Stabilization of vesicular stomatitis virus L polymerase protein by P protein binding: a small deletion in the C-terminal domain of L abrogates binding.
We showed previously that cells expressing the vesicular stomatitis virus (VSV) L polymerase gene via the vaccinia-T7 RNA polymerase system accumulated 2- to 5-fold more L protein when the P protein was coexpressed (Canter et al., 1993, Virology 194, 518-529). The results presented here provide an explanation for this phenomenon. Pulse-chase analysis revealed that L was unstable with a half-life of 3 to 6 hr if expressed in the absence of P protein, but was stable for at least 16 hr when coexpressed with a 10- to 15-fold molar excess of P. The P protein, in contrast, was stable under both conditions. Stabilization correlated with formation of a P:L polymerase complex evidenced both by coimmunoprecipitation and by glycerol gradient sedimentation analyses. A mutant L protein, lacking amino acids 1638 to 1673, was not stabilized by coexpression and showed no binding to P protein. Its anomalous sedimentation, however, suggested misfolding and/or aggregation as the cause for the failure to bind P. Transcription reconstitution in vitro, using extracts from cells expressing excess of P over L protein, strongly depended on coexpression of the proteins for optimal activity. We propose that the coexpression dependence for polymerase reconstitution documented here for VSV, as well as that reported previously for the Sendai paramyxovirus, reflects the protective effect of P protein on L protein stability.
Canter
D M
DM
Molecular Biology Institute, San Diego State University, California 92182, USA.
Perrault
J
J
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004795:Enzyme Stability; D010750:Phosphoproteins; D011233:Precipitin Tests; D012324:RNA-Dependent RNA Polymerase; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic; D014721:Vesicular stomatitis Indiana virus / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D015678:Viral Structural Proteins / Q000235:genetics / Q000378:metabolism*
C081250:P protein, Vesicular stomatitis virus; D010750:Phosphoproteins; D014764:Viral Proteins; D015678:Viral Structural Proteins; C040530:L protein, vesicular stomatitis virus; D012324:RNA-Dependent RNA Polymerase; D012321:DNA-Directed RNA Polymerases