Elongation activity of poliovirus RNA polymerase derived from Sabin type 1 sequence is not temperature sensitive Free

Abstract

Determinants of attenuation in the Sabin type 1 strain of poliovirus are located in the 5′ noncoding region, the capsid coding region and the viral RNA-dependent RNA polymerase (3D) coding region. These mutations also contribute to a temperature sensitive phenotype of virus replication. We have cloned and expressed the Sabin 1 virus 3D protein which contains three amino acid differences from the wild-type (Mahoney) sequence, as well as a wild-type polymerase containing only a single Sabin amino acid substitution at nt 6203. These enzymes have been examined and compared for temperature sensitive polymerase activity. Wild-type and mutated polymerases demonstrated identical specific activities at 30, 35 and 39 °C. All three showed the same kinetics of heat inactivation after pre-incubation at elevated temperatures. Thus the contribution of Sabin 3D sequences to the inability of the virus to grow at elevated temperatures must lie in a function or activity of the enzyme other than RNA polymerization. A likely reaction is the initiation step of RNA chain synthesis.

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1995-08-01
2024-03-28
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References

  1. Burns C. C., Lawson M. A., Semler B. L., Ehrenfeld E. 1989; Effects of mutations in poliovirus 3D pol on RNA polymerase activity and on polyprotein cleavage. Journal of Virology 63:4866–4874
    [Google Scholar]
  2. Christodoulou C., Colbere-Garapin F., Macadam A., Taffs L. F., Marsden S., Minor P., Horaud F. 1990; Mapping of mutations associated with neurovirulence in monkey infected with Sabin 1 poliovirus revertants selected at high temperature. Journal of Virology 64:4922–1929
    [Google Scholar]
  3. Hey T. D., Richards O. C., Ehrenfeld E. 1986; Synthesis of plus and minus-strand RNA from poliovirion RNA template in vitro . Journal of Virology 58:790–796
    [Google Scholar]
  4. Ho S. N., Hunt H. D., Horton R. M., Pullen J. K., Pease L. R. 1989; Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77:51–59
    [Google Scholar]
  5. Macadam A. J., Arnold C., Howlett J., John A., Marsden S., Taffs F., Reeve P., Hamada N., Wareham K., Almond J., Cammack N., Minor P. D. 1989; Reversion of the attenuated and temperature-sensitive phenotypes of the Sabin type 3 strain of poliovirus in vaccinees. Virology 172:408–414
    [Google Scholar]
  6. Macadam A. J., Ferguson G., Arnold C., Minor P. D. 1991a; An assembly defect as a result of an attenuating mutation in the capsid protein of the poliovirus type 3 vaccine strain. Journal of Virology 72:2475–2481
    [Google Scholar]
  7. Macadam A. J., Pollard S. R., Ferguson G., Dunn G., Skuce R., Almond J. W., Minor P. D. 19916; The 5′ non-coding region of the type 2 poliovirus vaccine strain contains determinants of attenuation and temperature sensitivity. Virology 181:451–458
    [Google Scholar]
  8. Minor P. D. 1992; The molecular biology of poliovaccines. Journal of General Virology 73:3065–3077
    [Google Scholar]
  9. Neufeld K. L., Richards O. C., Ehrenfeld E. 1991; Purification, characterization, and comparison of poliovirus RNA polymerase from native and recombinant sources. Journal of Biological Chemistry 266:24212–24219
    [Google Scholar]
  10. Richards O. C., Ivanoff L. A., Bienkowska-Szewczyk K., Butt B., Petteway S. R., Rothstein M. A., Ehrenfeld E. 1987; Formation of poliovirus RNA polymerase 3D in Escherichia coli by cleavage of fusion proteins expressed from cloned viral cDNA. Virology 161:348–356
    [Google Scholar]
  11. Richards O. C., Yu P., Neufeld K. L., Ehrenfeld E. 1992; Nucleotide binding by the poliovirus RNA polymerase. Journal of Biological Chemistry 267:17141–17146
    [Google Scholar]
  12. Rothstein M. A., Richards O. C., Amin C., Ehrenfeld E. 1988; Enzymatic activity of poliovirus RNA polymerase synthesized in Escherichia coli from viral cDNA. Virology 164:301–308
    [Google Scholar]
  13. Sabin A. B. 1957; Properties and behavior of orally administered attenuated poliovirus vaccine. Journal of the American Medical Association 164:1216–1223
    [Google Scholar]
  14. Tardy-Panit M., Blondel B., Martin A., Tekaia F., Horaud F., Del Peyroux F. 1993; A mutation in the RNA polymerase of poliovirus type 1 contributes to attenuation in mice. Journal of Virology 67:4630–4638
    [Google Scholar]
  15. Toyoda H., Yang C.-F., Takeda N., Nomoto A., Wimmer E. 1987; Analysis of RNA synthesis of type 1 poliovirus by using an in vitro molecular genetic approach. Journal of Virology 61:2816–2822
    [Google Scholar]
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