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

Mapping Intimacies ◽

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.

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.

Effective Lethal Mutagenesis of Influenza Virus by Three Nucleoside Analogs

Journal of Virology ◽

10.1128/jvi.03483-14 ◽

2015 ◽

Vol 89 (7) ◽

pp. 3584-3597 ◽

Cited By ~ 37

Author(s):

Matthew D. Pauly ◽

Adam S. Lauring

Keyword(s):

Influenza Virus ◽

Mutation Rate ◽

Broad Spectrum ◽

Rna Viruses ◽

Nucleoside Analogs ◽

High Mutation Rate ◽

Induced Mutations ◽

Genetic Barrier ◽

Defective Interfering Particles ◽

Lethal Mutagenesis

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.

Viral Mutation Rates

Journal of Virology ◽

10.1128/jvi.00694-10 ◽

2010 ◽

Vol 84 (19) ◽

pp. 9733-9748 ◽

Cited By ~ 634

Author(s):

Rafael Sanjuán ◽

Miguel R. Nebot ◽

Nicola Chirico ◽

Louis M. Mansky ◽

Robert Belshaw

Keyword(s):

Mutation Rate ◽

Rna Viruses ◽

Experimental Testing ◽

Mutation Rates ◽

Cell Infection ◽

Nucleotide Substitutions ◽

Data Set ◽

Dna Viruses ◽

Double Stranded Dna ◽

Viral Mutation

ABSTRACT Accurate estimates of virus mutation rates are important to understand the evolution of the viruses and to combat them. However, methods of estimation are varied and often complex. Here, we critically review over 40 original studies and establish criteria to facilitate comparative analyses. The mutation rates of 23 viruses are presented as substitutions per nucleotide per cell infection (s/n/c) and corrected for selection bias where necessary, using a new statistical method. The resulting rates range from 10−8 to10−6 s/n/c for DNA viruses and from 10−6 to 10−4 s/n/c for RNA viruses. Similar to what has been shown previously for DNA viruses, there appears to be a negative correlation between mutation rate and genome size among RNA viruses, but this result requires further experimental testing. Contrary to some suggestions, the mutation rate of retroviruses is not lower than that of other RNA viruses. We also show that nucleotide substitutions are on average four times more common than insertions/deletions (indels). Finally, we provide estimates of the mutation rate per nucleotide per strand copying, which tends to be lower than that per cell infection because some viruses undergo several rounds of copying per cell, particularly double-stranded DNA viruses. A regularly updated virus mutation rate data set will be available at www.uv.es/rsanjuan/virmut .

Upper-limit mutation rate estimation for a plant RNA virus

Biology Letters ◽

10.1098/rsbl.2008.0762 ◽

2009 ◽

Vol 5 (3) ◽

pp. 394-396 ◽

Cited By ~ 28

Author(s):

Rafael Sanjuán ◽

Patricia Agudelo-Romero ◽

Santiago F. Elena

Keyword(s):

Mutation Rate ◽

Rna Viruses ◽

Rna Virus ◽

Plant Viruses ◽

Mutation Rates ◽

Methodological Error ◽

Direct Estimate ◽

Rate Estimation ◽

Mutation Rate Estimation

It is generally accepted that mutation rates of RNA viruses are inherently high due to the lack of proofreading mechanisms. However, direct estimates of mutation rate are surprisingly scarce, in particular for plant viruses. Here, based on the analysis of in vivo mutation frequencies in tobacco etch virus , we calculate an upper-bound mutation rate estimation of 3×10 −5 per site and per round of replication; a value which turns out to be undistinguishable from the methodological error. Nonetheless, the value is barely on the lower side of the range accepted for RNA viruses, although in good agreement with the only direct estimate obtained for other plant viruses. These observations suggest that, perhaps, differences in the selective pressures operating during plant virus evolution may have driven their mutation rates towards values lower than those characteristic of other RNA viruses infecting bacteria or animals.

A Novel, Drug Resistance-Independent, Fluorescence-Based Approach To Measure Mutation Rates in Microbial Pathogens

mBio ◽

10.1128/mbio.00120-19 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 6

Author(s):

Erika Shor ◽

Jessica Schuyler ◽

David S. Perlin

Keyword(s):

Drug Resistance ◽

Mutation Rate ◽

Fungal Pathogen ◽

Candida Glabrata ◽

Drug Tolerance ◽

Clinical Isolates ◽

Mutation Rates ◽

Mismatch Repair Gene ◽

Repair Gene ◽

Content Type

ABSTRACT All evolutionary processes are underpinned by a cellular capacity to mutate DNA. To identify factors affecting mutagenesis, it is necessary to compare mutation rates between different strains and conditions. Drug resistance-based mutation reporters are used extensively to measure mutation rates, but they are suitable only when the compared strains have identical drug tolerance levels—a condition that is not satisfied under many “real-world” circumstances, e.g., when comparing mutation rates among a series of environmental or clinical isolates. Candida glabrata is a fungal pathogen that shows a high degree of genetic diversity and fast emergence of antifungal drug resistance. To enable meaningful comparisons of mutation rates among C. glabrata clinical isolates, we developed a novel fluorescence-activated cell sorting-based approach to measure the mutation rate of a chromosomally integrated GFP gene. We found that in Saccharomyces cerevisiae this approach recapitulated the reported mutation rate of a wild-type strain and the mutator phenotype of a shu1Δ mutant. In C. glabrata, the GFP reporter captured the mutation rate increases caused either by a genotoxic agent or by deletion of DNA mismatch repair gene MSH2, as well as the specific mutational signature associated with msh2Δ. Finally, the reporter was used to measure the mutation rates of C. glabrata clinical isolates carrying different alleles of MSH2. Together, these results show that fluorescence-based mutation reporters can be used to measure mutation rates in microbes under conditions of unequal drug susceptibility to reveal new insights about drivers of mutagenesis. IMPORTANCE Measurements of mutation rates—i.e., how often proliferating cells acquire mutations in their DNA—are essential for understanding cellular processes that maintain genome stability. Many traditional mutation rate measurement assays are based on detecting mutations that cause resistance to a particular drug. Such assays typically work well for laboratory strains but have significant limitations when comparing clinical or environmental isolates that have various intrinsic levels of drug tolerance, which confounds the interpretation of results. Here we report the development and validation of a novel method of measuring mutation rates, which detects mutations that cause loss of fluorescence rather than acquisition of drug resistance. Using this method, we measured the mutation rates of clinical isolates of fungal pathogen Candida glabrata. This assay can be adapted to other organisms and used to compare mutation rates in contexts where unequal drug sensitivity is anticipated.

Comparison of the Mutation Rates of Human Influenza A and B Viruses

Journal of Virology ◽

10.1128/jvi.80.7.3675-3678.2006 ◽

2006 ◽

Vol 80 (7) ◽

pp. 3675-3678 ◽

Cited By ~ 151

Author(s):

Eri Nobusawa ◽

Katsuhiko Sato

Keyword(s):

Genetic Diversity ◽

Mutation Rate ◽

Influenza A ◽

Mdck Cells ◽

Evolutionary Rates ◽

Mutation Rates ◽

Influenza B ◽

Human Influenza ◽

Influenza A Viruses ◽

Infectious Cycle

ABSTRACT Human influenza A viruses evolve more rapidly than influenza B viruses. To clarify the cause of this difference, we have evaluated the mutation rate of the nonstructural gene as revealed by the genetic diversity observed during the growth of individual plaques in MDCK cells. Six plaques were studied, representing two strains each of type A and B viruses. A total of 813,663 nucleotides were sequenced, giving rates of 2.0 × 10−6 and 0.6 × 10−6 mutations per site per infectious cycle, which, when extended to 1 year, agree well with the published annual evolutionary rates.

Viral Quasispecies and Lethal Mutagenesis

European Review ◽

10.1017/s1062798715000411 ◽

2016 ◽

Vol 24 (1) ◽

pp. 39-48 ◽

Cited By ~ 1

Author(s):

Esteban Domingo ◽

Celia Perales

Keyword(s):

Nucleotide Sequence ◽

Genetic Information ◽

Rna Viruses ◽

Viral Disease ◽

Mutation Rates ◽

Current Status ◽

Viral Quasispecies ◽

Lethal Mutagenesis ◽

Dna And Rna ◽

A Genome

Virology has undergone a profound transformation with the incorporation of quasispecies theory to the understanding of the composition and dynamics of viral populations as they cause disease. RNA viral populations do not consist of a genome class with a defined nucleotide sequence but of a cloud or swarm or related mutants due to high mutation rates (number of incorrect nucleotides introduced per nucleotide copied) during replication. DNA and RNA viruses whose multiplication is catalysed by a low fidelity polymerase replicate close to an error threshold for maintenance of their genetic information. This means that modest increases in mutation rate jeopardize their genetic stability. Realization of this important corollary of quasispecies theory has opened new approaches to combating viral disease. One of these approaches is lethal mutagenesis that consists of forcing virus extinction by an excess of mutations evoked by virus-specific mutagenic agents. This article summarizes the origin and current status of this new antiviral approach.

A speed-fidelity trade-off determines the mutation rate and virulence of an RNA virus

10.1101/309880 ◽

2018 ◽

Author(s):

William Fitzsimmons ◽

Robert J. Woods ◽

John T. McCrone ◽

Andrew Woodman ◽

Jamie J. Arnold ◽

...

Keyword(s):

Mutation Rate ◽

Experimental Evolution ◽

Rna Viruses ◽

Fundamental Problem ◽

Rna Virus ◽

Mutation Rates ◽

Direct Selection ◽

Growth Defect ◽

Infection Model ◽

Selection For

AbstractMutation rates can evolve through genetic drift, indirect selection due to genetic hitchhiking, or direct selection on the physicochemical cost of high fidelity. However, for many systems, it has been difficult to disentangle the relative impact of these forces empirically. In RNA viruses, an observed correlation between mutation rate and virulence has led many to argue that their extremely high mutation rates are advantageous, because they may allow for increased adaptability. This argument has profound implications, as it suggests that pathogenesis in many viral infections depends on rare orde novomutations. Here we present data for an alternative model whereby RNA viruses evolve high mutation rates as a byproduct of selection for increased replicative speed. We find that a poliovirus antimutator, 3DG64S, has a significant replication defect and that wild type and 3DG64Spopulations have similar adaptability in two distinct cellular environments. Experimental evolution of 3DG64Sunder r-selection led to reversion and compensation of the fidelity phenotype. Mice infected with 3DG64Sexhibited delayed morbidity at doses well above the LD50, consistent with attenuation by slower growth as opposed to reduced mutational supply. Furthermore, compensation of the 3DG64Sgrowth defect restored virulence, while compensation of the fidelity phenotype did not. Our data are consistent with the kinetic proofreading model for biosynthetic reactions and suggest that speed is more important than accuracy. In contrast to what has been suggested for many RNA viruses, we find that within host spread is associated with viral replicative speed and not standing genetic diversity.Author SummaryMutation rate evolution has long been a fundamental problem in evolutionary biology. The polymerases of RNA viruses generally lack proofreading activity and exhibit extremely high mutation rates. Since most mutations are deleterious and mutation rates are tuned by natural selection, we asked why hasn’t the virus evolved to have a lower mutation rate? We used experimental evolution and a murine infection model to show that RNA virus mutation rates may actually be too high and are not necessarily adaptive. Rather, our data indicate that viral mutation rates are driven higher as a result of selection for viruses with faster replication kinetics. We suggest that viruses have high mutation rates, not because they facilitate adaption, but because it is hard to be both fast and accurate.

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.

The Rate and Character of Spontaneous Mutation in an RNA Virus

Genetics ◽

10.1093/genetics/162.4.1505 ◽

2002 ◽

Vol 162 (4) ◽

pp. 1505-1511 ◽

Cited By ~ 1

Author(s):

José M Malpica ◽

Aurora Fraile ◽

Ignacio Moreno ◽

Clara I Obies ◽

John W Drake ◽

...

Keyword(s):

Mutation Rate ◽

Movement Protein ◽

Rna Viruses ◽

Rna Virus ◽

Spontaneous Mutation ◽

Mutation Rates ◽

Lower Side ◽

Mutation Process ◽

Spontaneous Mutation Rate ◽

Mutant Frequency

Abstract Estimates of spontaneous mutation rates for RNA viruses are few and uncertain, most notably due to their dependence on tiny mutation reporter sequences that may not well represent the whole genome. We report here an estimate of the spontaneous mutation rate of tobacco mosaic virus using an 804-base cognate mutational target, the viral MP gene that encodes the movement protein (MP). Selection against newly arising mutants was countered by providing MP function from a transgene. The estimated genomic mutation rate was on the lower side of the range previously estimated for lytic animal riboviruses. We also present the first unbiased riboviral mutational spectrum. The proportion of base substitutions is the same as that in a retrovirus but is lower than that in most DNA-based organisms. Although the MP mutant frequency was 0.02-0.05, 35% of the sequenced mutants contained two or more mutations. Therefore, the mutation process in populations of TMV and perhaps of riboviruses generally differs profoundly from that in populations of DNA-based microbes and may be strongly influenced by a subpopulation of mutator polymerases.