REGULATION OF ALPHAVIRUS 26S MESSENGER-RNA TRANSCRIPTION BY REPLICASECOMPONENT NSP2

Semliki Forest virus (SFV) mutant ts4 has a reversible temperature-sen sitive defect in the synthesis the subgenomic 26S mRNA. The viral nons tructural protein nsP2 was identified as a regulator of 26S synthesis by transferring nsP2 coding sequences from ts4 into the infectious SFV cDNA clone (SFoto) to create SFots4. Sequencing identified the causal mutation as C4038U, predicting the amino acid change M781T in nsP2. A revertant was isolated in which a back mutation of U to C restored th e wild-type phenotype. Compared to Sindbis virus nsP2 mutants ts15, ts 17, ts18, ts24 and ts133, which also exhibit temperature-sensitive 26S RNA synthesis, ts4 and SFots4 reduced 26S RNA synthesis faster and to lower levels after temperature shift. Under these conditions, ts4 and SFots4 also displayed complete conversion of RFII + RFI II into RFI a nd reactivated minus-strand synthesis, After shift to 39 degrees C, ts 4 nsP2 was released from a crude RNA polymerase preparation consisting of membranes sedimenting at 15000g (P15) and the remaining, unrelease d nsP2 was capable of being cross-linked in almost equimolar ratio wit h nsP1: and nsP3. This supports the hypothesis that nsP3 binds directl y or undirectly to the promoter for 26S RNA and that it is also an ess ential component of the viral replicase synthesizing 42S RNA plus stra nds, Only the former activity is temperature-sensitive in ts4 mutant.