POLYPEPTIDE REQUIREMENTS FOR ASSEMBLY OF FUNCTIONAL SINDBIS VIRUS-REPLICATION COMPLEXES - A MODEL FOR THE TEMPORAL REGULATION OF MINUS-STRAND AND PLUS-STRAND RNA-SYNTHESIS

Proteolytic processing of the Sindbis virus non-structural polyprotein s (P123 and P1234) and synthesis of minus-and plus-strand RNAs are hig hly regulated during virus infection. Although their precise roles hav e not been defined, these polyproteins, processing intermediates or ma ture cleavage products (nsP1-4) are believed to be essential component s of viral replication and transcription complexes. In this study, we have shown that nsP4 can function as the polymerase for both minus- an d plus-strand RNA synthesis. Mutations inactivating the nsP2 proteinas e, resulting in uncleaved P123, led to enhanced accumulation of minus- strand RNAs and reduced accumulation of genomic and subgenomic plus-st rand RNAs. In contrast, no RNA synthesis was observed with a mutation which increased the efficiency of P123 processing. Inclusion of this m utation in a P123 polyprotein with cleavage sites 1/2 and 2/3 blocked allowed synthesis of both minus- and plus-strand RNAs. We conclude tha t nsP4 and uncleaved P123 normally function as the minus-strand replic ation complex, and propose that processing of P123 switches the templa te preference of the complex to minus-strands, resulting in efficient synthesis of plus-strand genomic and subgenomic RNAs and shut-off of m inus-strand RNA synthesis.