A single mutation in poliovirus RNA-dependent RNA polymerase confers resistance to mutagenic nucleotide analogs via increased fidelity

Ribavirin is a nucleotide analog that can be incorporated by viral polymerases, causing mutations by allowing base mismatches. It is currently used therapeutically as an antiviral drug during hepatitis C virus infections. During the amplification of poliovirus genomic RNA or hepatitis C replicons, error frequency is known to increase upon ribavirin treatment. This observation has led to the hypothesis that ribavirin's antiviral activity results from error catastrophe caused by increased mutagenesis of viral genomes. Here, we describe the generation of ribavirin-resistant poliovirus by serial viral passage in the presence of increasing concentrations of the drug. Ribavirin resistance can be caused by a single amino acid change, G64S, in the viral polymerase in an unresolved portion of the fingers domain. Compared with wild-type virus, ribavirin-resistant poliovirus displays increased fidelity of RNA synthesis in the absence of ribavirin and increased survival both in the presence of ribavirin and another mutagen, 5-azacytidine. Ribavirin-resistant poliovirus represents an unusual class of viral drug resistance: resistance to a mutagen through increased fidelity.

[1]  J. Arnold,et al.  Poliovirus RNA-dependent RNA Polymerase (3Dpol) , 2000, The Journal of Biological Chemistry.

[2]  D. Baltimore,et al.  Molecular cloning of poliovirus cDNA and determination of the complete nucleotide sequence of the viral genome. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[3]  A. Bruchfeld,et al.  Dosage of Ribavirin in Patients With Hepatitis C Should Be Based on Renal Function: A Population Pharmacokinetic Analysis , 2002, Therapeutic drug monitoring.

[4]  E. Wimmer,et al.  Production of guanidine-resistant and -dependent poliovirus mutants from cloned cDNA: mutations in polypeptide 2C are directly responsible for altered guanidine sensitivity , 1986, Journal of virology.

[5]  F. Gillin,et al.  Control of mutation frequency by bacteriophage T4 DNA polymerase. I. The CB120 antimutator DNA polymerase is defective in strand displacement. , 1976, The Journal of biological chemistry.

[6]  R. Schaaper.  Antimutator mutants in bacteriophage T4 and Escherichia coli. , 1998, Genetics.

[7]  Pedro R. Lowenstein,et al.  Response of Foot-and-Mouth Disease Virus to Increased Mutagenesis: Influence of Viral Load and Fitness in Loss of Infectivity , 2000, Journal of Virology.

[8]  E. Wimmer,et al.  Guanidine-dependent mutants of poliovirus: identification of three classes with different growth requirements. , 1987, Virology.

[9]  L. Loeb,et al.  Prokaryotic DNA polymerase I: evolution, structure, and "base flipping" mechanism for nucleotide selection. , 2001, Journal of molecular biology.

[10]  G L Verdine,et al.  Structure of a covalently trapped catalytic complex of HIV-1 reverse transcriptase: implications for drug resistance. , 1998, Science.

[11]  E. Ehrenfeld,et al.  Identification of nucleotide binding sites in the poliovirus RNA polymerase. , 1995, Biochemistry.

[12]  E. Ehrenfeld,et al.  One of Two NTP Binding Sites in Poliovirus RNA Polymerase Required for RNA Replication* , 1997, The Journal of Biological Chemistry.

[13]  E. Ehrenfeld,et al.  Mutation of lysine residues in the nucleotide binding segments of the poliovirus RNA-dependent RNA polymerase , 1996, Journal of virology.

[14]  C. Cameron,et al.  RNA virus error catastrophe: Direct molecular test by using ribavirin , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[15]  S D Kemp,et al.  Multiple mutations in HIV-1 reverse transcriptase confer high-level resistance to zidovudine (AZT). , 1989, Science.

[16]  J. Arnold,et al.  The broad-spectrum antiviral ribonucleoside ribavirin is an RNA virus mutagen , 2000, Nature Medicine.

[17]  B. Berkhout,et al.  Increased polymerase fidelity of lamivudine-resistant HIV-1 variants does not limit their evolutionary potential. , 1999, AIDS.

[18]  W. C. Drosopoulos,et al.  The influence of 3TC resistance mutation M184I on the fidelity and error specificity of human immunodeficiency virus type 1 reverse transcriptase. , 1998, Nucleic acids research.

[19]  J. Hansen,et al.  Structure of the RNA-dependent RNA polymerase of poliovirus. , 1997, Structure.

[20]  N. Muzyczka,et al.  Studies on the biochemical basis of spontaneous mutation. I. A comparison of the deoxyribonucleic acid polymerases of mutator, antimutator, and wild type strains of bacteriophage T4. , 1972, The Journal of biological chemistry.

[21]  V. Agol,et al.  Genetic studies on the poliovirus 2C protein, an NTPase. A plausible mechanism of guanidine effect on the 2C function and evidence for the importance of 2C oligomerization. , 1994, Journal of molecular biology.

[22]  D. Pearl,et al.  Combination of Drugs and Drug-Resistant Reverse Transcriptase Results in a Multiplicative Increase of Human Immunodeficiency Virus Type 1 Mutant Frequencies , 2002, Journal of Virology.

[23]  G. Bessard,et al.  Ribavirin Quantification in Combination Treatment of Chronic Hepatitis C , 2003, Antimicrobial Agents and Chemotherapy.

[24]  E. Schmidt,et al.  Viral RNA Mutations Are Region Specific and Increased by Ribavirin in a Full-Length Hepatitis C Virus Replication System , 2002, Journal of Virology.

[25]  J. Drake,et al.  Rates of spontaneous mutation. , 1998, Genetics.

[26]  K. Kirkegaard,et al.  The mechanism of RNA recombination in poliovirus , 1986, Cell.

[27]  K. Kirkegaard,et al.  Oligomeric structures of poliovirus polymerase are important for function , 2001, The EMBO journal.

[28]  W. C. Drosopoulos,et al.  Increased Misincorporation Fidelity Observed for Nucleoside Analog Resistance Mutations M184V and E89G in Human Immunodeficiency Virus Type 1 Reverse Transcriptase Does Not Correlate with the Overall Error Rate Measured In Vitro , 1998, Journal of Virology.

[29]  M. Wainberg,et al.  Enhanced Fidelity of 3TC-Selected Mutant HIV-1 Reverse Transcriptase , 1996, Science.

[30]  E. Wimmer,et al.  Guanidine-selected mutants of poliovirus: mapping of point mutations to polypeptide 2C , 1986, Journal of virology.

[31]  E. Domingo,et al.  RNA virus mutations and fitness for survival. , 1997, Annual review of microbiology.