GENETIC-ANALYSIS OF THE NSP3 REGION OF SINDBIS VIRUS - EVIDENCE FOR ROLES IN MINUS-STRAND AND SUBGENOMIC RNA-SYNTHESIS

Sindbis virus nonstructural polyproteins and their cleavage products a re believed to be essential components of viral RNA replication and tr anscription complexes. Although numerous studies have investigated the effect of mutations in nsP1-, nsP2-, and nsP4-coding regions on Sindb is virus-specific RNA synthesis, relatively little is known about the function of the region encoding nsP3. nsP3 is a phosphoprotein compris ing two regions: an N-terminal portion which is highly conserved among alphaviruses and a C-terminal portion which is not conserved, varying both in sequence and in length. We have constructed a library of rand om linker insertion mutations in the nsP3-coding region and characteri zed selected viable mutants. Initially, 126 mutants containing inserti ons in the conserved region and 23 with insertions in the nonconserved region were screened for temperature-sensitive (ts) plaque formation or for significant differences in plaque morphology. All nonconserved- region mutants were similar to the parental virus, whereas 13 of those in the conserved region were either ts or exhibited altered plaque ph enotypes. Ten of these 13 mutants were ts for plaque formation as well as RNA accumulation at 40 degrees C. Highly ts mutants CR3.36 and CR3 .39 were defective in their ability to synthesize minus-strand RNAs at the nonpermissive temperature. The CR3.36 and CR3.39 insertion mutati ons localized to different regions near nsP3 residues 58 and 226, resp ectively. CR3.39 was able to complement ts mutants from Sindbis virus complementation groups A, B, F, and G. Another mutant isolated from th e library, CR3.34, while not ts for plaque formation or RNA synthesis, formed smaller plaques and was defective in subgenomic RNA synthesis at all temperatures examined. These results suggest a role for nsP3 or nsP3-containing polyproteins in the synthesis of viral minus-strand a nd subgenomic RNAs.