Both NS and L proteins are required for in vitro RNA synthesis by vesicular stomatitis virus.
Vesicular stomatitis virions, Indiana serotype, were solubilized with high salt solubilizer and separated by ultracentrifugation into a supernatant fraction containing L, G, NS, and M proteins and pellet fraction containing the RNA complexed with N protein. NS protein was purified from the supernatnat fluid by sequential chromatography on phosphocellulose and diethylaminoethyl cellulose columns. The purified NS protein was assayed in a standard transcription system in combination with purified L protein and purified template (pellet fraction) prepared by renografin or CsCl banding. Results of the polymerase assays indicate that both L and NS proteins are required to reconstitute transcription activity with a highly purified template composed of only RNA and N protein. The NS protein polymerase activity is destroyed by trypsin but withstands 90 C temperatures for 10 min. Cytoplasmic NS protein can substitute for virion NS protein in the in vitro transcription assay.
D002474:Cell-Free System; D002499:Centrifugation, Density Gradient; D002852:Chromatography, Ion Exchange; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D003973:Diatrizoate; D004591:Electrophoresis, Polyacrylamide Gel; D006358:Hot Temperature; D009711:Nucleotides / Q000378:metabolism; D012367:RNA, Viral / Q000096:biosynthesis*; D012965:Sodium Chloride; D012995:Solubility; D013698:Templates, Genetic; D014158:Transcription, Genetic; D014316:Tritium; D014357:Trypsin; D014721:Vesicular stomatitis Indiana virus / Q000201:enzymology / Q000378:metabolism*; D014764:Viral Proteins / Q000302:isolation & purification / Q000378:metabolism*
D009711:Nucleotides; D012367:RNA, Viral; D014764:Viral Proteins; D014316:Tritium; D003973:Diatrizoate; D012965:Sodium Chloride; D012321:DNA-Directed RNA Polymerases; D014357:Trypsin