Mutations in conserved domain II of the large (L) subunit of the Sendai virus RNA polymerase abolish RNA synthesis

The large (L) protein of Sendai virus complexes with the phosphoprotein (P) to form the active RNA-dependent RNA polymerase. The I,protein is believed to be responsible for all of the catalytic activities of the polymerase as sociated with transcription and replication. Sequence alignment of the L pr oteins of negative-strand RNA viruses has revealed six conserved domains (I -VI) thought to be responsible for the enzymatic activities. Charged-to-ala nine mutagenesis was carried out in a highly charged, conserved region (ami no acids 533-569) within domain II to test the hypothesis of Muller et al. [J. Gen. Virol. 75, 1345-1352 (1994)] that this region may contribute to th e template binding domain of the viral RNA polymerase. The mutant proteins were tested for expression and stability, the ability to synthesize viral R NA in vitro and in vivo, and protein-protein interactions. Five of the seve n mutants were completely defective in all Viral RNA synthesis, whereas two mutants showed significant levels of both mRNA and leader RNA synthesis. O ne of the transcriptionally active mutants also gave genome replication in vitro although not in vivo. The other mutant was defective in all the repli cation assays and thus the mutation uncoupled transcription and replication . Because the completely inactive L mutants can bind to the P protein to fo rm the polymerase complex and the polymerases bind to the viral nucleocapsi d template, these amino acids are essential for the activity of the L prote in, (C) 1999 Academic Press.