In vitro transcription of vesicular stomatitis virus. Incorporation of deoxyguanosine and deoxycytidine, and formation of deoxyguanosine caps.

Abstract

Detergent-disrupted vesicular stomatitis virus carried out in vitro transcription at a reduced rate in the presence of deoxyguanosine triphosphate. The transcripts annealed completely to excess vesicular stomatitis virus genomic RNA and consistent of the short leader RNA and even polyadenylated messenger RNA. Incubation with RNase T1 showed that the transcripts were resistant to cleavage. Nearest-neighbor analysis demonstrated that approximately two-thirds of the guanosine residues had been replaced by deoxyguanosine. The hybrid "deoxyguanosine-RNAs" carried cap structures which contained deoxyguanosine instead of guanosine. Competition experiments using both guanosine- and deoxyguanosine triphosphates indicated that GpppA and dGpppA cap structures were synthesized in approximately equal amounts at a ratio of 20 microM guanosine triphosphate to 50 microM deoxyguanosine triphosphate. Deoxyguanosine triphosphate was accepted by the polymerases of various strains and serotypes of vesicular stomatitis virus, demonstrating that its incorporation was a common characteristic. Deoxycytidine triphosphate could also substitute for cytidine triphosphate but to a lesser degree. Deoxyadenine, deoxyuridine-, and thymidine triphosphates were not or were very poorly accepted even at concentrations of 2 MM.