ALPHAVIRUS NSP3 FUNCTIONS TO FORM REPLICATION COMPLEXES TRANSCRIBING NEGATIVE-STRAND RNA

The alphavirus mutant Sindbis virus HR ts4, which has been assigned to the A complementation group, possessed a selective defect in negative -strand synthesis that was similar although not identical to that obse rved for the B complementation group mutant tsll (Y.-F. Wang, S. G. Sa wicki, and D. L. Sawicki, J. Virol. 65:985-988, 1991). The causal muta tion was identified as a change of a C to a U residue at nucleotide 49 03 in the nsP3 open reading frame that predicted a change of Ala-268 t o Val. Thus, both nsP3 and nsP1 play a role selectively in the transcr iption of negative strands early in infection. The assignment of the m utation carried by an A complementation group mutant of Sindbis virus HR to nsP3 was unexpected, as mutations in other A complementation gro up mutants studied to date mapped to nsP2. Another mutant with a condi tionally lethal mutation, ts7 of the G complementation group, also pos sessed a causal mutation resulting from a single-residue change in nsP 3. Negative-strand synthesis ceased more slowly after a shift to the n onpermissive temperature in ts7- than in ts4-infected cells, and ts7 c omplemented ts11, but ts4 did not. However, the nsP3 of both ts4 and t s7 allowed reactivation of negative-strand synthesis by stable replica tion complexes containing nsP4 from ts24. Therefore, mutations in nsP3 affected only early events in replication and probably prevent the fo rmation and/or function of the initial replication complex that synthe sizes its negative strand template. Because neither ts4 nor ts7 comple mented 10 A complementation group mutants, the genes for nsP2 and nsP3 function initially as a single cistron. We interpret these findings a nd present a model to suggest that the initial alphavirus replication complex is formed from tightly associated nsP2 and nsP3, perhaps in th e form of P23, and proteolytically processed and trans-active nsP4 and nsP1.