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. 1988 Feb;62(2):558–562. doi: 10.1128/jvi.62.2.558-562.1988

Direct measurement of the poliovirus RNA polymerase error frequency in vitro.

C D Ward 1, M A Stokes 1, J B Flanegan 1
PMCID: PMC250568  PMID: 2826815

Abstract

The fidelity of RNA replication by the poliovirus-RNA-dependent RNA polymerase was examined by copying homopolymeric RNA templates in vitro. The poliovirus RNA polymerase was extensively purified and used to copy poly(A), poly(C), or poly(I) templates with equimolar concentrations of noncomplementary and complementary ribonucleotides. The error frequency was expressed as the amount of a noncomplementary nucleotide incorporated divided by the total amount of complementary and noncomplementary nucleotide incorporated. The polymerase error frequencies were very high and ranged from 7 x 10(-4) to 5.4 x 10(-3), depending on the specific reaction conditions. There were no significant differences among the error frequencies obtained with different noncomplementary nucleotide substrates on a given template or between the values determined on two different templates for a specific noncomplementary substrate. The activity of the polymerase on poly(U) and poly(G) was too low to measure error frequencies on these templates. A fivefold increase in the error frequency was observed when the reaction conditions were changed from 3.0 mM Mg2+ (pH 7.0) to 7.0 mM Mg2+ (pH 8.0). This increase in the error frequency correlates with an eightfold increase in the elongation rate that was observed under the same conditions in a previous study.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Battula N., Loeb L. A. The infidelity of avian myeloblastosis virus deoxyribonucleic acid polymerase in polynucleotide replication. J Biol Chem. 1974 Jul 10;249(13):4086–4093. [PubMed] [Google Scholar]
  2. Clewley J. P., Bishop D. H., Kang C. Y., Coffin J., Schnitzlein W. M., Reichmann M. E., Shope R. E. Oligonucleotide fingerprints of RNA species obtained from rhabdoviruses belonging to the vesicular stomatitis virus subgroup. J Virol. 1977 Jul;23(1):152–166. doi: 10.1128/jvi.23.1.152-166.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Dasgupta A., Baron M. H., Baltimore D. Poliovirus replicase: a soluble enzyme able to initiate copying of poliovirus RNA. Proc Natl Acad Sci U S A. 1979 Jun;76(6):2679–2683. doi: 10.1073/pnas.76.6.2679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Domingo E., Dávila M., Ortín J. Nucleotide sequence heterogeneity of the RNA from a natural population of foot-and-mouth-disease virus. Gene. 1980 Nov;11(3-4):333–346. doi: 10.1016/0378-1119(80)90073-6. [DOI] [PubMed] [Google Scholar]
  5. Domingo E., Flavell R. A., Weissmann C. In vitro site-directed mutagenesis: generation and properties of an infectious extracistronic mutant of bacteriophage Qbeta. Gene. 1976;1(1):3–25. doi: 10.1016/0378-1119(76)90003-2. [DOI] [PubMed] [Google Scholar]
  6. Domingo E., Sabo D., Taniguchi T., Weissmann C. Nucleotide sequence heterogeneity of an RNA phage population. Cell. 1978 Apr;13(4):735–744. doi: 10.1016/0092-8674(78)90223-4. [DOI] [PubMed] [Google Scholar]
  7. Fields S., Winter G. Nucleotide-sequence heterogeneity and sequence rearrangements in influenza virus cDNA. Gene. 1981 Nov;15(2-3):207–214. doi: 10.1016/0378-1119(81)90130-x. [DOI] [PubMed] [Google Scholar]
  8. Flanegan J. B., Baltimore D. Poliovirus polyuridylic acid polymerase and RNA replicase have the same viral polypeptide. J Virol. 1979 Jan;29(1):352–360. doi: 10.1128/jvi.29.1.352-360.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Flanegan J. B., Baltimore D. Poliovirus-specific primer-dependent RNA polymerase able to copy poly(A). Proc Natl Acad Sci U S A. 1977 Sep;74(9):3677–3680. doi: 10.1073/pnas.74.9.3677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Flanegan J. B., Van Dyke T. A. Isolation of a soluble and template-dependent poliovirus RNA polymerase that copies virion RNA in vitro. J Virol. 1979 Oct;32(1):155–161. doi: 10.1128/jvi.32.1.155-161.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gopinathan K. P., Weymouth L. A., Kunkel T. A., Loeb L. A. Mutagenesis in vitro by DNA polymerase from an RNA tumour virus. Nature. 1979 Apr 26;278(5707):857–859. doi: 10.1038/278857a0. [DOI] [PubMed] [Google Scholar]
  12. Hall Z. W., Lehman I. R. An in vitro transversion by a mutationally altered T4-induced DNA polymerase. J Mol Biol. 1968 Sep 28;36(3):321–333. doi: 10.1016/0022-2836(68)90158-7. [DOI] [PubMed] [Google Scholar]
  13. Harris T. J., Robson K. H., Brown F. A study of the level of nucleotide sequence conservation between the RNAs of two types serotypes of foot-and-mouth disease virus. J Gen Virol. 1980 Oct;50(2):403–418. doi: 10.1099/0022-1317-50-2-403. [DOI] [PubMed] [Google Scholar]
  14. Holland J. J., Kohne D., Doyle M. V. Analysis of virus replication in ageing human fibroblast cultures. Nature. 1973 Oct 12;245(5424):316–319. doi: 10.1038/245316a0. [DOI] [PubMed] [Google Scholar]
  15. Holland J., Spindler K., Horodyski F., Grabau E., Nichol S., VandePol S. Rapid evolution of RNA genomes. Science. 1982 Mar 26;215(4540):1577–1585. doi: 10.1126/science.7041255. [DOI] [PubMed] [Google Scholar]
  16. King A. M., Underwood B. O., McCahon D., Newman J. W., Brown F. Biochemical identification of viruses causing the 1981 outbreaks of foot and mouth disease in the UK. Nature. 1981 Oct 8;293(5832):479–480. doi: 10.1038/293479a0. [DOI] [PubMed] [Google Scholar]
  17. Kunkel T. A., Schaaper R. M., Beckman R. A., Loeb L. A. On the fidelity of DNA replication. Effect of the next nucleotide on proofreading. J Biol Chem. 1981 Oct 10;256(19):9883–9889. [PubMed] [Google Scholar]
  18. Lubeck M. D., Schulman J. L., Palese P. Antigenic variants of influenza viruses: marked differences in the frequencies of variants selected with different monoclonal antibodies. Virology. 1980 Apr 30;102(2):458–462. doi: 10.1016/0042-6822(80)90114-2. [DOI] [PubMed] [Google Scholar]
  19. Minor P. D., John A., Ferguson M., Icenogle J. P. Antigenic and molecular evolution of the vaccine strain of type 3 poliovirus during the period of excretion by a primary vaccinee. J Gen Virol. 1986 Apr;67(Pt 4):693–706. doi: 10.1099/0022-1317-67-4-693. [DOI] [PubMed] [Google Scholar]
  20. Minor P. D., Schild G. C., Ferguson M., Mackay A., Magrath D. I., John A., Yates J. P., Spitz M. Genetic and antigenic variation in type 3 polioviruses: characterization of strains by monoclonal antibodies and T1 oligonucleotide mapping. J Gen Virol. 1982 Aug;61(Pt 2):167–176. doi: 10.1099/0022-1317-61-2-167. [DOI] [PubMed] [Google Scholar]
  21. Nottay B. K., Kew O. M., Hatch M. H., Heyward J. T., Obijeski J. F. Molecular variation of type 1 vaccine-related and wild polioviruses during replication in humans. Virology. 1981 Jan 30;108(2):405–423. doi: 10.1016/0042-6822(81)90448-7. [DOI] [PubMed] [Google Scholar]
  22. Parvin J. D., Moscona A., Pan W. T., Leider J. M., Palese P. Measurement of the mutation rates of animal viruses: influenza A virus and poliovirus type 1. J Virol. 1986 Aug;59(2):377–383. doi: 10.1128/jvi.59.2.377-383.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Portner A., Webster R. G., Bean W. J. Similar frequencies of antigenic variants in Sendai, vesicular stomatitis, and influenza A viruses. Virology. 1980 Jul 15;104(1):235–238. doi: 10.1016/0042-6822(80)90382-7. [DOI] [PubMed] [Google Scholar]
  24. Prabhakar B. S., Haspel M. V., McClintock P. R., Notkins A. L. High frequency of antigenic variants among naturally occurring human Coxsackie B4 virus isolates identified by monoclonal antibodies. Nature. 1982 Nov 25;300(5890):374–376. doi: 10.1038/300374a0. [DOI] [PubMed] [Google Scholar]
  25. Rose J. K. Heterogneeous 5'-terminal structures occur on vesicular stomatitis virus mRNAs. J Biol Chem. 1975 Oct 25;250(20):8098–8104. [PubMed] [Google Scholar]
  26. Sobrino F., Dávila M., Ortín J., Domingo E. Multiple genetic variants arise in the course of replication of foot-and-mouth disease virus in cell culture. Virology. 1983 Jul 30;128(2):310–318. doi: 10.1016/0042-6822(83)90258-1. [DOI] [PubMed] [Google Scholar]
  27. Steinhauer D. A., Holland J. J. Direct method for quantitation of extreme polymerase error frequencies at selected single base sites in viral RNA. J Virol. 1986 Jan;57(1):219–228. doi: 10.1128/jvi.57.1.219-228.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Takeda N., Miyamura K., Ogino T., Natori K., Yamazaki S., Sakurai N., Nakazono N., Ishii K., Kono R. Evolution of enterovirus type 70: oligonucleotide mapping analysis of RNA genome. Virology. 1984 Apr 30;134(2):375–388. doi: 10.1016/0042-6822(84)90305-2. [DOI] [PubMed] [Google Scholar]
  29. Tuschall D. M., Hiebert E., Flanegan J. B. Poliovirus RNA-dependent RNA polymerase synthesizes full-length copies of poliovirion RNA, cellular mRNA, and several plant virus RNAs in vitro. J Virol. 1982 Oct;44(1):209–216. doi: 10.1128/jvi.44.1.209-216.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Van Dyke T. A., Rickles R. J., Flanegan J. B. Genome-length copies of poliovirion RNA are synthesized in vitro by the poliovirus RNA-dependent RNA polymerase. J Biol Chem. 1982 Apr 25;257(8):4610–4617. [PubMed] [Google Scholar]
  31. Webster R. G., Laver W. G. Determination of the number of nonoverlapping antigenic areas on Hong Kong (H3N2) influenza virus hemagglutinin with monoclonal antibodies and the selection of variants with potential epidemiological significance. Virology. 1980 Jul 15;104(1):139–148. doi: 10.1016/0042-6822(80)90372-4. [DOI] [PubMed] [Google Scholar]
  32. Yewdell J. W., Webster R. G., Gerhard W. U. Antigenic variation in three distinct determinants of an influenza type A haemagglutinin molecule. Nature. 1979 May 17;279(5710):246–248. doi: 10.1038/279246a0. [DOI] [PubMed] [Google Scholar]
  33. Young D. C., Dunn B. M., Tobin G. J., Flanegan J. B. Anti-VPg antibody precipitation of product RNA synthesized in vitro by the poliovirus polymerase and host factor is mediated by VPg on the poliovirion RNA template. J Virol. 1986 Jun;58(3):715–723. doi: 10.1128/jvi.58.3.715-723.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]

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