Macromolecular synthesis in cells infected by frog virus 3 IX. Two temporal classes of early viral RNA

Abstract

By selectively inhibiting functional viral protein synthesis with cycloheximide or fluorophenylalanine (FPA) and analyzing the resultant viral RNAs by polyacrylamide gel electrophoresis and hybridization, we have shown that early frog virus 3 (FV3) RNA can be subdivided into two sequentially synthesized classes. The polyacrylamide gel pattern of viral RNA synthesized 6 hr postinfection (p.i.) in cells treated with FPA was identical to that of the RNA synthesized at 1 hr p.i. in the absence of drug. RNA synthesized at 6 hr p.i. in the presence of FPA hybridized to 36% of the viral genome, whereas RNA synthesized 6 hr p.i. in its absence contained many additional species and hybridized to 50% of the genome. Therefore, the FPA RNAs appeared to represent the same sequences as the early viral RNA. In contrast, viral RNAs synthesized when protein synthesis was inhibited by cycloheximide did not contain all of the species present at 1 hr p.i. or in FPA RNA. The cycloheximide RNAs, which hybridized to only 16% of the genome, comprised a subset of the FPA RNAs. One of the viral RNA species was synthesized at a very high rate in cells exposed to cycloheximide, but at a much lower rate during infection in the presence or absence of FPA. When we delayed the addition of cycloheximide until 1 or 2 hr p.i., all of the remaining early viral RNA species were synthesized and the rate of synthesis of the overproduced RNA decreased. We could, therefore, divide FV3 early RNA synthesis into two temporal classes: “immediate early,” which took place in the absence of protein synthesis (cycloheximide), and “delayed early,” which occurred under conditions of restricted protein synthesis (FPA). In addition, our data suggest that at least one species of immediate early viral RNA was subject to negative transcriptional control.