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.