Stabilities of Avian Sarcoma Virus RNAs: Comparison of Subgenomic and Genomic Species with Cellular mRNAs

Abstract

The stabilities of B77 avian sarcoma virus intracellular RNA were compared to the stability of the total cellular poly(A)-containing RNA by labeling infected chicken embryo fibroblasts with [3H]uridine for 15 h, adding actinomycin D (1 .mu.g/ml) to block further transcription of viral RNA, and selecting virus-specific RNA from the total cellular poly(A)-containing RNA at 3 hourly intervals. The 3 virus-specific RNA species (9.3, 3.3 and 5.4 kilobases [kb]) decayed with half-lives of 7.5, 10 and 15 h, respectively, whereas the bulk of the cellular mRNA decayed with a half-life of 13 h. To correlate these decay rates with the disappearance of mRNA activities, the actinomycin D-treated cells were pulse-labeled with [3H]leucine at 3 hourly intervals after the addition of the drug and virus-specific protein synthesis was assayed by immunoprecipitation. The mRNA activity for the precursor to the non-glycosylated viral structural proteins (Pr76gag) decayed with a half-life of .apprx. 6 h; the mRNA activity coding for the precursor to the envelope proteins (gPr92env) decayed with a half-life of 14 h. Thus, the rate of decay of the individual mRNA species corresponded reasonably well with the decay rate for the synthesis of 2 of the corresponding gene products. The results indicated that the 6.4 kb env mNA is more stable under these conditions than the 9.3 kb gag mRNA but was not significantly more stable than the bulk of the cellular mRNA. Virus particle production following the addition of actinomycin D was determined by the reverse transcriptase assay and by the incorporation of viral genomic 70S RNA into extracellular virions. Both assays yielded similar results and indicated that particle production was inhibited at a rate (t1/2 = 4 h) somewhat faster than the decay of Pr76gag synthesis or the disappearance of 9.3 kb RNA. It was established by 2 independent methods (pulse and chase, and approach to isotope equilibrium), however, that the intracellular half-life of the RNA that is packaged into virions is 6-7 h. Thus, these results suggest that a single metabolic pool of 9.3 kb RNA exists in avian sarcoma virus-infected cells and is used both as mRNA and as genome RNA.