Intragenic complementation and oligomerization of the L subunit of the sendai virus RNA polymerase.
The RNA-dependent RNA polymerase of Sendai virus consists of two subunits, the L and P proteins, where L is thought to be responsible for all the catalytic activities necessary for viral RNA synthesis. Sequence alignment of the L proteins of a variety of negative-stranded RNA viruses revealed six regions of good conservation, designated domains I-VI, which are thought to correspond to functional domains of the protein. Analysis of a number of site-directed mutants within the six domains of L allowed us to conclude that the activities of the polymerase are not simply compartmentalized and that each domain contributes to multiple steps in viral RNA synthesis. Nevertheless these domains can function in trans since we demonstrate here that intragenic complementation between pairs of coexpressed inactive L mutants can restore viral RNA synthesis on an added template. Although intragenic complementation is typically very inefficient, complementation to restore leader RNA synthesis was surprisingly very efficient for some pairs and complementation of mRNA synthesis and genome replication was less, but still significant. Complementation occurred with L mutants in five of the six domains, the exception being a domain III mutant, and required the cotranslation of the two L mutants. C-terminal truncations deleting up to half of L were capable of restoring transcription of an inactive domain I L mutant at amino acid 379. Oligomerization of L in the polymerase complex was demonstrated directly by the co-immunoprecipitation of differentially epitope-tagged full-length and truncated L proteins. These data are consistent with L protein being an oligomer with multiple independent domains each of which exhibits several functions.
Smallwood
Sherin
S
Department of Molecular Genetics and Microbiology, University of Florida College of Medicine, Gainesville 32610, USA.
Cevik
Bayram
B
Moyer
Sue A
SA
D006801:Humans; D014176:Protein Biosynthesis; D021122:Protein Subunits; D012367:RNA, Viral / Q000096:biosynthesis; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry* / Q000502:physiology; D029082:Sendai virus / Q000201:enzymology / Q000235:genetics*; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured; D014779:Virus Replication
D021122:Protein Subunits; D012367:RNA, Viral; D012324:RNA-Dependent RNA Polymerase