Sendai virus wild-type and mutant C proteins show a direct correlation between L polymerase binding and inhibition of viral RNA synthesis

The Sendai virus C proteins, C', C, Y1, and Y2, are a nested set of four in dependently initiated carboxy-coterminal proteins encoded on the P mRNA fro m an alternate reading frame. Together the C proteins have been shown to in hibit viral transcription and replication in vivo and in vitro and C' binds the Sendai virus L protein, the presumed catalytic subunit of the viral RN A polymerase. To identify amino acids within the C' protein that are import ant for binding L, site-directed mutagenesis of the gstC' gene was used to change conserved charged amino acids to alanine, generating nine mutants, A dditionally, a tenth natural mutant, gstF170S, was also constructed. Six of the gstC' mutants, primarily in the C-terminal half of C', exhibited a def ect in the ability to bind L protein. The mutants were assayed for their ef fect on in vitro transcription and replication from the antigenomic promote r, and the data suggest in all but one case a direct correlation between th e ability of C to bind L and to inhibit these steps in RNA synthesis. Furth er studies with two nonfusion C mutants showed that this correlation was sp ecifically due to the C' portion, and not the gst portion, of the fusion pr oteins. To study their individual functions, each of the four C proteins wa s fused downstream of glutathione S-transferase. The gstC', gstC, gstY1, an d gstY1 fusion proteins were all able to bind L protein and to inhibit vira l mRNA and (+)-leader RNA synthesis, and antigenome replication in vitro. I n addition, the nonfusion C, Y1, and Y2 proteins all inhibited transcriptio n, The inhibition of (+)-leader RNA and mRNA synthesis by wt C proteins (no nfusion) showed nearly identical dose-response curves, suggesting that inhi bition occurs early in RNA synthesis. (C) 2001 Academic Press.