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Journal of Virology, September 2002, p. 8632-8640, Vol. 76, No. 17
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.17.8632-8640.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Alphavirus Minus-Strand RNA Synthesis: Identification of a Role for Arg183 of the nsP4 Polymerase

Cori L. Fata,,{dagger} Stanley G. Sawicki, and Dorothea L. Sawicki*

Department of Microbiology and Immunology, Medical College of Ohio, Toledo, Ohio

Received 21 March 2002/ Accepted 29 May 2002

A partially conserved region spanning amino acids 142 to 191 of the Sindbis virus (SIN) nsP4 core polymerase is implicated in host restriction, elongation, and promoter recognition. We extended the analysis of this region by substituting Ser, Ala, or Lys for a highly conserved Arg183 residue immediately preceding its absolutely conserved Ser184-Ala-Val-Pro-Ser188 sequence. In chicken cells, the nsP4 Arg183 mutants had a nonconditionally lethal, temperature-sensitive (ts) growth phenotype caused by a ts defect in minus-strand synthesis whose extent varied with the particular amino acid substituted (Ser>Ala>Lys). Plus-strand synthesis by nsP4 Arg183 mutant polymerases was unaffected when corrected for minus-strand numbers, although 26S mRNA synthesis was enhanced at the elevated temperature compared to wild type. The ts defect was not due to a failure to form or accumulate nsP4 at 40°C. In contrast to their growth in chicken cells, the nsP4 Arg183 mutants replicated equally poorly, if at all, in mosquito cells. We conclude that Arg183 within the Pro180-Asn-Ile-Arg-Ser184 sequence of the SIN nsP4 polymerase contributes to the efficient initiation of minus strands or the formation of its replicase and that a host factor(s) participates in this event.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Medical College of Ohio, 3055 Arlington Ave., Toledo, OH 43614. Phone: (419) 383-4337 or (419) 383-4342. Fax: (419) 383-3002. E-mail: dsawicki{at}mco.edu.

{dagger} Present address: Department of Biochemistry, Institute of Molecular Virology, University of Wisconsin—Madison, Madison, Wis.

Journal of Virology, September 2002, p. 8632-8640, Vol. 76, No. 17
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.17.8632-8640.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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