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 polyproteins (P123 and P1234) and synthesis of minus-and plus-strand RNAs are highly regulated during virus infection. Although their precise roles have not been defined, these polyproteins, processing intermediates or mature cleavage products (nsP1-4) are believed to be essential components of viral replication and transcription complexes. In this study, we have shown that nsP4 can function as the polymerase for both minus- and plus-strand RNA synthesis. Mutations inactivating the nsP2 proteinase, resulting in uncleaved P123, led to enhanced accumulation of minus-strand RNAs and reduced accumulation of genomic and subgenomic plus-strand RNAs. In contrast, no RNA synthesis was observed with a mutation which increased the efficiency of P123 processing. Inclusion of this mutation in a P123 polyprotein with cleavage sites 1/2 and 2/3 blocked allowed synthesis of both minus- and plus-strand RNAs. We conclude that nsP4 and uncleaved P123 normally function as the minus-strand replication complex, and propose that processing of P123 switches the template preference of the complex to minus-strands, resulting in efficient synthesis of plus-strand genomic and subgenomic RNAs and shut-off of minus-strand RNA synthesis.