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Journal of Virology, September 2004, p. 9645-9651, Vol. 78, No. 18
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.18.9645-9651.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

A Mutant of Sindbis Virus Which Is Able To Replicate in Cells with Reduced CTP Makes a Replicase/Transcriptase with a Decreased Km for CTP

Mei-Ling Li,1 Yen-Huei Lin,1,{dagger} H. Anne Simmonds,2 and Victor Stollar1*

Department of Molecular Genetics, Microbiology and Immunology, UMDNJ-Robert Wood Johnson Medical School, Piscataway, New Jersey,1 Purine Research Unit, Guy's Hospital, London Bridge, Great Britain2

Received 30 December 2003/ Accepted 13 April 2004

We reported earlier the isolation and characterization of a Sindbis virus mutant, SVPZF, that can grow in mosquito cells treated with pyrazofurin (PZF), a compound that interferes with pyrimidine biosynthesis (Y. H. Lin, P. Yadav, R. Ravatn, and V. Stollar, Virology 272:61-71, 2000; Y. H. Lin, H. A. Simmonds, and V. Stollar, Virology 292:78-86, 2002). Three amino acid changes in nsP4, the viral RNA polymerase, were required to produce this phenotype. We now describe a mutant of Sindbis virus, SVCPC, that is resistant to cyclopentenylcytosine (CPC), a compound that interferes only with the synthesis of CTP. Thus, in contrast to SVPZF, which was selected for its ability to grow in mosquito cells with low levels of UTP and CTP, SVCPC was selected for its ability to grow in cells in which only the level of CTP was reduced. Although SVPZF was cross-resistant to CPC, SVCPC was not resistant to PZF. Only one amino acid change in nsP4, Leu 585 to Phe, was required for the CPC resistance phenotype. The viral replicase/transcriptase generated in SVCPC-infected mosquito cells had a lower Km for CTP (but not for UTP) than did the enzyme made in SVSTD-infected mosquito cells. SVPZF and SVCPC represent the first examples of viral mutants selected for the ability to grow in cells with low levels of ribonucleoside triphosphates (rNTPs). Further study of these mutants and determination of the structure of nsP4 should demonstrate how alterations in an RNA-dependent RNA polymerase permit it to function in cells with abnormally low levels of rNTPs.

* Corresponding author. Mailing address: Department of Molecular Genetics, Microbiology and Immunology, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ 08854. Phone: (732) 235-4596. Fax: (732) 235-5223. E-mail: stollar{at}umdnj.edu.

{dagger} Present address: Wyeth-Lederle Vaccines, Pearl River, NY 10965.

Journal of Virology, September 2004, p. 9645-9651, Vol. 78, No. 18
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.18.9645-9651.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Li, M.-L., Kwan, T.-Y., Simmonds, H. A., Stollar, V. (2008). Synthesis of Genomic and Subgenomic RNA in Mosquito Cells Infected with Two Sindbis Virus nsP4 Mutants: Influence of Intracellular Nucleoside Triphosphate Concentrations. J. Virol. 82: 6880-6888 [Abstract] [Full Text]  


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