Synthesis of reovirus-specific polypeptides in cells pretreated with cycloheximide

When L cells are infected with reovirus in the presence of cycloheximide neither virus-specific polypeptides nor viral double-stranded RNA are synthesized. There is some synthesis of viral single-stranded RNA, transcribed mainly from segments L1, M3, S3, and S4 of the 10 viral genomic segments, and in previous work this has been termed the early mRNA pattern. In an attempt to determine whether these early transcripts are functional mRNA's, the transcripts were allowed to accumulate for a period of 17.5 h at 31 C in cycloheximide-treated cells. The cycloheximide was removed and the cells were exposed for various periods to radioactive amino acids to label any virus-specific polypeptides that might be synthesized. An immunoprecipitation technique was used to separate the viral polypeptides from cellular extracts and this precipitate was then analyzed on sodium dodecyl sulfate-polyacrylamide gels. Within 30 min of cycloheximide removal, four major polypeptides (lambda2, mu0, sigma2a, and sigma3) and two minor polypeptides (lambda1 and mu2) were found. In infected cells without cycloheximide eight viral polypeptides (lambda1, lambda2, mu0, mu2, sigma1, sigma2, sigma2a, sigma3) were found at 17.5 h after infection and the same pattern was found between 3 to 4 h after removal of cycloheximide which had been present for 17.5 h after infection. The latter result shows that the cycloheximide inhibition is reversible and that the cells readily recovered and synthesized the normal complement of viral polypeptides. In one set of experiments cordycepin was added to infected cells immediately after the removal of cycloheximide at 17.5 h to inhibit the synthesis of new viral transcripts. During the succeeding 4 h in the presence of cordycepin, the pattern of protein synthesis was the same as that obtained during the 30 min after cycloheximide removal. It is concluded that the polypeptides formed right after removal of cycloheximide are the translation products of transcripts accumulated during cycloheximide treatment and, therefore, that these transcripts are functional viral mRNA's.