title: Mutations in conserved domain I of the Sendai virus L polymerase protein uncouple transcription and replication.
abstract: To begin to map functional domains of the Sendai P-L RNA polymerase complex we wanted to characterize the P binding site on the Sendai L protein. Analysis of in vitro and in vivo P-L polymerase complex formation with carboxyl-truncations of the L protein showed that the N-terminal half of the protein was required. Site-directed mutagenesis of the Sendai virus L gene was employed to change amino acids within a highly conserved region of the N-terminal domain I from amino acids (aa) 348-379 singly or in pairs from the Sendai to the corresponding measles L sequence or to alanine. The mutant L proteins coexpressed with the viral P and NP proteins in mammalian cells were assayed for their ability to form the P-L complex and to synthesize RNA in vitro and showed a variety of defective phenotypes. While most of the mutant L proteins still formed the P-L polymerase complex, a change from serine to arginine at aa 368 and a three-amino-acid insertion at aa 379 virtually abolished both complex formation and RNA synthesis. Changes of aas 370 and 376-377 in the L protein gave only small decreases in viral RNA synthesis. Substitutions at either aas 349-350 or aas 354-355 and a three-amino-acid insertion at aa 348 in the L protein yielded enzymes that catalyzed significant transcription, but were defective in DI RNA replication, thus differentially affecting the two processes. Since DI leader RNA, but not genome RNA, was still synthesized by this class of mutants, the defect in replication appears to be in the ability of the mutant enzyme to package newly synthesized nascent RNA. Single changes at aas 362, 363, and 366 in the L protein gave enzymes with severely decreased overall RNA synthesis, although some leader RNA was synthesized, suggesting that they cannot transcribe or replicate past the leader gene. These studies have identified a region in conserved domain I critical for multiple functions of the Sendai virus L protein.
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D017124:Conserved Sequence; D017931:DNA Primers; D004279:DNA, Viral / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D003673:Defective Viruses / Q000235:genetics; D008459:Measles virus / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D010222:Parainfluenza Virus 1, Human / Q000235:genetics* / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000096:biosynthesis*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013045:Species Specificity; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D017931:DNA Primers; D004279:DNA, Viral; C055859:P protein, Sendai virus; D010750:Phosphoproteins; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C064365:L protein, Sendai virus; D012321:DNA-Directed RNA Polymerases