence homology to members of the genus Partitivirus, and grouped together with PsV-S in phylogenetic analysis. But immunoblot analysis showed that the capsid protein (P3) of AoV-F virus component did not reacted against PsV-S antiserum. These evidences suggest that the cross serological relationship between AoV-F and PsV-S previously observed may have been due to the RdRps of the respective viruses rather than between their respective capsid proteins as was assumed in 1985.

Comparative mechanistic studies of de novo RNA synthesis by flavivirus RNA-dependent RNA polymerases

Flavivirus protein NS5 harbors the RNA-dependent RNA polymerase (RdRp) activity. In contrast to the RdRps of hepaci- and pestiviruses, which belong to the same family of Flaviviridae, NS5 carries two activities, a methyltransferase (MTase) and a RdRp. RdRp domains of Dengue virus (DV) and West Nile virus (WNV) NS5 were purified in high yield relative to full-length NS5 and showed full RdRp activity. Steady-state enzymatic parameters were determined on homopolymeric template poly(rC). The presence of the MTase domain does not affect the RdRp activity. Flavivirus RdRp domains might bear more than one GTP binding site displaying positive cooperativity. The kinetics of RNA synthesis by four Flaviviridae RdRps were compared. In comparison to Hepatitis C RdRp, DV and WNV as well as Bovine Viral Diarrhea virus RdRps show less rate limitation by early steps of short-product formation. This suggests that they display a higher conformational flexibility upon the transition from initiation to elongation.

Bacterial Expression and Characterization of a Single‐Chain Variable Fragment Antibody Specific to Several Replicases of Plant (+)RNA Viruses

AbstractThe use of synthetic antibodies may be an alternative tool for plant protection. RNA‐dependent RNA polymerases (RdRps) play a key role in the process of virus replication. If recombinant antibodies like single‐chain variable fragment antibodies (scFv) are directed against RdRps, they might be used to create virus‐resistant plants. To this end, a recombinant antibody from a phage display library directed against the conserved amino acid domain (glycine‐aspartate‐aspartate, GDD) in the active centre of the RdRp of plant (+)RNA viruses was selected. High expression level in Escherichia coli enabled the purification of scFv which was used to raise polyclonal anti‐scFv antibodies. The scFv was characterized and assessed for specificity to the RdRps of viruses belonging to different families. It reacted with the RdRps of numerous distinct viruses, showing its potential in generating resistance to a large number of (+)RNA viruses containing the GDD motif.

Heterologous RNA replication enhancer stimulates in vitro RNA synthesis and template-switching by the carmovirus, but not by the tombusvirus, RNA-dependent RNA polymerase: Implication for modular evolution of RNA viruses

The viral RNA plays multiple roles during replication of RNA viruses, serving as a template for complementary RNA synthesis and facilitating the assembly of the viral replicase complex. These roles are coordinated by cis-acting regulatory elements, such as promoters and replication enhancers (REN). To test if these RNA elements can be used by related viral RNA-dependent RNA polymerases (RdRp), we compared the potential stimulatory effects of homologous and heterologous REN elements on complementary RNA synthesis and template-switching by the tombus- ( Cucumber necrosis virus, CNV), carmovirus ( Turnip crinkle virus, TCV) and hepatitis C virus (HCV) RdRps in vitro. The CNV RdRp selectively utilized its cognate REN, while discriminating against the heterologous TCV REN. On the contrary, RNA synthesis by the TCV RdRp was stimulated by the TCV REN and the heterologous tombusvirus REN with comparable efficiency. The heterologous REN elements also promoted in vitro template-switching by the TCV and HCV RdRps. Based on these observations, we propose that REN elements could facilitate intervirus recombination and postrecombinational amplification of new recombinant viruses.

De novo RNA synthesis and homology modeling of the classical swine fever virus RNA polymerase

Classical swine fever virus (CSFV) non-structural protein 5B (NS5B) encodes an RNA-dependent RNA polymerase (RdRp), a key enzyme which initiates RNA replication by a de novo mechanism without a primer and is a potential target for anti-virus therapy. We expressed the NS5B protein in Escherichia coli. The rGTP can stimulate de novo initiation of RNA synthesis and mutation of the GDD motif to Gly–Asp–Asp (GAA) abolishes the RNA synthesis. To better understand the mechanism of viral RNA synthesis in CSFV, a three-dimensional model was built by homology modeling based on the alignment with several virus RdRps. The model contains 605 residues folded in the characteristic fingers, palm and thumb domains. The fingers domain contains an N-terminal region that plays an important role in conformational change. We propose that the experimentally observed promotion of polymerase efficiency by rGTP is probably due to the conformational changes of the polymerase caused by binding the rGTP. Mutation of the GDD to GAA interferes with the interaction between the residues at the polymerase active site and metal ions, and thus renders the polymerase inactive.

Single-chain antibodies against a plant viral RNA-dependent RNA polymerase confer virus resistance.

Crop loss due to viral diseases is still a major problem for agriculture today. We present a strategy to achieve virus resistance based on the expression of single-chain Fv fragments (scFvs) against a conserved domain in a plant viral RNA-dependent RNA polymerase (RdRp), a key enzyme in virus replication. The selected scFvs inhibited complementary RNA synthesis of different plant virus RdRps in vitro and virus replication in planta. Moreover, the scFvs also bound to the RdRp of the distantly related hepatitis C virus. T(1) and T(2) progeny of transgenic lines of Nicotiana benthamiana expressing different scFvs either in the cytosol or in the endoplasmic reticulum showed varying degrees of resistance against four plant viruses from different genera, three of which belong to the Tombusviridae family. Virus resistance based on antibodies to RdRps adds another tool to the repertoire for combating plant viruses.