scholarly journals Poliovirus Polymerase Leu420 Facilitates RNA Recombination and Ribavirin Resistance

2016 ◽  
Vol 90 (19) ◽  
pp. 8410-8421 ◽  
Author(s):  
Brian J. Kempf ◽  
Olve B. Peersen ◽  
David J. Barton
Keyword(s):  
Molecular Mechanisms ◽  
Antiviral Drug ◽  
Rna Replication ◽  
Viral Rna ◽  
Rna Recombination ◽  
Elongation Complex ◽  
Species Groups ◽  
Nascent Rna ◽  
Substitution Mutations ◽  
And Function

ABSTRACTRNA recombination is important in the formation of picornavirus species groups and the ongoing evolution of viruses within species groups. In this study, we examined the structure and function of poliovirus polymerase, 3Dpol, as it relates to RNA recombination. Recombination occurs when nascent RNA products exchange one viral RNA template for another during RNA replication. Because recombination is a natural aspect of picornavirus replication, we hypothesized that some features of 3Dpolmay exist, in part, to facilitate RNA recombination. Furthermore, we reasoned that alanine substitution mutations that disrupt 3Dpol-RNA interactions within the polymerase elongation complex might increase and/or decrease the magnitudes of recombination. We found that an L420A mutation in 3Dpoldecreased the frequency of RNA recombination, whereas alanine substitutions at other sites in 3Dpolincreased the frequency of recombination. The 3DpolLeu420 side chain interacts with a ribose in the nascent RNA product 3 nucleotides from the active site of the polymerase. Notably, the L420A mutation that reduced recombination also rendered the virus more susceptible to inhibition by ribavirin, coincident with the accumulation of ribavirin-induced G→A and C→U mutations in viral RNA. We conclude that 3DpolLeu420 is critically important for RNA recombination and that RNA recombination contributes to ribavirin resistance.IMPORTANCERecombination contributes to the formation of picornavirus species groups and the emergence of circulating vaccine-derived polioviruses (cVDPVs). The recombinant viruses that arise in nature are occasionally more fit than either parental strain, especially when the two partners in recombination are closely related, i.e., members of characteristic species groups, such as enterovirus species groups A to H or rhinovirus species groups A to C. Our study shows that RNA recombination requires conserved features of the viral polymerase. Furthermore, a polymerase mutation that disables recombination renders the virus more susceptible to the antiviral drug ribavirin, suggesting that recombination contributes to ribavirin resistance. Elucidating the molecular mechanisms of RNA replication and recombination may help mankind achieve and maintain poliovirus eradication.

scholarly journals CRISPR/Cas9-Mediated Knockout of DNAJC14 Verifies This Chaperone as a Pivotal Host Factor for RNA Replication of Pestiviruses

2018 ◽  
Vol 93 (5) ◽  
Author(s):  
O. Isken ◽  
A. Postel ◽  
B. Bruhn ◽  
E. Lattwein ◽  
P. Becher ◽  
...  
Keyword(s):  
Nonstructural Protein ◽  
Rna Replication ◽  
Viral Rna ◽  
Temporal Control ◽  
Rna Recombination ◽  
Diarrhea Virus ◽  
Nonstructural Protein 2 ◽  
Persistent Infections ◽  
Cellular Cofactor ◽  
Viral Diarrhea Virus

ABSTRACTPestiviruses like bovine viral diarrhea virus (BVDV) are a threat to livestock. For pestiviruses, cytopathogenic (cp) and noncytopathogenic (noncp) strains are distinguished in cell culture. The noncp biotype of BVDV is capable of establishing persistent infections, which is a major problem in disease control. The noncp biotype rests on temporal control of viral RNA replication, mediated by regulated cleavage of nonstructural protein 2-3 (NS2-3). This cleavage is catalyzed by the autoprotease in NS2, the activity of which depends on its cellular cofactor, DNAJC14. Since this chaperone is available in small amounts and binds tightly to NS2, NS2-3 translated later in infection is no longer cleaved. As NS3 is an essential constituent of the viral replicase, this shift in polyprotein processing correlates with downregulation of RNA replication. In contrast, cp BVDV strains arising mostly by RNA recombination show highly variable genome structures and display unrestricted NS3 release. The functional importance of DNAJC14 for noncp pestiviruses has been established so far only for BVDV-1. It was therefore enigmatic whether replication of other noncp pestiviruses is also DNAJC14 dependent. By generating bovine and porcine DNAJC14 knockout cells, we could show that (i) replication of 6 distinct noncp pestivirus species (A to D, F, and G) depends on DNAJC14, (ii) the pestiviral replicase NS3-5B can assemble into functional complexes in the absence of DNAJC14, and (iii) all cp pestiviruses replicate their RNA and generate infectious progeny independent of host DNAJC14. Together, these findings confirm DNAJC14 as a pivotal cellular cofactor for the replication and maintenance of the noncp biotype of pestiviruses.IMPORTANCEOnly noncp pestivirus strains are capable of establishing life-long persistent infections to generate the virus reservoir in the field. The molecular basis for this biotype is only partially understood and only investigated in depth for BVDV-1 strains. Temporal control of viral RNA replication correlates with the noncp biotype and is mediated by limiting amounts of cellular DNAJC14 that activate the viral NS2 protease to catalyze the release of the essential replicase component NS3. Here, we demonstrate that several species of noncp pestiviruses depend on DNAJC14 for their RNA replication. Moreover, all cp pestiviruses, in sharp contrast to their noncp counterparts, replicate independently of DNAJC14. The generation of a cp BVDV in the persistently infected animal is causative for onset of mucosal disease. Therefore, the observed strict biotype-specific difference in DNAJC14 dependency should be further examined for its role in cell type/tissue tropism and the pathogenesis of this lethal disease.

scholarly journals An Extended Primer Grip of Picornavirus Polymerase Facilitates Sexual RNA Replication Mechanisms

2019 ◽  
Author(s):  
Brian J. Kempf ◽  
Colleen L. Watkins ◽  
Olve B. Peersen ◽  
David J Barton
Keyword(s):  
Rna Synthesis ◽  
Rna Replication ◽  
Serial Passage ◽  
Resistant Virus ◽  
Rna Recombination ◽  
Resistance Mutations ◽  
Error Catastrophe ◽  
Sequence Complementarity ◽  
Species Groups ◽  
Codon Mutation

ABSTRACTPicornaviruses have both asexual and sexual RNA replication mechanisms. Asexual RNA replication mechanisms involve one parental template whereas sexual RNA replication mechanisms involve two or more parental templates. Because sexual RNA replication mechanisms counteract ribavirin-induced error catastrophe, we selected for ribavirin-resistant poliovirus to identify polymerase residues that facilitate sexual RNA replication mechanisms. We used serial passage in ribavirin, beginning with a variety of ribavirin-sensitive and ribavirin-resistant parental viruses. Ribavirin-sensitive virus contained an L420A polymerase mutation while ribavirin-resistant virus contained a G64S polymerase mutation. A G64 codon mutation (G64Fix) was used to inhibit emergence of G64S-mediated ribavirin resistance. Revertants (L420) or pseudo-revertants (L420V, L420I) were selected from all independent lineages of L420A, G64Fix L420A and G64S L420A parental viruses. Ribavirin-resistant G64S mutations were selected in two independent lineages and novel ribavirin-resistance mutations were selected in the polymerase in other lineages (M299I, M323I, M392V, T353I). The structural orientation of M392, immediately adjacent to L420 and the polymerase primer grip region, led us to engineer additional polymerase mutations into poliovirus (M392A, M392L & M392V and K375R & R376K). L420A revertants and pseudorevertants (L420V, L420I) restored efficient sexual RNA replication mechanisms, confirming that ribavirin-induced error catastrophe coincides with defects in sexual RNA replication mechanisms. Viruses containing M392 mutations (M392A, M392L & M392V) and primer grip mutations (K375R & R376K) exhibited divergent RNA recombination, ribavirin sensitivity and biochemical phenotypes, consistent with changes in the fidelity of RNA synthesis. We conclude that an extended primer grip of the polymerase, including L420, M392, K375 & R376, contributes to the fidelity of RNA synthesis and to efficient sexual RNA replication mechanisms.IMPORTANCEPicornaviruses have both asexual and sexual RNA replication mechanisms. Sexual RNA replication shapes picornavirus species groups, contributes to the emergence of vaccine-derived polioviruses and counteracts error catastrophe. Can viruses distinguish between homologous and non-homologous partners during sexual RNA replication? We implicate an extended primer grip of the viral polymerase in sexual RNA replication mechanisms. By sensing RNA sequence complementarity near the active site, the extended primer grip of the polymerase has the potential to distinguish between homologous and non-homologous RNA templates during sexual RNA replication.

scholarly journals Role of Mitochondrial Membrane Spherules in Flock House Virus Replication

2016 ◽  
Vol 90 (7) ◽  
pp. 3676-3683 ◽  
Author(s):  
James R. Short ◽  
Jeffrey A. Speir ◽  
Radhika Gopal ◽  
Logan M. Pankratz ◽  
Jason Lanman ◽  
...  
Keyword(s):  
Host Defense ◽  
Rna Synthesis ◽  
Molecular Mechanisms ◽  
Rna Viruses ◽  
Rna Replication ◽  
Viral Rna ◽  
Flock House Virus ◽  
Double Stranded Rna ◽  
Content Type ◽  
Positive Sense

ABSTRACTViruses that generate double-stranded RNA (dsRNA) during replication must overcome host defense systems designed to detect this infection intermediate. All positive-sense RNA viruses studied to date modify host membranes to help facilitate the sequestration of dsRNA from host defenses and concentrate replication factors to enhance RNA production. Flock House virus (FHV) is an attractive model for the study of these processes since it is well characterized and infectsDrosophilacells, which are known to have a highly effective RNA silencing system. During infection, FHV modifies the outer membrane of host mitochondria to form numerous membrane invaginations, called spherules, that are ∼50 nm in diameter and known to be the site of viral RNA replication. While previous studies have outlined basic structural features of these invaginations, very little is known about the mechanism underlying their formation. Here we describe the optimization of an experimental system for the analysis of FHV host membrane modifications using crude mitochondrial preparations from infectedDrosophilacells. These preparations can be programmed to synthesize both single- and double-stranded FHV RNA. The system was used to demonstrate that dsRNA is protected from nuclease digestion by virus-induced membrane invaginations and that spherules play an important role in stimulating RNA replication. Finally, we show that spherules generated during FHV infection appear to be dynamic as evidenced by their ability to form or disperse based on the presence or absence of RNA synthesis.IMPORTANCEIt is well established that positive-sense RNA viruses induce significant membrane rearrangements in infected cells. However, the molecular mechanisms underlying these rearrangements, particularly membrane invagination and spherule formation, remain essentially unknown. How the formation of spherules enhances viral RNA synthesis is also not understood, although it is assumed to be partly a result of evading host defense pathways. To help interrogate some of these issues, we optimized a cell-free replication system consisting of mitochondria isolated from Flock House virus-infectedDrosophilacells for use in biochemical and structural studies. Our data suggest that spherules generated during Flock House virus replication are dynamic, protect double-stranded RNA, and enhance RNA replication in general. Cryo-electron microscopy suggests that the samples are amenable to detailed structural analyses of spherules engaged in RNA synthesis. This system thus provides a foundation for understanding the molecular mechanisms underlying spherule formation, maintenance, and function during positive-sense viral RNA replication.

scholarly journals The Heat Shock Protein 70 Cochaperone YDJ1 Is Required for Efficient Membrane-Specific Flock House Virus RNA Replication Complex Assembly and Function in Saccharomyces cerevisiae

2007 ◽  
Vol 82 (4) ◽  
pp. 2004-2012 ◽  
Author(s):  
Spencer A. Weeks ◽  
David J. Miller
Keyword(s):  
Rna Replication ◽  
Viral Rna ◽  
Flock House Virus ◽  
Complex Activity ◽  
Complex Assembly ◽  
Replication Complex ◽  
Hsp90 Chaperone ◽  
Chaperone Complex ◽  
And Function

ABSTRACT The assembly of RNA replication complexes on intracellular membranes is an essential step in the life cycle of positive-sense RNA viruses. We have previously shown that Hsp90 chaperone complex activity is essential for efficient Flock House virus (FHV) RNA replication in Drosophila melanogaster S2 cells. To further explore the role of cellular chaperones in viral RNA replication, we used both pharmacologic and genetic approaches to examine the role of the Hsp90 and Hsp70 chaperone systems in FHV RNA replication complex assembly and function in Saccharomyces cerevisiae. In contrast to results with insect cells, yeast deficient in Hsp90 chaperone complex activity showed no significant decrease in FHV RNA replication. However, yeast with a deletion of the Hsp70 cochaperone YDJ1 showed a dramatic reduction in FHV RNA replication that was due in part to reduced viral RNA polymerase accumulation. Furthermore, the absence of YDJ1 did not reduce FHV RNA replication when the viral RNA polymerase and replication complexes were retargeted from the mitochondria to the endoplasmic reticulum. These results identify YDJ1 as an essential membrane-specific host factor for FHV RNA replication complex assembly and function in S. cerevisiae and are consistent with known differences in the role of distinct chaperone complexes in organelle-specific protein targeting between yeast and higher eukaryotes.

scholarly journals An Extended Primer Grip of Picornavirus Polymerase Facilitates Sexual RNA Replication Mechanisms

2020 ◽  
Vol 94 (16) ◽  
Author(s):  
Brian J. Kempf ◽  
Colleen L. Watkins ◽  
Olve B. Peersen ◽  
David J. Barton
Keyword(s):  
Rna Synthesis ◽  
Rna Replication ◽  
Serial Passage ◽  
Resistant Virus ◽  
Rna Recombination ◽  
Resistance Mutations ◽  
Error Catastrophe ◽  
Sequence Complementarity ◽  
Species Groups ◽  
Codon Mutation

ABSTRACT Picornaviruses have both asexual and sexual RNA replication mechanisms. Asexual RNA replication mechanisms involve one parental template, whereas sexual RNA replication mechanisms involve two or more parental templates. Because sexual RNA replication mechanisms counteract ribavirin-induced error catastrophe, we selected for ribavirin-resistant poliovirus to identify polymerase residues that facilitate sexual RNA replication mechanisms. We used serial passage in ribavirin, beginning with a variety of ribavirin-sensitive and ribavirin-resistant parental viruses. Ribavirin-sensitive virus contained an L420A polymerase mutation, while ribavirin-resistant virus contained a G64S polymerase mutation. A G64 codon mutation (G64Fix) was used to inhibit emergence of G64S-mediated ribavirin resistance. Revertants (L420) or pseudorevertants (L420V and L420I) were selected from all independent lineages of L420A, G64Fix L420A, and G64S L420A parental viruses. Ribavirin resistance G64S mutations were selected in two independent lineages, and novel ribavirin resistance mutations were selected in the polymerase in other lineages (M299I, M323I, M392V, and T353I). The structural orientation of M392, immediately adjacent to L420 and the polymerase primer grip region, led us to engineer additional polymerase mutations into poliovirus (M392A, M392L, M392V, K375R, and R376K). L420A revertants and pseudorevertants (L420V and L420I) restored efficient viral RNA recombination, confirming that ribavirin-induced error catastrophe coincides with defects in sexual RNA replication mechanisms. Viruses containing M392 mutations (M392A, M392L, and M392V) and primer grip mutations (K375R and R376K) exhibited divergent RNA recombination, ribavirin sensitivity, and biochemical phenotypes, consistent with changes in the fidelity of RNA synthesis. We conclude that an extended primer grip of the polymerase, including L420, M392, K375, and R376, contributes to the fidelity of RNA synthesis and to efficient sexual RNA replication mechanisms. IMPORTANCE Picornaviruses have both asexual and sexual RNA replication mechanisms. Sexual RNA replication shapes picornavirus species groups, contributes to the emergence of vaccine-derived polioviruses, and counteracts error catastrophe. Can viruses distinguish between homologous and nonhomologous partners during sexual RNA replication? We implicate an extended primer grip of the viral polymerase in sexual RNA replication mechanisms. By sensing RNA sequence complementarity near the active site, the extended primer grip of the polymerase has the potential to distinguish between homologous and nonhomologous RNA templates during sexual RNA replication.

scholarly journals Picornavirus RNA Recombination Counteracts Error Catastrophe

2019 ◽  
Vol 93 (14) ◽  
Author(s):  
Brian J. Kempf ◽  
Colleen L. Watkins ◽  
Olve B. Peersen ◽  
David J. Barton
Keyword(s):  
Rna Viruses ◽  
Rna Replication ◽  
Viral Rna ◽  
Common Mechanism ◽  
Negative Consequences ◽  
Rna Recombination ◽  
Viral Polymerase ◽  
Advantages And Disadvantages ◽  
Error Catastrophe ◽  
Positive Strand Rna

ABSTRACTTemplate-dependent RNA replication mechanisms render picornaviruses susceptible to error catastrophe, an overwhelming accumulation of mutations incompatible with viability. Viral RNA recombination, in theory, provides a mechanism for viruses to counteract error catastrophe. We tested this theory by exploiting well-defined mutations in the poliovirus RNA-dependent RNA polymerase (RDRP), namely, a G64S mutation and an L420A mutation. Our data reveal two distinct mechanisms by which picornaviral RDRPs influence error catastrophe: fidelity of RNA synthesis and RNA recombination. A G64S mutation increased the fidelity of the viral polymerase and rendered the virus resistant to ribavirin-induced error catastrophe, but only when RNA recombination was at wild-type levels. An L420A mutation in the viral polymerase inhibited RNA recombination and exacerbated ribavirin-induced error catastrophe. Furthermore, when RNA recombination was substantially reduced by an L420A mutation, a high-fidelity G64S polymerase failed to make the virus resistant to ribavirin. These data indicate that viral RNA recombination is required for poliovirus to evade ribavirin-induced error catastrophe. The conserved nature of L420 within RDRPs suggests that RNA recombination is a common mechanism for picornaviruses to counteract and avoid error catastrophe.IMPORTANCEPositive-strand RNA viruses produce vast amounts of progeny in very short periods of time via template-dependent RNA replication mechanisms. Template-dependent RNA replication, while efficient, can be disadvantageous due to error-prone viral polymerases. The accumulation of mutations in viral RNA genomes leads to error catastrophe. In this study, we substantiate long-held theories regarding the advantages and disadvantages of asexual and sexual replication strategies among RNA viruses. In particular, we show that picornavirus RNA recombination counteracts the negative consequences of asexual template-dependent RNA replication mechanisms, namely, error catastrophe.

scholarly journals Mechanism of molnupiravir-induced SARS-CoV-2 mutagenesis

2021 ◽  
Author(s):  
Florian Kabinger ◽  
Carina Stiller ◽  
Jana Schmitzová ◽  
Christian Dienemann ◽  
Goran Kokic ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Active Form ◽  
Antiviral Drug ◽  
Viral Rna ◽  
Phase Iii ◽  
Drug Candidate ◽  
Base Pairs ◽  
Biochemical Assays ◽  
Phase Iii Trials ◽  
Rna Complexes

AbstractMolnupiravir is an orally available antiviral drug candidate currently in phase III trials for the treatment of patients with COVID-19. Molnupiravir increases the frequency of viral RNA mutations and impairs SARS-CoV-2 replication in animal models and in humans. Here, we establish the molecular mechanisms underlying molnupiravir-induced RNA mutagenesis by the viral RNA-dependent RNA polymerase (RdRp). Biochemical assays show that the RdRp uses the active form of molnupiravir, β-d-N4-hydroxycytidine (NHC) triphosphate, as a substrate instead of cytidine triphosphate or uridine triphosphate. When the RdRp uses the resulting RNA as a template, NHC directs incorporation of either G or A, leading to mutated RNA products. Structural analysis of RdRp–RNA complexes that contain mutagenesis products shows that NHC can form stable base pairs with either G or A in the RdRp active center, explaining how the polymerase escapes proofreading and synthesizes mutated RNA. This two-step mutagenesis mechanism probably applies to various viral polymerases and can explain the broad-spectrum antiviral activity of molnupiravir.

scholarly journals Role of 1’-Ribose Cyano Substitution for Remdesivir to Effectively Inhibit Nucleotide Addition and Proofreading in SARS-CoV-2 Viral RNA Replication

2020 ◽  
Author(s):  
Lu Zhang ◽  
Dong Zhang ◽  
Xiaowei Wang ◽  
Congmin Yuan ◽  
Yongfang Li ◽  
...  
Keyword(s):  
Rational Design ◽  
Molecular Mechanisms ◽  
Cyano Group ◽  
Rna Replication ◽  
Viral Rna ◽  
Upstream Site ◽  
Health Crisis ◽  
Nucleotide Addition

ABSTRACTCOVID-19 has recently caused a global health crisis and an effective interventional therapy is urgently needed. SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) is a promising but challenging drug target due to its intrinsic proofreading exoribonuclease (ExoN). Remdesivir targeting SARS-CoV-2 RdRp exerts high drug efficacy in vitro and in vivo. However, its underlying inhibitory mechanisms remain elusive. Here, we performed all-atom molecular dynamics simulations with an accumulated simulation time of 24 microseconds to elucidate the molecular mechanisms underlying the inhibitory effects of Remdesivir. We found that Remdesivir’s 1’-cyano group of possesses the dual role of inhibiting nucleotide addition and proofreading. The presence of its polar 1’-cyano group at an upstream site in RdRp causes instability and hampers RdRp translocation. This leads to a delayed chain termination of RNA extension, which may also subsequently reduce the likelihood for Remdesivir to be cleaved by ExoN acting on the 3’-terminal nucleotide. In addition, our simulations suggest that Remdesivir’s 1’-cyano group can also disrupt the cleavage active site of ExoN via steric interactions, leading to a further reduced cleavage efficiency. Our work provides plausible molecular mechanisms on how Remdesivir inhibits viral RNA replication and may guide rational design for new treatments of COVID-19 targeting viral replication.

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