Mutations in domain V of the sendai virus L polymerase protein uncouple transcription and replication and differentially affect replication in vitro and in vivo

The Sendai virus L and P proteins comprise the viral RNA-dependent RNA poly merase. The L subunit is thought to be responsible for all the catalytic ac tivities necessary for viral RNA synthesis. Sequence alignment of the L pro teins of negative-stranded RNA viruses revealed six regions of good conserv ation, domains I-VI, which are thought to correspond to functional domains of the protein. Domain V, amino acids 1129-1378, has no recognizable motifs , and to date its function is unknown. Site-directed mutagenesis was used t o construct mutations across domain V. The mutant L proteins were all stabl y expressed and were tested for activity in several aspects of RNA synthesi s. One set of mutants could synthesize more le+ RNA than mRNA, while two mu tants showed the opposite phenotype, synthesizing more mRNA than le+ RNA. T he majority of the mutants could synthesize mRNA, but not genome RNA in vit ro, thus uncoupling transcription and replication. Several mutants could re plicate in vivo, but not in vitro, at nearly wildtype L levels, suggesting the importance of the intact host cell for replication in some instances. O ne L mutant, SS24, was virtually inactive in all viral RNA synthesis. SS24 L was able to form a polymerase complex that recognized the nucleocapsid te mplate, and thus these amino acids are essential for the initiation of RNA synthesis, (C) 2000 Academic Press.