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Journal of Virology, September 2002, p. 8641-8649, Vol. 76, No. 17
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.17.8641-8649.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Modification of Asn374 of nsP1 Suppresses a Sindbis Virus nsP4 Minus-Strand Polymerase Mutant

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

Department of Microbiology and Immunology, Medical College of Ohio, Toledo, Ohio 43614

Received 21 March 2002/ Accepted 29 May 2002

Our recent study (C. L. Fata, S. G. Sawicki, and D. L. Sawicki, J. Virol. 76:8632-8640, 2002) found minus-strand synthesis to be temperature sensitive in vertebrate and invertebrate cells when the Arg183 residue of the Sindbis virus nsP4 polymerase was changed to Ser, Ala, or Lys. Here we report the results of studies identifying an interacting partner of the region of the viral polymerase containing Arg183 that suppresses the Ser183 codon mutation. Large-plaque revertants were observed readily following growth of the nsP4 Ser183 mutant at 40°C. Fifteen revertants were characterized, and all had a mutation in the Asn374 codon of nsP1 that changed it to either a His or an Ile codon. When combined with nsP4 Ser183, substitution of either His374 or Ile374 for Asn374 restored wild-type growth in chicken embryo fibroblast (CEF) cells at 40°C. In Aedes albopictus cells at 34.5°C, neither nsP1 substitution suppressed the nsP4 Ser183 defect in minus-strand synthesis. This argued that the nsP4 Arg183 residue itself is needed for minus-strand replicase assembly or function in the mosquito environment. The nsP1 His374 suppressor when combined with the wild-type nsP4 gave greater than wild-type levels of viral RNA synthesis in CEF cells at 40°C (~140%) and in Aedes cells at 34.5°C (200%). Virus producing nsP1 His374 and wild-type nsP4 Arg183 made more minus strands during the early period of infection and before minus-strand synthesis ceased at about 4 h postinfection. Shirako et al. (Y. Shirako, E. G. Strauss, and J. H. Strauss, Virology 276:148-160, 2000) identified amino acid substitutions in nsP1 and nsP4 that suppressed mutations that changed the N-terminal Tyr of nsP4. The nsP4 N-terminal mutants were defective also in minus-strand synthesis. Our study implicates an interaction between another conserved nsP1 region and an internal region, predicted to be in the finger domain, of nsP4 for the formation or activity of the minus-strand polymerase. Finally, the observation that a single point mutation in nsP1 results in minus-strand synthesis at greater than wild-type levels supports the concept that the wild-type nsP sequences are evolutionary compromises.

* 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. 8641-8649, Vol. 76, No. 17
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.17.8641-8649.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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