Alphavirus positive and negative strand RNA synthesis and the role of polyproteins in formation of viral replication complexes.

The genome of alphaviruses is translated into polyproteins that are processed into a viral replicase that produces both negative and positive strands. In infected cells, negative strand synthesis is short-lived and occurs only early, whereas positive strand synthesis is stable and occurs both early and late. Analysis of temperature sensitive mutants indicated: nsP1 functioned in the initiation of transcription; nsP3 acted to form initial transcription complexes; and nsP2 and nsP4 first recognized positive strands as templates and then made negative strands the preferred templates. While nsP4 and nsP1 individually rescued early defects in transcription, nsP2 and nsP3 acted initially in cis. We interpret our results to suggest nsP1234 was cleaved to nsP4, nsP1 and nsP23, bound a positive strand and synthesized a negative strand. Cleavage of P23 or other modifications to nsP2 and nsP4 convert the initial transcription complex to a stable complex that synthesizes positive strands. Negative strand synthesis is unstable because of the failure to form initial transcription complexes after host factors that are part of the replicase are depleted or the half-life of polyprotein precursors like P23 is shortened.