Deletion and duplication mutations in the C-terminal nonconserved region of Sindbis virus nsP3: effects on phosphorylation and on virus replication in vertebrate and invertebrate cells.

Little is known concerning the function of the C-terminal nonconserved region of Sindbis virus nsP3. In this report, we created a number of in-frame deletions and duplications (from 12 to 159 residues) in this region and examined their effects on Sindbis virus replication. Sindbis RNA transcripts containing these mutations were infectious and gave rise to viable virus after transfection of chicken embryo fibroblasts (CEF). In CEF, the rate of virus release and accumulation of viral RNAs were similar for the mutants and the parental virus, although larger deletions resulted in decreased total viral RNA synthesis and lower virus yields early in infection. For some mutants, dramatic differences in nsP3 phosphorylation were noted, both in the level of phosphorylation and in the pattern of electrophoretically distinct forms. The two largest deletions resulted in only trace levels of nsP3 phosphorylation, which indicates that highly phosphorylated nsP3 is not necessary for efficient SIN replication in CEF. In the C7-10 mosquito cell line, the largest deletion mutants were defective at initiating a productive infection, generating plaques at only 1-2% the efficiency of the parental virus. However, once infection was established normal virus yields were produced. Thus, although nonessential, the nsP3 nonconserved region may be important for optimal virus replication in diverse host cells. The ability to engineer viable in-frame insertion mutations in this region of the genome provides yet another strategy for expression of heterologous polypeptides and RNAs using alphavirus vectors.