scholarly journals Low-Fidelity Polymerases of Alphaviruses Recombine at Higher Rates To Overproduce Defective Interfering Particles

2015 ◽  
Vol 90 (5) ◽  
pp. 2446-2454 ◽  
Author(s):  
Enzo Z. Poirier ◽  
Bryan C. Mounce ◽  
Kathryn Rozen-Gagnon ◽  
Peter Jan Hooikaas ◽  
Kenneth A. Stapleford ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Viruses ◽  
Rna Virus ◽  
Point Mutations ◽  
Viral Fitness ◽  
Dependent Manner ◽  
Rna Dependent Rna Polymerase ◽  
Live Attenuated Vaccine ◽  
Defective Interfering Particles

ABSTRACTLow-fidelity RNA-dependent RNA polymerases for many RNA virus mutators have been shown to confer attenuated phenotypes, presumably due to increased mutation rates. Additionally, for many RNA viruses, replication to high titers results in the production of defective interfering particles (DIs) that also attenuate infection. We hypothesized that fidelity, recombination, and DI production are tightly linked. We show that a Sindbis virus mutator replicating at a high multiplicity of infection manifests an earlier and greater accumulation of DIs than its wild-type counterpart. The isolated DIs interfere with the replication of full-length virus in a dose-dependent manner. Importantly, the ability of the mutator virus to overproduce DIs could be linked to an increased recombination frequency. These data confirm that RNA-dependent RNA polymerase fidelity and recombination are inversely correlated for this mutator. Our findings suggest that defective interference resulting from higher recombination rates may be more detrimental to RNA virus mutators than the increase in mutational burden.IMPORTANCEReplication, adaptation, and evolution of RNA viruses rely in large part on their low-fidelity RNA-dependent RNA polymerase. Viruses artificially modified in their polymerases to decrease fidelity (mutator viruses) are attenuatedin vivo, demonstrating the important role of fidelity in viral fitness. However, attenuation was attributed solely to the modification of the viral mutation rate and the accumulation of detrimental point mutations. In this work, we described an additional phenotype of mutator viruses: an increased recombination rate leading to defective interfering particle (DI) overproduction. Because DIs are known for their inhibitory effect on viral replication, our work suggests that fidelity variants may be attenuatedin vivovia several mechanisms. This has important implications in the development of fidelity variants as live attenuated vaccine strains.

scholarly journals Point mutations in the West Nile virus (Flaviviridae; Flavivirus) RNA-dependent RNA polymerase alter viral fitness in a host-dependent manner in vitro and in vivo

Virology ◽  
2012 ◽  
Vol 427 (1) ◽  
pp. 18-24 ◽  
Author(s):  
Greta A. Van Slyke ◽  
Alexander T. Ciota ◽  
Graham G. Willsey ◽  
Joachim Jaeger ◽  
Pei-Yong Shi ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Rna Polymerase ◽  
Point Mutations ◽  
Viral Fitness ◽  
Dependent Manner ◽  
The West ◽  
Rna Dependent Rna Polymerase ◽  
West Nile

scholarly journals Plant Virus Genome Is Shaped by Specific Dinucleotide Restrictions That Influence Viral Infection

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Alfonso González de Prádena ◽  
Adrián Sánchez Jimenez ◽  
David San León ◽  
Peter Simmonds ◽  
Juan Antonio García ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
High Throughput Sequencing ◽  
Rna Viruses ◽  
Rna Virus ◽  
Virus Genome ◽  
Viral Fitness ◽  
Plum Pox Virus ◽  
Dependent Manner ◽  
Viral Restriction ◽  
Rna Accumulation

ABSTRACT The presence of CpG and UpA dinucleotides is restricted in the genomes of animal RNA viruses to avoid specific host defenses. We wondered whether a similar phenomenon exists in nonanimal RNA viruses. Here, we show that these two dinucleotides, especially UpA, are underrepresented in the family Potyviridae, the most important group of plant RNA viruses. Using plum pox virus (PPV; Potyviridae family) as a model, we show that an increase in UpA frequency strongly diminishes virus accumulation. Remarkably, unlike previous observations in animal viruses, PPV variants harboring CpG-rich fragments display just faint (or no) attenuation. The anticorrelation between UpA frequency and viral fitness additionally demonstrates the relevance of this particular dinucleotide: UpA-high mutants are attenuated in a dose-dependent manner, whereas a UpA-low variant displays better fitness than its parental control. Using high-throughput sequencing, we also show that UpA-rich PPV variants are genetically stable, without apparent changes in sequence that revert and/or compensate for the dinucleotide modification despite its attenuation. In addition, we also demonstrate here that the PPV restriction of UpA-rich variants works independently of the classical RNA silencing pathway. Finally, we show that the anticorrelation between UpA frequency and RNA accumulation applies to mRNA-like fragments produced by the host RNA polymerase II. Together, our results inform us about a dinucleotide-based system in plant cells that controls diverse RNAs, including RNA viruses. IMPORTANCE Dinucleotides (combinations of two consecutive nucleotides) are not randomly present in RNA viruses; in fact, the presence of CpG and UpA is significantly repressed in their genomes. Although the meaning of this phenomenon remains obscure, recent studies with animal-infecting viruses have revealed that their low CpG/UpA frequency prevents virus restriction via a host antiviral system that recognizes, and promotes the degradation of, CpG/UpA-rich RNAs. Whether similar systems act in organisms from other life kingdoms has been unknown. To fill this gap in our knowledge, we built several synthetic variants of a plant RNA virus with deoptimized dinucleotide frequencies and analyzed their viral fitness and genome adaptation. In brief, our results inform us for the first time about an effective dinucleotide-based system that acts in plants against viruses. Remarkably, this viral restriction in plants is reminiscent of, but not identical to, the equivalent antiviral response in animals.

scholarly journals Late Male-Killing Viruses in Homona magnanima Identified as Osugoroshi Viruses, Novel Members of Partitiviridae

2021 ◽  
Vol 11 ◽  
Author(s):  
Ryosuke Fujita ◽  
Maki N. Inoue ◽  
Takumi Takamatsu ◽  
Hiroshi Arai ◽  
Mayu Nishino ◽  
...  
Keyword(s):  
Mathematical Modeling ◽  
Rna Polymerase ◽  
Rna Viruses ◽  
Rna Virus ◽  
Horizontal Transmission ◽  
Rna Dependent Rna Polymerase ◽  
Double Stranded Rna ◽  
First Report ◽  
Male Killing ◽  
Male Specific

Late male-killing, a male-specific death after hatching, is a unique phenotype found in Homona magnanima, oriental tea tortrix. The male-killing agent was suspected to be an RNA virus, but details were unknown. We herein successfully isolated and identified the putative male-killing virus as Osugoroshi viruses (OGVs). The three RNA-dependent RNA polymerase genes detected were phylogenetically related to Partitiviridae, a group of segmented double-stranded RNA viruses. Purified dsRNA from a late male-killing strain of H. magnanima revealed 24 segments, in addition to the RdRps, with consensus terminal sequences. These segments included the previously found male-killing agents MK1068 (herein OGV-related RNA16) and MK1241 (OGV-related RNA7) RNAs. Ultramicroscopic observation of purified virions, which induced late male-killing in the progeny of injected moths, showed sizes typical of Partitiviridae. Mathematical modeling showed the importance of late male-killing in facilitating horizontal transmission of OGVs in an H. magnanima population. This study is the first report on the isolation of partiti-like virus from insects, and one thought to be associated with late male-killing, although the viral genomic contents and combinations in each virus are still unknown.

scholarly journals NeoRdRp: A comprehensive dataset for identifying RNA-dependent RNA polymerase of various RNA viruses from metatranscriptomic data

2022 ◽  
Author(s):  
Shoichi Sakaguchi ◽  
Syun-ichi Urayama ◽  
Yoshihiro Takaki ◽  
Hong Wu ◽  
Youichi Suzuki ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Viruses ◽  
Rna Virus ◽  
Sequence Similarity ◽  
Virus Detection ◽  
Detection Methods ◽  
Amino Acid Sequence Similarity ◽  
Sequencing Data ◽  
Rna Dependent Rna Polymerase ◽  
Multiple Sequence

RNA viruses are distributed in various environments, and most RNA viruses have been recently identified by metatranscriptome sequencing. However, due to the high nucleotide diversity of RNA viruses, it is still challenging to identify their sequences. Therefore, this study generated a dataset of RNA-dependent RNA polymerase (RdRp) domains essential for all RNA viruses belonging to Orthornavirae. Also, the collected genes with RdRp domains from various RNA viruses were clustered by amino acid sequence similarity. For each cluster, a multiple sequence alignment was generated, and a hidden Markov model (HMM) profile was created if the number of sequences was greater than five. Using the 1,467 HMM profiles, we detected RdRp domains in the RefSeq RNA virus sequences, combined the hit sequences with the RdRp domains, and reconstructed the HMM profiles. As a result, 2,234 HMM profiles were generated from 12,316 RdRp domain sequences, and the dataset was named NeoRdRp. Additionally, using the UniProt dataset, we confirmed that almost all NeoRdRp HMM profiles could specifically detect RdRps in Orthornavirae. Furthermore, we compared the NeoRdRp dataset with two previously reported RNA virus detection methods to detect RNA virus sequences from metatranscriptome sequencing data. Our methods can identify most of the RNA viruses in the datasets; however, some RNA viruses were not detected, similar to the other two methods. The NeoRdRp can be improved by repeatedly adding new RdRp sequences and can be expected to be widely applied as a system for detecting various RNA viruses from metatranscriptome data.

scholarly journals Isolation and Analysis of Rare Norovirus Recombinants from Coinfected Mice Using Drop-Based Microfluidics

2015 ◽  
Vol 89 (15) ◽  
pp. 7722-7734 ◽  
Author(s):  
Huidan Zhang ◽  
Shelley K. Cockrell ◽  
Abimbola O. Kolawole ◽  
Assaf Rotem ◽  
Adrian W. R. Serohijos ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Viruses ◽  
Phylogenetic Analyses ◽  
Low Frequency ◽  
Template Switching ◽  
Murine Norovirus ◽  
Rna Dependent Rna Polymerase ◽  
Infectious Gastroenteritis

ABSTRACTHuman noroviruses (HuNoVs) are positive-sense RNA viruses that can cause severe, highly infectious gastroenteritis. HuNoV outbreaks are frequently associated with recombination between circulating strains. Strain genotyping and phylogenetic analyses show that noroviruses often recombine in a highly conserved region near the junction of the viral polyprotein (open reading frame 1 [ORF1]) and capsid (ORF2) genes and occasionally within the RNA-dependent RNA polymerase (RdRP) gene. Although genotyping methods are useful for tracking changes in circulating viral populations, they report only the dominant recombinant strains and do not elucidate the frequency or range of recombination events. Furthermore, the relatively low frequency of recombination in RNA viruses has limited studies to cell culture orin vitrosystems, which do not reflect the complexities and selective pressures present in an infected organism. Using two murine norovirus (MNV) strains to model coinfection, we developed a microfluidic platform to amplify, detect, and recover individual recombinants followingin vitroandin vivocoinfection. One-step reverse transcriptase PCR (RT-PCR) was performed in picoliter drops with primers that identified the wild-type and recombinant progenies and scanned for recombination breakpoints at ∼1-kb intervals. We detected recombination between MNV strains at multiple loci spanning the viral protease, RdRP, and capsid ORFs and isolated individual recombinant RNA genomes that were present at a frequency of 1/300,000 or higher. This study is the first to examine norovirus recombination following coinfection of an animal and suggests that the exchange of RNA among viral genomes in an infected host occurs in multiple locations and is an important driver of genetic diversity.IMPORTANCERNA viruses increase diversity and escape host immune barriers by genomic recombination. Studies using a number of viral systems indicate that recombination occurs via template switching by the virus-encoded RNA-dependent RNA polymerase (RdRP). However, factors that govern the frequency and positions of recombination in an infected organism remain largely unknown. This work leverages advances in the applied physics of drop-based microfluidics to isolate and sequence rare recombinants arising from the coinfection of mice with two distinct strains of murine norovirus. This study is the first to detect and analyze norovirus recombination in an animal model.

In Silico Identification of a Potent Arsenic Based Approved Drug Darinaparsin against SARS-CoV-2: Inhibitor of RNA Dependent RNA polymerase (RdRp) and Essential Proteases

2020 ◽  
Vol 20 ◽  
Author(s):  
Trinath Chowdhury ◽  
Gourisankar Roymahapatra ◽  
Santi M. Mandal
Keyword(s):  
Rna Polymerase ◽  
Viral Entry ◽  
In Silico ◽  
Rna Dependent Rna Polymerase ◽  
Replication Complex ◽  
In Silico Study ◽  
Life Threatening ◽  
In Vivo Analysis ◽  
3Cl Protease

Background: COVID-19 is a life threatening novel corona viral infection to our civilization and spreading rapidly. Terrific efforts are generous by the researchers to search for a drug to control SARS-CoV-2. Methods: Here, a series of arsenical derivatives were optimized and analyzed with in silico study to search the inhibitor of RNA dependent RNA polymerase (RdRp), the major replication factor of SARS-CoV-2. All the optimized derivatives were blindly docked with RdRp of SARS-CoV-2 using iGEMDOCK v2.1. Results: Based on the lower idock score in the catalytic pocket of RdRp, darinaparsin (-82.52 kcal/mol) revealed most effective among them. Darinaparsin strongly binds with both Nsp9 replicase protein (-8.77 kcal/mol) and Nsp15 endoribonuclease (-8.3 kcal/mol) of SARS-CoV-2 as confirmed from the AutoDock analysis. During infection, the ssRNA of SARS-CoV2 is translated into large polyproteins forming viral replication complex by specific proteases like 3CL protease and papain protease. This is also another target to control the virus infection where darinaparsin also perform the inhibitory role to proteases of 3CL protease (-7.69 kcal/mol) and papain protease (-8.43 kcal/mol). Conclusion: In host cell, the furin protease serves as a gateway to the viral entry and darinaparsin docked with furin protease which revealed a strong binding affinity. Thus, screening of potential arsenic drugs would help in providing the fast invitro to in-vivo analysis towards development of therapeutics against SARS-CoV-2.

scholarly journals Essential role of IPS-1 in innate immune responses against RNA viruses

2006 ◽  
Vol 203 (7) ◽  
pp. 1795-1803 ◽  
Author(s):  
Himanshu Kumar ◽  
Taro Kawai ◽  
Hiroki Kato ◽  
Shintaro Sato ◽  
Ken Takahashi ◽  
...  
Keyword(s):  
Rna Viruses ◽  
Rna Virus ◽  
Critical Role ◽  
Nuclear Factor Κb ◽  
Type I ◽  
Innate Immune Responses ◽  
Antiviral Responses ◽  
Deficient Mice

IFN-β promoter stimulator (IPS)-1 was recently identified as an adapter for retinoic acid–inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (Mda5), which recognize distinct RNA viruses. Here we show the critical role of IPS-1 in antiviral responses in vivo. IPS-1–deficient mice showed severe defects in both RIG-I– and Mda5-mediated induction of type I interferon and inflammatory cytokines and were susceptible to RNA virus infection. RNA virus–induced interferon regulatory factor-3 and nuclear factor κB activation was also impaired in IPS-1–deficient cells. IPS-1, however, was not essential for the responses to either DNA virus or double-stranded B-DNA. Thus, IPS-1 is the sole adapter in both RIG-I and Mda5 signaling that mediates effective responses against a variety of RNA viruses.

scholarly journals Cotranslational Coat Protein-Mediated Inhibition of Potyviral RNA Translation

2015 ◽  
Vol 89 (8) ◽  
pp. 4237-4248 ◽  
Author(s):  
Jane Besong-Ndika ◽  
Konstantin I. Ivanov ◽  
Anders Hafrèn ◽  
Thierry Michon ◽  
Kristiina Mäkinen
Keyword(s):  
Coat Protein ◽  
Stop Codon ◽  
Rna Virus ◽  
Viral Rna ◽  
Dependent Manner ◽  
Content Type ◽  
Virion Assembly ◽  
Rna Translation ◽  
Intrinsic Capacity

ABSTRACTPotato virus A(PVA) is a single-stranded positive-sense RNA virus and a member of the familyPotyviridae. The PVA coat protein (CP) has an intrinsic capacity to self-assemble into filamentous virus-like particles, but the mechanism responsible for the initiation of viral RNA encapsidationin vivoremains unclear. Apart from virion assembly, PVA CP is also involved in the inhibition of viral RNA translation. In this study, we show that CP inhibits PVA RNA translation in a dose-dependent manner, through a mechanism involving the CP-encoding region. Analysis of this region, however, failed to identify any RNA secondary structure(s) preferentially recognized by CP, suggesting that the inhibition depends on CP-CP rather than CP-RNA interactions. In agreement with this possibility, insertion of an in-frame stop codon upstream of the CP sequence led to a marked decrease in the inhibition of viral RNA translation. Based on these results, we propose a model in which the cotranslational interactions between excess CP accumulating intransand CP translated from viral RNA incisare required to initiate the translational repression. This model suggests a mechanism for how viral RNA can be sequestered from translation and specifically selected for encapsidation at the late stages of viral infection.IMPORTANCEThe main functions of the CP during potyvirus infection are to protect viral RNA from degradation and to transport it locally, systemically, and from host to host. Although virion assembly is a key step in the potyviral infectious cycle, little is known about how it is initiated and how viral RNA is selected for encapsidation. The results presented here suggest that CP-CP rather than CP-RNA interactions are predominantly involved in the sequestration of viral RNA away from translation. We propose that the cotranslational nature of these interactions may represent a mechanism for the selection of viral RNA for encapsidation. A better understanding of the mechanism of virion assembly may lead to development of crops resistant to potyviruses at the level of viral RNA encapsidation, thereby reducing the detrimental effects of potyvirus infections on food production.

scholarly journals Structural and Nonstructural Genes Contribute to the Genetic Diversity of RNA Viruses

mBio ◽  
2018 ◽  
Vol 9 (5) ◽  
Author(s):  
Natalie D. Collins ◽  
Andrew S. Beck ◽  
Steven G. Widen ◽  
Thomas G. Wood ◽  
Stephen Higgs ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Yellow Fever ◽  
Yellow Fever Virus ◽  
Rna Viruses ◽  
Rna Virus ◽  
Infectious Clone ◽  
Vaccine Virus ◽  
Wild Type ◽  
Rna Dependent Rna Polymerase ◽  
Replication Complex

ABSTRACT One paradigm to explain the complexity of viral RNA populations is that the low fidelity of the RNA-dependent RNA polymerase (RdRp) drives high mutation rates and consequently genetic diversity. Like most RNA viruses, wild-type yellow fever virus (YFV) replication is error-prone due to the lack of proofreading by the virus-encoded RdRp. However, there is evidence that replication of the live attenuated YF vaccine virus 17D, derived from wild-type strain Asibi, is less error-prone than wild-type RNA viruses. Recent studies comparing the genetic diversity of wild-type Asibi and 17D vaccine virus found that wild-type Asibi has the typical heterogeneous population of an RNA virus, while there is limited intra- and interpopulation variability of 17D vaccine virus. Utilizing chimeric and mutant infectious clone-derived viruses, we show that high and low genetic diversity profiles of wild-type Asibi virus and vaccine virus 17D, respectively, are multigenic. Introduction of either structural (pre-membrane and envelope) genes or NS2B or NS4B substitutions into the Asibi and 17D backbone resulted in altered variant population, nucleotide diversity, and mutation frequency compared to the parental viruses. Additionally, changes in genetic diversity of the chimeric and mutant viruses correlated with the phenotype of multiplication kinetics in human alveolar A549 cells. Overall, the paradigm that only the error-prone RdRp controls genetic diversity needs to be expanded to address the role of other genes in genetic diversity, and we hypothesize that it is the replication complex as a whole and not the RdRp alone that controls genetic diversity. IMPORTANCE With the advent of advanced sequencing technology, studies of RNA viruses have shown that genetic diversity can contribute to both attenuation and virulence and the paradigm is that this is controlled by the error-prone RNA-dependent RNA polymerase (RdRp). Since wild-type yellow fever virus (YFV) strain Asibi has genetic diversity typical of a wild-type RNA virus, while 17D virus vaccine has limited diversity, it provides a unique opportunity to investigate RNA population theory in the context of a well-characterized live attenuated vaccine. Utilizing infectious clone-derived viruses, we show that genetic diversity of RNA viruses is complex and that multiple genes, including structural genes and NS2B and NS4B genes also contribute to genetic diversity. We suggest that the replication complex as a whole, rather than only RdRp, drives genetic diversity, at least for YFV.

scholarly journals In vitro analysis of a transcription termination site for RNA polymerase II.

1990 ◽  
Vol 10 (11) ◽  
pp. 5782-5795 ◽  
Author(s):  
D K Wiest ◽  
D K Hawley
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Dna Sequences ◽  
Nuclear Extract ◽  
Transcription Unit ◽  
Dependent Manner ◽  
Wide Range ◽  
Vitro Analysis

Transcription from the adenovirus major late (ML) promoter has previously been shown to pause or terminate prematurely in vivo and in vitro at a site within the first intron of the major late transcription unit. We are studying the mechanism of elongation arrest at this site in vitro to define the DNA sequences and proteins that determine the elongation behavior of RNA polymerase II. Our assay system consists of a nuclear extract prepared from cultured human cells. With standard reaction conditions, termination is not observed downstream of the ML promoter. However, in the presence of Sarkosyl, up to 80% of the transcripts terminate 186 nucleotides downstream of the start site. Using this assay, we showed that the DNA sequences required to promote maximal levels of termination downstream of the ML promoter reside within a 65-base-pair region and function in an orientation-dependent manner. To test whether elongation complexes from the ML promoter were functionally homogeneous, we determined the termination efficiency at each of two termination sites placed in tandem. We found that the behavior of the elongation complexes was different at these sites, with termination being greater at the downstream site over a wide range of Sarkosyl concentrations. This result ruled out a model in which the polymerases that read through the first site were stably modified to antiterminate. We also demonstrated that the ability of the elongation complexes to respond to the ML termination site was promoter specific, as the site did not function efficiently downstream of a heterologous promoter. Taken together, the results presented here are not consistent with the simplest class of models that have been proposed previously for the mechanism of Sarkosyl-induced termination.

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