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

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Abstract
The synthesis of viral RNA by wild-type vesicular stomatitis virus (L1VSV) and a small, plaque-size mutant (S2VSV) 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 L1 or S2VSV at low multiplicity was of the same size classes, 12 to 15S, 28S, and 38S. The major differences were in the proportion of RNA produced of each size class. L1VSV always synthesized larger proportions of 38S RNA, and S2VSV produced larger proportions of 12 to 15S RNA. Both S2 and L1VSV exhibited RNA transcriptase activity in vitro and in cell culture. The products of the in vitro reaction were the same, 12 to 15S 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 28S and 12 to 15S 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 S2 and L1VSV to initiate and maintain the normal rate of viral RNA synthesis. However, the overall rate of RNA synthesis in L1VSV infections became independent of protein synthesis after 2 h whereas the rate in S2VSV infections did not. With either virus, synthesis of 38S RNA did not occur in the absence of protein synthesis. Moreover, continuous 38S RNA production required continuous protein synthesis. Production of 38S RNA ceased within 30 min after addition of cycloheximide to S2− or L1VSV-infected CE or L cells that had already begun to synthesize the 38S form. The cycloheximide-induced cessation of 38S RNA synthesis was accompanied by a marked increase in production of 12 to 15S and 28S RNA in L1VSV-infected cells, but no increase in synthesis of small RNA species occurred in S2VSV-infected cells.