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 polymerase. The L subunit is thought to be responsible for all the catalytic activities necessary for viral RNA synthesis. Sequence alignment of the L proteins of negative-stranded RNA viruses revealed six regions of good conservation, 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 to construct mutations across domain V. The mutant L proteins were all stably expressed and were tested for activity in several aspects of RNA synthesis. One set of mutants could synthesize more le+ RNA than mRNA, while two mutants showed the opposite phenotype, synthesizing more mRNA than le+ RNA. The majority of the mutants could synthesize mRNA, but not genome RNA in vitro, thus uncoupling transcription and replication. Several mutants could replicate in vivo, but not in vitro, at nearly wildtype L levels, suggesting the importance of the intact host cell for replication in some instances. One L mutant, SS24, was virtually inactive in all viral RNA synthesis. SS24 L was able to form a polymerase complex that recognized the nucleocapsid template, and thus these amino acids are essential for the initiation of RNA synthesis.
Cortese
C K
CK
Department of Molecular Genetics and Microbiology, University of Florida College of Medicine, Gainesville, Florida 32610, USA.
Feller
J A
JA
Moyer
S A
SA
D012321:DNA-Directed RNA Polymerases; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010750:Phosphoproteins / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis; D012367:RNA, Viral / Q000096:biosynthesis; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics* / Q000378:metabolism; D018108:Respirovirus / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism; D014779:Virus Replication
C055859:P protein, Sendai virus; D010750:Phosphoproteins; D012333:RNA, Messenger; D012367:RNA, Viral; D014764:Viral Proteins; C064365:L protein, Sendai virus; D012324:RNA-Dependent RNA Polymerase; D012321:DNA-Directed RNA Polymerases