No abstract

SUMMARYThe stabilities of B77 avian sarcoma virus intracellular RNAs were compared to the stability of the total cellular poly(A)-containing RNA by labelling infected chicken embryo fibroblasts with [3H]uridine for 15 h, adding actinomycin D (1 ~tg per 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 three virus-specific RNA species (9-3, 3.3 and 5.4 kilobases) 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-labelled 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 (Pr76g ag) decayed with a half-life of approximately 6 h, whereas the mRNA activity coding for the precursor to the envelope proteins (gPr92 env) 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 two of the corresponding gene products. The results indicated that the 5-4 kb env mRNA 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 (t~ = 4 h) somewhat faster than the decay of Pr769ag synthesis or the disappearance of 9.3 kb RNA. It was established by two independent methods (pulse and chase, and approach to isotope equilibrium), however, that the intracellular halflife of the RNA that is packaged into virions is 6 to 7 h. Tt~us, 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.

The level of RNA transcripts in human foreskin fibroblast (HFF) cells initiated from the avian sarcoma virus (ASV) long terminal repeat (LTR) promoter was stimulated more than 10-fold when the cells were also infected with human cytomegalovirus (HCMV). HCMV was able to stimulate transcription from the ASV LTR promoter even when all the LTR sequence upstream of the TATA box was deleted, suggesting that only the basal LTR promoter is required for the effect. There were no significant changes in the ASV RNA splicing pattern in stimulated and unstimulated HFF cells. The mRNAs showing an increase during HCMV stimulation included aberrantly spliced ASV RNA species as well as unspliced gag-pol, single-spliced env and single-spliced src mRNAs. This pattern was quite different from ASV splicing in chicken embryo fibroblasts (CEF) but typical of that seen in other mammalian cells. A dramatic increase in infectious ASV production from the normally non-permissive HFF was correlated with the increase in amount of ASV RNA in response to HCMV. Thus, there is not an absolute block to ASV production in human cells. However, infectious ASV production was inefficent in HCMV-stimulated HFF compared to that in CEF cells.