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Journal of Virology, July 2008, p. 6880-6888, Vol. 82, No. 14
0022-538X/08/$08.00+0     doi:10.1128/JVI.00517-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Synthesis of Genomic and Subgenomic RNA in Mosquito Cells Infected with Two Sindbis Virus nsP4 Mutants: Influence of Intracellular Nucleoside Triphosphate Concentrations{triangledown}

Mei-Ling Li,1 Tzu-Yu Kwan,1 H. Anne Simmonds,3 and Victor Stollar1,2*

Department of Molecular Genetics, Microbiology and Immunology, UMDNJ-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854,1 Cancer Institute of New Jersey, New Brunswick, New Jersey 08903,2 Purine Research Unit, Guy's Hospital, London SE1 9RT, Great Britain3

Received 7 March 2008/ Accepted 30 April 2008

Cells infected with Sindbis virus (SV) make two positive-strand RNAs, a genomic-length RNA (G) RNA and a subgenomic (SG) RNA. In cells infected with SVstd, and in general in cells infected with wt alphaviruses, more SG RNA is made than G RNA. How the balance between synthesis of G RNA and SG RNA is regulated is not known. SVpzf and SVcpc are nsP4 mutants of SV which, in mosquito cells, make more G RNA than SG RNA. When low concentrations of pyrazofurin (inhibits the synthesis of UTP and CTP) were added to SVpzf-infected cells, the yield of virus was increased, and the ratio of SG/G RNA was changed from <1 to >1. These effects were reversed by uridine. In SVcpc-infected cells, but not in SVstd-infected cells, synthesis of viral RNA was inhibited by the addition of either uridine or cytidine, and viral yields were lowered. Our findings suggest that the activities of the viral RNA-synthesizing complexes in cells infected with SVpzf or SVcpc, in contrast to those in SVstd-infected cells, are sensitive to high concentrations of UTP or CTP. Using a cell-free system that synthesizes both SG and G RNA, we measured viral RNA synthesis as a function of the UTP/CTP concentrations. The results indicated that the presence of the SVpzf mutations in nsP4 and the SG promoter produced a pattern quite different from that seen with the SVstd nsP4 and SG promoter. As the UTP/CTP concentrations were increased, the SVpzf system, in contrast to the SVstd system, made more G RNA than SG RNA, reflecting the situation in cells infected with SVpzf.

* Corresponding author. Mailing address: Department of Molecular Genetics, Microbiology and Immunology, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ 08854. Phone: (908) 235-4596. Fax: (908) 235-5223. E-mail: stollar{at}umdnj.edu

{triangledown} Published ahead of print on 28 May 2008.

Journal of Virology, July 2008, p. 6880-6888, Vol. 82, No. 14
0022-538X/08/$08.00+0     doi:10.1128/JVI.00517-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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