Applications of High-Fidelity Sequencing Protocol to RNA Viruses

ABSTRACT The coronavirus (CoV) RNA genome is the largest among the single-stranded positive-sense RNA viruses. CoVs encode a proofreading 3′-to-5′ exoribonuclease within nonstructural protein 14 (nsp14-ExoN) that is responsible for CoV high-fidelity replication. Alanine substitution of ExoN catalytic residues [ExoN(-)] in severe acute respiratory syndrome-associated coronavirus (SARS-CoV) and murine hepatitis virus (MHV) disrupts ExoN activity, yielding viable mutant viruses with defective replication, up to 20-fold-decreased fidelity, and increased susceptibility to nucleoside analogues. To test the stability of the ExoN(-) genotype and phenotype, we passaged MHV-ExoN(-) 250 times in cultured cells (P250), in parallel with wild-type MHV (WT-MHV). Compared to MHV-ExoN(-) P3, MHV-ExoN(-) P250 demonstrated enhanced replication and increased competitive fitness without reversion at the ExoN(-) active site. Furthermore, MHV-ExoN(-) P250 was less susceptible than MHV-ExoN(-) P3 to multiple nucleoside analogues, suggesting that MHV-ExoN(-) was under selection for increased replication fidelity. We subsequently identified novel amino acid changes within the RNA-dependent RNA polymerase and nsp14 of MHV-ExoN(-) P250 that partially accounted for the reduced susceptibility to nucleoside analogues. Our results suggest that increased replication fidelity is selected in ExoN(-) CoVs and that there may be a significant barrier to ExoN(-) reversion. These results also support the hypothesis that high-fidelity replication is linked to CoV fitness and indicate that multiple replicase proteins could compensate for ExoN functions during replication. IMPORTANCE Uniquely among RNA viruses, CoVs encode a proofreading exoribonuclease (ExoN) in nsp14 that mediates high-fidelity RNA genome replication. Proofreading-deficient CoVs with disrupted ExoN activity [ExoN(-)] either are nonviable or have significant defects in replication, RNA synthesis, fidelity, fitness, and virulence. In this study, we showed that ExoN(-) murine hepatitis virus can adapt during long-term passage for increased replication and fitness without reverting the ExoN-inactivating mutations. Passage-adapted ExoN(-) mutants also demonstrate increasing resistance to nucleoside analogues that is explained only partially by secondary mutations in nsp12 and nsp14. These data suggest that enhanced resistance to nucleoside analogues is mediated by the interplay of multiple replicase proteins and support the proposed link between CoV fidelity and fitness. IMPORTANCE Uniquely among RNA viruses, CoVs encode a proofreading exoribonuclease (ExoN) in nsp14 that mediates high-fidelity RNA genome replication. Proofreading-deficient CoVs with disrupted ExoN activity [ExoN(-)] either are nonviable or have significant defects in replication, RNA synthesis, fidelity, fitness, and virulence. In this study, we showed that ExoN(-) murine hepatitis virus can adapt during long-term passage for increased replication and fitness without reverting the ExoN-inactivating mutations. Passage-adapted ExoN(-) mutants also demonstrate increasing resistance to nucleoside analogues that is explained only partially by secondary mutations in nsp12 and nsp14. These data suggest that enhanced resistance to nucleoside analogues is mediated by the interplay of multiple replicase proteins and support the proposed link between CoV fidelity and fitness.

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Proofreading-deficient coronaviruses adapt over long-term passage for increased fidelity and fitness without reversion of exoribonuclease-inactivating mutations

10.1101/175562 ◽

2017 ◽

Cited By ~ 3

Author(s):

Kevin W. Graepel ◽

Xiaotao Lu ◽

James Brett Case ◽

Nicole R. Sexton ◽

Everett Clinton Smith ◽

...

Keyword(s):

Rna Viruses ◽

Hepatitis Virus ◽

Nucleoside Analogs ◽

High Fidelity ◽

Murine Hepatitis Virus ◽

Replication Fidelity ◽

Competitive Fitness ◽

Rna Genome ◽

Inactivating Mutations

ABSTRACTThe coronavirus (CoV) RNA genome is the largest among single-stranded positive sense RNA viruses. CoVs encode a proofreading 3′→5′exoribonuclease within nonstructural protein 14 (nsp14-ExoN) that is responsible for CoV high-fidelity replication. Alanine substitution of ExoN catalytic residues [ExoN(-)] in SARS-CoV and murine hepatitis virus (MHV) disrupts ExoN activity, yielding viable mutant viruses with defective replication, up to 20-fold decreased fidelity, and increased susceptibility to nucleoside analogs. To test the stability of the ExoN(-) genotype and phenotype, we passaged MHV-ExoN(-) 250 times in cultured cells (P250), in parallel with WT-MHV. Compared to MHV-ExoN(-) P3, MHV-ExoN(-) P250 demonstrated enhanced replication, reduced susceptibility to nucleoside analogs, and increased competitive fitness. However, passage did not select for complete or partial reversion at the ExoN-inactivating mutations. We identified novel amino acid changes within the RNA-dependent RNA polymerase (nsp12-RdRp) and nsp14 of MHV-ExoN(-) P250 that partially account for the observed changes in replication, susceptibility to nucleoside analogs, and competitive fitness observed in the passaged virus population, indicating that additional determinants can compensate for the activities of nsp14-ExoN. Our results suggest that while selection favors restoration of replication fidelity in ExoN(-) CoVs, there may be a significant barrier to ExoN(-) reversion. These results also support the hypothesis that high-fidelity replication is linked to CoV fitness and identify additional candidate proteins that may regulate CoV replication fidelity.IMPORTANCEUnique among RNA viruses, CoVs encode a proofreading exoribonuclease (ExoN) in nsp14 that mediates high-fidelity RNA genome replication. Proofreading-deficient CoVs with disrupted ExoN activity [ExoN(-)] are either non-viable or have significant defects in replication, RNA synthesis, fidelity, fitness, and virulence. In this study, we show that ExoN(-) murine hepatitis virus can adapt over long-term passage for increased replication and fitness without reverting the ExoN-inactivating mutations. Passage-adapted ExoN(-) mutants also demonstrate increasing resistance to nucleoside analogs that is only partially explained by secondary mutations in nsp12 and nsp14. These data suggest that enhanced resistance to nucleoside analogs is mediated by the interplay of multiple replicase proteins and support the proposed link between CoV fidelity and fitness.

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Lethal Mutagenesis of Poliovirus Mediated by a Mutagenic Pyrimidine Analogue

Journal of Virology ◽

10.1128/jvi.01028-07 ◽

2007 ◽

Vol 81 (20) ◽

pp. 11256-11266 ◽

Cited By ~ 36

Author(s):

Jason D. Graci ◽

Daniel A. Harki ◽

Victoria S. Korneeva ◽

Jocelyn P. Edathil ◽

Kathleen Too ◽

...

Keyword(s):

Antiviral Activity ◽

Mechanism Of Action ◽

Rna Viruses ◽

Rna Virus ◽

Virus Titer ◽

High Fidelity ◽

Mutational Robustness ◽

Lethal Mutagenesis ◽

The Impact

ABSTRACT Lethal mutagenesis is the mechanism of action of ribavirin against poliovirus (PV) and numerous other RNA viruses. However, there is still considerable debate regarding the mechanism of action of ribavirin against a variety of RNA viruses. Here we show by using T7 RNA polymerase-mediated production of PV genomic RNA, PV polymerase-catalyzed primer extension, and cell-free PV synthesis that a pyrimidine ribonucleoside triphosphate analogue (rPTP) with ambiguous base-pairing capacity is an efficient mutagen of the PV genome. The in vitro incorporation properties of rPTP are superior to ribavirin triphosphate. We observed a log-linear relationship between virus titer reduction and the number of rPMP molecules incorporated. A PV genome encoding a high-fidelity polymerase was more sensitive to rPMP incorporation, consistent with diminished mutational robustness of high-fidelity PV. The nucleoside (rP) did not exhibit antiviral activity in cell culture, owing to the inability of rP to be converted to rPMP by cellular nucleotide kinases. rP was also a poor substrate for herpes simplex virus thymidine kinase. The block to nucleoside phosphorylation could be bypassed by treatment with the P nucleobase, which exhibited both antiviral activity and mutagenesis, presumably a reflection of rP nucleotide formation by a nucleotide salvage pathway. These studies provide additional support for lethal mutagenesis as an antiviral strategy, suggest that rPMP prodrugs may be highly efficacious antiviral agents, and provide a new tool to determine the sensitivity of RNA virus genomes to mutagenesis as well as interrogation of the impact of mutational load on the population dynamics of these viruses.

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Chikungunya Virus Fidelity Variants Exhibit Differential Attenuation and Population Diversity in Cell Culture and Adult Mice

Journal of Virology ◽

10.1128/jvi.01606-18 ◽

2018 ◽

Vol 93 (3) ◽

Cited By ~ 8

Author(s):

Kasen K. Riemersma ◽

Cody Steiner ◽

Anil Singapuri ◽

Lark L. Coffey

Keyword(s):

Global Health ◽

Chikungunya Virus ◽

Rna Viruses ◽

Population Diversity ◽

Mutation Rates ◽

High Fidelity ◽

Health Threat ◽

Arthritic Disease ◽

Adult Mice

ABSTRACTChikungunya virus (CHIKV) is a reemerging global health threat that produces debilitating arthritis in people. Like other RNA viruses with high mutation rates, CHIKV produces populations of genetically diverse genomes within a host. While several known CHIKV mutations influence disease severity in vertebrates and transmission by mosquitoes, the role of intrahost diversity in chikungunya arthritic disease has not been studied. In this study, high- and low-fidelity CHIKV variants, previously characterized by alteredin vitropopulation mutation frequencies, were used to evaluate how intrahost diversity influences clinical disease, CHIKV replication, and antibody neutralization in immunocompetent adult mice inoculated in the rear footpads. Both high- and low-fidelity mutations were hypothesized to attenuate CHIKV arthritic disease, replication, and neutralizing antibody levels compared to wild-type (WT) CHIKV. Unexpectedly, high-fidelity mutants elicited more severe arthritic disease than the WT despite comparable CHIKV replication, whereas a low-fidelity mutant produced attenuated disease and replication. Serum antibody developed against both high- and low-fidelity CHIKV exhibited reduced neutralization of WT CHIKV. Using next-generation sequencing (NGS), the high-fidelity mutations were demonstrated to be genetically stable but produced more genetically diverse populations than WT CHIKV in mice. This enhanced diversification was subsequently reproduced after serialin vitropassage. The NGS results contrast with previously reported population diversities for fidelity variants, which focused mainly on part of the E1 gene, and highlight the need for direct measurements of mutation rates to clarify CHIKV fidelity phenotypes.IMPORTANCECHIKV is a reemerging global health threat that elicits debilitating arthritis in humans. There are currently no commercially available CHIKV vaccines. Like other RNA viruses, CHIKV has a high mutation rate and is capable of rapid intrahost diversification during an infection. In other RNA viruses, virus population diversity associates with disease progression; however, potential impacts of intrahost viral diversity on CHIKV arthritic disease have not been studied. Using previously characterized CHIKV fidelity variants, we addressed whether CHIKV population diversity influences the severity of arthritis and host antibody response in an arthritic mouse model. Our findings show that CHIKV populations with greater genetic diversity can cause more severe disease and stimulate antibody responses with reduced neutralization of low-diversity virus populationsin vitro. The discordant high-fidelity phenotypes in this study highlight the complexity of inferring replication fidelity indirectly from population diversity.

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High Fidelity of Murine Hepatitis Virus Replication Is Decreased in nsp14 Exoribonuclease Mutants

Journal of Virology ◽

10.1128/jvi.01296-07 ◽

2007 ◽

Vol 81 (22) ◽

pp. 12135-12144 ◽

Cited By ~ 151

Author(s):

Lance D. Eckerle ◽

Xiaotao Lu ◽

Steven M. Sperry ◽

Leena Choi ◽

Mark R. Denison

Keyword(s):

Active Site ◽

Rna Viruses ◽

Hepatitis Virus ◽

Nonstructural Protein ◽

Critical Role ◽

Wild Type Virus ◽

High Fidelity ◽

Wild Type ◽

Murine Hepatitis Virus ◽

Replication Fidelity

ABSTRACT Replication fidelity of RNA virus genomes is constrained by the opposing necessities of generating sufficient diversity for adaptation and maintaining genetic stability, but it is unclear how the largest viral RNA genomes have evolved and are maintained under these constraints. A coronavirus (CoV) nonstructural protein, nsp14, contains conserved active-site motifs of cellular exonucleases, including DNA proofreading enzymes, and the severe acute respiratory syndrome CoV (SARS-CoV) nsp14 has 3′-to-5′ exoribonuclease (ExoN) activity in vitro. Here, we show that nsp14 ExoN remarkably increases replication fidelity of the CoV murine hepatitis virus (MHV). Replacement of conserved MHV ExoN active-site residues with alanines resulted in viable mutant viruses with growth and RNA synthesis defects that during passage accumulated 15-fold more mutations than wild-type virus without changes in growth fitness. The estimated mutation rate for ExoN mutants was similar to that reported for other RNA viruses, whereas that of wild-type MHV was less than the established rates for RNA viruses in general, suggesting that CoVs with intact ExoN replicate with unusually high fidelity. Our results indicate that nsp14 ExoN plays a critical role in prevention or repair of nucleotide incorporation errors during genome replication. The established mutants are unique tools to test the hypothesis that high replication fidelity is required for the evolution and stability of large RNA genomes.

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Oncogenesis and the Lucké Virus

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100100743 ◽

1968 ◽

Vol 26 ◽

pp. 200-201

Author(s):

A. E. Vatter ◽

J. Zambernard

Keyword(s):

Rna Viruses ◽

Vegetative State ◽

Temperature Sensitive ◽

Structural Level ◽

Oncogenic Viruses ◽

Dna Viruses ◽

Leopard Frogs ◽

Renal Adenocarcinoma ◽

Induced Tumors ◽

Northern Leopard Frogs

Oncogenic viruses, like viruses in general, can be divided into two classes, those that contain deoxyribonucleic acid (DNA) and those that contain ribonucleic acid (RNA). The RNA viruses have been recovered readily from the tumors which they cause whereas, the DNA-virus induced tumors have not yielded the virus. Since DNA viruses cannot be recovered, the bulk of present day investigations have been concerned with RNA viruses.The Lucké renal adenocarcinoma is a spontaneous tumor which occurs in northern leopard frogs (Rana pipiens) and has received increased attention in recent years because of its probable viral etiology. This hypothesis was first advanced by Lucké after he observed intranuclear inclusions in some of the tumor cells. Tumors with inclusions were examined at the fine structural level by Fawcett who showed that they contained immature and mature virus˗like particles.The use of this system in the study of oncogenic tumors offers several unique features, the virus has been shown to contain DNA and it can be recovered from the tumor, also, it is temperature sensitive. This latter feature is of importance because the virus can be transformed from a latent to a vegetative state by lowering or elevating the environmental temperature.

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Using the Theory of Planned Behavior to Predict Faking in Selection Exercises Varying in Fidelity

Journal of Personnel Psychology ◽

10.1027/1866-5888/a000211 ◽

2018 ◽

Vol 17 (3) ◽

pp. 155-160 ◽

Cited By ~ 3

Author(s):

Daniel Dürr ◽

Ute-Christine Klehe

Keyword(s):

Theory Of Planned Behavior ◽

Planned Behavior ◽

Predictive Validity ◽

Group Discussion ◽

Role Play ◽

Selection Procedure ◽

High Fidelity ◽

Selection Research ◽

Play Group

Abstract. Faking has been a concern in selection research for many years. Many studies have examined faking in questionnaires while far less is known about faking in selection exercises with higher fidelity. This study applies the theory of planned behavior (TPB; Ajzen, 1991 ) to low- (interviews) and high-fidelity (role play, group discussion) exercises, testing whether the TPB predicts reported faking behavior. Data from a mock selection procedure suggests that candidates do report to fake in low- and high-fidelity exercises. Additionally, the TPB showed good predictive validity for faking in a low-fidelity exercise, yet not for faking in high-fidelity exercises.

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