Effective Lethal Mutagenesis of Influenza Virus by Three Nucleoside Analogs

ABSTRACTLethal mutagenesis is a broad-spectrum antiviral strategy that exploits the high mutation rate and low mutational tolerance of many RNA viruses. This approach uses mutagenic drugs to increase viral mutation rates and burden viral populations with mutations that reduce the number of infectious progeny. We investigated the effectiveness of lethal mutagenesis as a strategy against influenza virus using three nucleoside analogs, ribavirin, 5-azacytidine, and 5-fluorouracil. All three drugs were active against a panel of seasonal H3N2 and laboratory-adapted H1N1 strains. We found that each drug increased the frequency of mutations in influenza virus populations and decreased the virus' specific infectivity, indicating a mutagenic mode of action. We were able to drive viral populations to extinction by passaging influenza virus in the presence of each drug, indicating that complete lethal mutagenesis of influenza virus populations can be achieved when a sufficient mutational burden is applied. Population-wide resistance to these mutagenic agents did not arise after serial passage of influenza virus populations in sublethal concentrations of drug. Sequencing of these drug-passaged viral populations revealed genome-wide accumulation of mutations at low frequency. The replicative capacity of drug-passaged populations was reduced at higher multiplicities of infection, suggesting the presence of defective interfering particles and a possible barrier to the evolution of resistance. Together, our data suggest that lethal mutagenesis may be a particularly effective therapeutic approach with a high genetic barrier to resistance for influenza virus.IMPORTANCEInfluenza virus is an RNA virus that causes significant morbidity and mortality during annual epidemics. Novel therapies for RNA viruses are needed due to the ease with which these viruses evolve resistance to existing therapeutics. Lethal mutagenesis is a broad-spectrum strategy that exploits the high mutation rate and the low mutational tolerance of most RNA viruses. It is thought to possess a higher barrier to resistance than conventional antiviral strategies. We investigated the effectiveness of lethal mutagenesis against influenza virus using three different drugs. We showed that influenza virus was sensitive to lethal mutagenesis by demonstrating that all three drugs induced mutations and led to an increase in the generation of defective viral particles. We also found that it may be difficult for resistance to these drugs to arise at a population-wide level. Our data suggest that lethal mutagenesis may be an attractive anti-influenza strategy that warrants further investigation.

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Epistatic interactions within the influenza virus polymerase complex mediate mutagen resistance and replication fidelity

10.1101/131417 ◽

2017 ◽

Author(s):

Matthew D. Pauly ◽

Daniel M. Lyons ◽

Adam S. Lauring

Keyword(s):

Genetic Diversity ◽

Drug Resistance ◽

Influenza Virus ◽

Mutation Rate ◽

Rna Viruses ◽

Nucleoside Analogs ◽

Mutation Rates ◽

Epistatic Interactions ◽

Polymerase Complex ◽

Lethal Mutagenesis

AbstractLethal mutagenesis is a broad-spectrum antiviral strategy that employs mutagenic nucleoside analogs to exploit the high mutation rate and low mutational tolerance of many RNA viruses. Studies of mutagen-resistant viruses have identified determinants of replicative fidelity and the importance of mutation rate to viral population dynamics. We have previously demonstrated the effective lethal mutagenesis of influenza virus using three nucleoside analogs as well as the virus’s high genetic barrier to mutagen resistance. Here, we investigate the mutagen-resistant phenotypes of mutations that were enriched in drug-treated populations. We find that PB1 T123A has higher replicative fitness than the wild type, PR8, and maintains its level of genome production during 5-fluorouracil treatment. Surprisingly, this mutagen-resistant variant also has an increased baseline rate of C to U and G to A mutations. A second drug-selected mutation, PA T97I, interacts epistatically with PB T123A to mediate high-level mutagen resistance, predominantly by limiting the inhibitory effect of nucleosides on polymerase activity. Consistent with the importance of epistatic interactions in the influenza polymerase, we find that nucleoside analog resistance and replication fidelity are strain dependent. Two previously identified ribavirin-resistance mutations, PB1 V43I and PB1 D27N, do not confer drug resistance in the PR8 background, and the PR8-PB1 V43I polymerase exhibits a normal baseline mutation rate. Our results highlight the genetic complexity of the influenza virus polymerase and demonstrate that increased replicative capacity is a mechanism by which an RNA virus can counter the negative effects of elevated mutation rates.ImportanceRNA viruses exist as genetically diverse populations. This standing genetic diversity gives them the potential to adapt rapidly, evolve resistance to antiviral therapeutics, and evade immune responses. Viral mutants with altered mutation rates or mutational tolerance have provided insights into how genetic diversity arises and how it affects the behavior of RNA viruses. To this end, we identified variants within the polymerase complex of influenza virus that are able tolerate drug-mediated increases in viral mutation rates. We find that drug resistance is highly dependent on interactions among mutations in the polymerase complex. In contrast to other viruses, influenza virus counters the effect of higher mutation rates primarily by maintaining high levels of genome replication. These findings suggest the importance of maintaining large population sizes for viruses with high mutation rates and show that multiple proteins can affect both mutation rate and genome synthesis.

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Epistatic Interactions within the Influenza A Virus Polymerase Complex Mediate Mutagen Resistance and Replication Fidelity

mSphere ◽

10.1128/msphere.00323-17 ◽

2017 ◽

Vol 2 (4) ◽

Cited By ~ 7

Author(s):

Matthew D. Pauly ◽

Daniel M. Lyons ◽

William J. Fitzsimmons ◽

Adam S. Lauring

Keyword(s):

Genetic Diversity ◽

Drug Resistance ◽

Influenza Virus ◽

Influenza A Virus ◽

Mutation Rate ◽

Influenza A ◽

Rna Viruses ◽

Nucleoside Analogs ◽

Mutation Rates ◽

Polymerase Complex

ABSTRACT RNA viruses exist as genetically diverse populations. This standing genetic diversity gives them the potential to adapt rapidly, evolve resistance to antiviral therapeutics, and evade immune responses. Viral mutants with altered mutation rates or mutational tolerance have provided insights into how genetic diversity arises and how it affects the behavior of RNA viruses. To this end, we identified variants within the polymerase complex of influenza virus that are able tolerate drug-mediated increases in viral mutation rates. We find that drug resistance is highly dependent on interactions among mutations in the polymerase complex. In contrast to other viruses, influenza virus counters the effect of higher mutation rates primarily by maintaining high levels of genome replication. These findings suggest the importance of maintaining large population sizes for viruses with high mutation rates and show that multiple proteins can affect both mutation rate and genome synthesis. Lethal mutagenesis is a broad-spectrum antiviral strategy that employs mutagenic nucleoside analogs to exploit the high mutation rate and low mutational tolerance of many RNA viruses. Studies of mutagen-resistant viruses have identified determinants of replicative fidelity and the importance of mutation rate to viral population dynamics. We have previously demonstrated the effective lethal mutagenesis of influenza A virus using three nucleoside analogs as well as the virus’s high genetic barrier to mutagen resistance. Here, we investigate the mutagen-resistant phenotypes of mutations that were enriched in drug-treated populations. We find that PB1 T123A has higher replicative fitness than the wild type, PR8, and maintains its level of genome production during 5-fluorouracil (2,4-dihydroxy-5-fluoropyrimidine) treatment. Surprisingly, this mutagen-resistant variant also has an increased baseline rate of C-to-U and G-to-A mutations. A second drug-selected mutation, PA T97I, interacts epistatically with PB1 T123A to mediate high-level mutagen resistance, predominantly by limiting the inhibitory effect of nucleosides on polymerase activity. Consistent with the importance of epistatic interactions in the influenza virus polymerase, our data suggest that nucleoside analog resistance and replication fidelity are strain dependent. Two previously identified ribavirin {1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-1H-1,2,4-triazole-3-carboxamide} resistance mutations, PB1 V43I and PB1 D27N, do not confer drug resistance in the PR8 background, and the PR8-PB1 V43I polymerase exhibits a normal baseline mutation rate. Our results highlight the genetic complexity of the influenza A virus polymerase and demonstrate that increased replicative capacity is a mechanism by which an RNA virus can counter the negative effects of elevated mutation rates. IMPORTANCE RNA viruses exist as genetically diverse populations. This standing genetic diversity gives them the potential to adapt rapidly, evolve resistance to antiviral therapeutics, and evade immune responses. Viral mutants with altered mutation rates or mutational tolerance have provided insights into how genetic diversity arises and how it affects the behavior of RNA viruses. To this end, we identified variants within the polymerase complex of influenza virus that are able to tolerate drug-mediated increases in viral mutation rates. We find that drug resistance is highly dependent on interactions among mutations in the polymerase complex. In contrast to other viruses, influenza virus counters the effect of higher mutation rates primarily by maintaining high levels of genome replication. These findings suggest the importance of maintaining large population sizes for viruses with high mutation rates and show that multiple proteins can affect both mutation rate and genome synthesis.

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Effect of Ribavirin on the Mutation Rate and Spectrum of Hepatitis C Virus In Vivo

Journal of Virology ◽

10.1128/jvi.00201-09 ◽

2009 ◽

Vol 83 (11) ◽

pp. 5760-5764 ◽

Cited By ~ 101

Author(s):

José M. Cuevas ◽

Fernando González-Candelas ◽

Andrés Moya ◽

Rafael Sanjuán

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Mutation Rate ◽

Rna Viruses ◽

Mutagenic Effect ◽

Significant Shift ◽

Interferon Treatment ◽

Lethal Mutagenesis

ABSTRACT Their extremely error-prone replication makes RNA viruses targets for lethal mutagenesis. In the case of hepatitis C virus (HCV), the standard treatment includes ribavirin, a base analog with an in vitro mutagenic effect, but the in vivo mode of action of ribavirin remains poorly understood. Here, we test the mutagenic effects of ribavirin plus interferon treatment in vivo using a new method to estimate mutation rates based on the analysis of nonsense mutations. We apply this methodology to a large HCV sequence database containing over 15,000 reverse transcription-PCR molecular clone sequences from 74 patients infected with HCV. We obtained an estimate of the spontaneous mutation rate of ca. 10−4 substitutions per site or lower, a value within the typically accepted range for RNA viruses. A roughly threefold increase in mutation rate and a significant shift in mutation spectrum were observed in samples from patients undergoing 6 months of interferon plus ribavirin treatment. This result is consistent with the known in vitro mutagenic effect of ribavirin and suggests that the antiviral effect of ribavirin plus interferon treatment is at least partly exerted through lethal mutagenesis.

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The Single-Stranded RNA Bacteriophage Qβ Adapts Rapidly to High Temperatures: An Evolution Experiment

Viruses ◽

10.3390/v12060638 ◽

2020 ◽

Vol 12 (6) ◽

pp. 638

Author(s):

Md. Tanvir Hossain ◽

Toma Yokono ◽

Akiko Kashiwagi

Keyword(s):

Escherichia Coli ◽

High Temperatures ◽

Mutation Rate ◽

Rna Viruses ◽

Point Mutations ◽

High Mutation Rate ◽

Short Period ◽

Evolution Experiment ◽

Novel Environments

Single-stranded (ss)RNA viruses are thought to evolve rapidly due to an inherently high mutation rate. However, it remains unclear how ssRNA viruses adapt to novel environments and/or how many and what types of substitutions are needed to facilitate this evolution. In this study, we followed the adaptation of the ssRNA bacteriophage Qβ using thermally adapted Escherichia coli as a host, which can efficiently grow at temperatures between 37.2 and 45.3 °C. This made it possible to evaluate Qβ adaptation to the highest known temperature that supports growth, 45.3 °C. We found that Qβ was capable of replication at this temperature; within 114 days (~1260 generations), we detected more than 34 novel point mutations in the genome of the thermally adapted Qβ population, representing 0.8% of the total Qβ genome. In addition, we returned the 45.3 °C-adapted Qβ populations to 37.2 °C and passaged them for 8 days (~124 generations). We found that the reverse-adapted Qβ population showed little to no decrease in fitness. These results indicate that Qβ can evolve in response to increasing temperatures in a short period of time with the accumulation of point mutations.

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Overcoming nonstructural protein 15-nidoviral uridylate-specific endoribonuclease (nsp15/NendoU) activity of SARS-CoV-2

Future Drug Discovery ◽

10.4155/fdd-2020-0012 ◽

2020 ◽

Vol 2 (3) ◽

pp. FDD42

Author(s):

Suranga L Senanayake

Keyword(s):

Broad Spectrum ◽

Nonstructural Protein ◽

Antiviral Agents ◽

Nucleoside Analogs ◽

Global Public Health ◽

Health Crisis ◽

Lethal Mutagenesis ◽

Double Hit ◽

Potential Inhibitors

COVID-19 has become the gravest global public health crisis since the Spanish Flu of 1918. Combination antiviral therapy with repurposed broad-spectrum antiviral agents holds a highly promising immediate treatment strategy, especially given uncertainties of vaccine efficacy and developmental timeline. Here, we describe a novel hypothetical approach: combining available broad-spectrum antiviral agents such as nucleoside analogs with potential inhibitors of NendoU, for example nsp15 RNA substrate mimetics. While only hypothesis-generating, this approach may constitute a ‘double-hit’ whereby two CoV-unique protein elements of the replicase–transcriptase complex are inhibited simultaneously; this may be an Achilles' heel and precipitate lethal mutagenesis in a coronavirus. It remains to be seen whether structurally optimized RNA substrate mimetics in combination with clinically approved and repurposed backbone antivirals can synergistically inhibit this endonuclease in vitro, thus fulfilling the ‘double-hit hypothesis’.

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Enhanced high mutation rate and natural selection to produce attenuated viral vaccine with CRISPR toolkit in RNA viruses especially SARS-CoV-2

Infection Genetics and Evolution ◽

10.1016/j.meegid.2021.105188 ◽

2021 ◽

pp. 105188

Author(s):

Saleh Jamehdor ◽

Sina Naserian ◽

Ali Teimoori

Keyword(s):

Natural Selection ◽

Mutation Rate ◽

Rna Viruses ◽

High Mutation Rate ◽

Viral Vaccine

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The mutation rates and mutational bias of influenza A virus

10.1101/110197 ◽

2017 ◽

Cited By ~ 4

Author(s):

Matthew D. Pauly ◽

Megan Procario ◽

Adam S. Lauring

Keyword(s):

Influenza Virus ◽

Influenza A Virus ◽

Mutation Rate ◽

Influenza A ◽

Rapid Evolution ◽

Viral Fitness ◽

High Mutation Rate ◽

Mutation Rates ◽

Mutational Bias ◽

Neutral Mutations

AbstractInfluenza virus has a high mutation rate, and this low replicative fidelity contributes to its capacity for rapid evolution. Clonal sequencing and fluctuation tests have suggested that the mutation rate of influenza A virus is 7.1 × 10−6− 4.5 × 10−5substitutions per nucleotide per cell infection cycle and 2.7 × 10−6− 3.0 × 10−5substitutions per nucleotide per strand copied (s/n/r). However, sequencing assays are biased toward mutations with minimal impacts on viral fitness and fluctuation tests typically investigate only a subset of the twelve mutational classes. We developed a fluctuation test based on reversion to fluorescence in a set of virally encoded mutant green fluorescent proteins. This method allowed us to measure the rates of selectively neutral mutations representative of all 12 mutational classes in the context of an unstructured RNA. We measured an overall mutation rate of 1.8 × 10−4s/n/r for PR8 (H1N1) and 2.5 × 10−4s/n/r for Hong Kong 2014 (H3N2). The replication mode was linear. The mutation rates of these divergent strains are significantly higher than previous estimates and suggest that each replicated genome will have an average of 2-3 mutations. The viral mutational spectrum is heavily biased toward A to G and U to C transitions, resulting in a transition to transversion bias of 2.7 and 3.6 for the two strains. These mutation rates were relatively constant over a range of physiological temperatures. Our high-resolution analysis of influenza virus mutation rates will enable more refined models of its molecular evolution.SignificanceThe rapid evolution of influenza virus is a major problem in public health. A key factor driving this rapid evolution is the virus’ very high mutation rate. We developed a new method for measuring the rates of all 12 mutational classes in influenza virus, which eliminates some of the biases of existing assays. We find that the influenza virus mutation rate is much higher than previously reported and is consistent across two distinct strains and a range of temperatures. Our data suggest that influenza viruses replicate at their maximally tolerable mutation rates, highlighting both the virus’ evolutionary potential and its significant constraints.

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