L protein requirement for in vitro RNA synthesis by vesicular stomatitis virus.
The endogenous transcriptase present in purified vesicular stomatitis (VS) virions was solubilized with a Triton X-100 high-salt solution. The polymerase activity was purified on glycerol gradients and by phosphocellulose column chromatography; the viral proteins present in the active enzyme fractions were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis. It was demonstrated that L protein, but not NS protein, was required for in vitro RNA synthesis on the VS viral nucleocapsid template. Solubilized L protein rebinds to the ribonucleoprotein template when the transcription complex is reconstituted, and the RNA synthesized in vitro by purified L protein hybridizes to virion RNA. Cyanogen bromide peptide fingerprints indicate that the large L protein is a unique polypeptide chain. It is concluded that the L protein functions as the transcriptase, and the nucleocapsid NS protein is not essential for in vitro RNA synthesis.
D002250:Carbon Radioisotopes; D002474:Cell-Free System; D002499:Centrifugation, Density Gradient; D002845:Chromatography; D012321:DNA-Directed RNA Polymerases / Q000032:analysis / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005990:Glycerol; D008715:Methionine / Q000378:metabolism; D009693:Nucleic Acid Hybridization; D009698:Nucleoproteins; D010455:Peptides / Q000032:analysis; D012367:RNA, Viral / Q000096:biosynthesis*; D012995:Solubility; D013501:Surface-Active Agents; D013698:Templates, Genetic; D014158:Transcription, Genetic; D014316:Tritium; D014500:Uracil Nucleotides / Q000378:metabolism; D014721:Vesicular stomatitis Indiana virus / Q000032:analysis / Q000201:enzymology / Q000378:metabolism*; D014764:Viral Proteins / Q000032:analysis / Q000378:metabolism*
D002250:Carbon Radioisotopes; D009698:Nucleoproteins; D010455:Peptides; D012367:RNA, Viral; D013501:Surface-Active Agents; D014500:Uracil Nucleotides; D014764:Viral Proteins; D014316:Tritium; D008715:Methionine; D012321:DNA-Directed RNA Polymerases; D005990:Glycerol