RNA Synthesis by Vesicular Stomatitis Virus and a Small Plaque Mutant: Effects of Cycloheximide

Abstract

The synthesis of viral RNA by wild-type vesicular stomatitis virus (L 1 VSV) and a small, plaque-size mutant (S 2 VSV) was studied in vitro and in chicken embryo (CE) and mouse L-cell cultures. Virus-specific RNA synthesized in CE or L cells infected with either L 1 or S 2 VSV at low multiplicity was of the same size classes, 12 to 15 S , 28 S , and 38 S . The major differences were in the proportion of RNA produced of each size class. L 1 VSV always synthesized larger proportions of 38 S RNA, and S 2 VSV produced larger proportions of 12 to 15 S RNA. Both S 2 and L 1 VSV exhibited RNA transcriptase activity in vitro and in cell culture. The products of the in vitro reaction were the same, 12 to 15 S for both. The products of the virion-associated transcriptase in CE or L-cell cultures in the presence of cycloheximide were also the same for both viruses but differed from the in vitro products in that 28 S and 12 to 15 S RNA were made. The effects of addition of cycloheximide at various times after infection demonstrated that new protein synthesis is required early (0-2 h) for both S 2 and L 1 VSV to initiate and maintain the normal rate of viral RNA synthesis. However, the overall rate of RNA synthesis in L 1 VSV infections became independent of protein synthesis after 2 h whereas the rate in S 2 VSV infections did not. With either virus, synthesis of 38 S RNA did not occur in the absence of protein synthesis. Moreover, continuous 38 S RNA production required continuous protein synthesis. Production of 38 S RNA ceased within 30 min after addition of cycloheximide to S 2 − or L 1 VSV-infected CE or L cells that had already begun to synthesize the 38 S form. The cycloheximide-induced cessation of 38 S RNA synthesis was accompanied by a marked increase in production of 12 to 15 S and 28 S RNA in L 1 VSV-infected cells, but no increase in synthesis of small RNA species occurred in S 2 VSV-infected cells.