ALPHAVIRUS POSITIVE AND NEGATIVE STRAND RNA-SYNTHESIS AND THE ROLE OFPOLYPROTEINS IN FORMATION OF VIRAL REPLICATION COMPLEXES

The genome of alphaviruses is translated into polyproteins that are pr ocessed into a viral replicase that produces both negative and positiv e strands. In infected cells, negative strand synthesis is short-lived and occurs only early, whereas positive strand synthesis is stable an d occurs both early and late. Analysis of temperature sensitive mutant s 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 stran ds the preferred templates. While nsP4 and nsP1 individually rescued e arly defects in transcription, nsP2 and nsP3 acted initially in cis. W e interpret our results to suggest nsP1234 was cleaved to nsP4, nsP1 a nd nsP23, bound a positive strand and synthesized a negative strand. C leavage of P23 or other modifications to nsP2 and nsP4 convert the ini tial transcription complex to a stable complex that synthesizes positi ve strands. Negative strand synthesis is unstable because of the failu re to form initial transcription complexes after host factors that are part of the replicase are depleted or the half-life of polyprotein pr ecursors like P23 is shortened.