The synthesis and processing of B77 avian sarcoma virus RNA in infected chicken embryo fibroblasts was followed in the presence and absence of cycloleucine, a competitive inhibitor of the synthesis of S-adenosylmethionine and thus an inhibitor of RNA methylations. An increase in the steady-state levels of genomelength RNA and a decrease in the steady-state levels of subgenomic RNA molecules were obtained in the S-adenosylmethionine-depleted avian sarcoma virus-infected cells after 24 h of treatment with the inhibitor. The total number of virus-specific RNA molecules per cell, however, remained relatively constant under either condition. The production of newly synthesized virus-specific RNA in cycloleucine-treated and untreated cells infected with a transformation-defective strain of B77 avian sarcoma virus was followed as a function of [3H]uridine labeling time. The accumulation of radioactive genome-length 8.4-kilobase (kb) RNA continued in cycloleucine-treated cells, and virus particle production proceeded at normal rates as previously shown by incorporation of labeled nucleoside precursors or amino acids. In contrast, newly synthesized 3.5-kb subgenomic mRNA, the putative mRNA for the envelope protein precursor, failed to accumulate in the treated cells. The extent of the inhibition in the appearance of the radioactive 3.5-kb RNA was correlated with the extent of the inhibition of viral genomic and cellular mRNA methylations and was a function of the cycloleucine concentration. Under conditions in which the accumulation of 3.5-kb envelope protein mRNA was blocked by the cycloleucine treatment, there were significant increases in the rate of synthesis of the polypeptide products of the genome-length RNA, the precursors to the non-glycosylated gag proteins (Pr76eag), and the reverse transcriptase (Pr 180gag P'l) relative to the rate of synthesis of the envelope protein precursor (gPr 92en). These results suggest that there is an S-adenosylmethionine requirement for the splicing, but not for the synthesis, packaging, or messenger function, of avian retrovirus genome-length RNA. Possible reasons for this requirement are discussed.

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We have previously shown that the inhibition of methylation reactions by the treatment of B77 avian sarcoma virus-infected cells with medium containing cycloleucine results in an inhibition in the intracellular accumulation of the spliced subgenomic mRNA for the virion envelope protein precursor, whereas the genome-size RNA accumulates in larger than normal amounts (C. M. Stoltzfus and R. W. Dane, J. Virol. 42:918-931, 1982). To measure the production of virus particles, we have now determined the reverse transcriptase activity in the culture fluid from infected cells treated with various concentrations of cycloleucine. The activity was somewhat greater in the fluid from the cycloleucine-treated cells than it was in the fluid from the control cells, suggesting an enhancement of particle production in the presence of cycloleucine. In contrast, the production of infectious virions, as determined by the focus assay, decreased when the cycloleucine concentration of the medium increased. We determined the polypeptide compositions of purified particles produced from infected cells treated with or without cycloleucine and labeled with [3H]leucine. The relative amounts of radioactivity associated with p19 and p27 were approximately the same in all of the preparations. In contrast, significant decreases were observed in the relative amounts of [3H]leucine radioactivity associated with the virion glycoproteins gp85 and gp37. The extent of the decrease in the ratio of gp85 to p27 was a function of the cycloleucine concentration and correlated well with the decrease in the infectivity of the virus particles. Therefore, it is probable that the observed reduction of specific infectivity results from the reduced amounts of envelope glycoproteins in the particles budding from cycloleucine-treated cells.We have previously shown that the treatment of avian sarcoma and leukosis virus-infected chicken embryo fibroblasts with cycloleucine results in the inhibition of some mRNA methylations (N-6 methyladenosine and 2'-O-methylribose) and little or no inhibition of other mRNA methylations (7-methylguanosine) (1). Under conditions where these methylations are inhibited more than 90%, virus particle production continues for extended periods of time (1; see below). We have also recently shown that the accumulation of spliced subgenomic mRNA is inhibited in cycloleucine-treated cells. This results in a decreased rate of synthesis of the envelope protein precursor gPr92env. In contrast, the accumulation of genome-size RNA continues in the treated cells, and the syntheses of the non-glycosylated internal proteins (Pr76sas, p27, etc.) as well as the presumptive reverse transcriptase precursor Pr1809ag9P pro-ceed at increased rates (6). As a working hypothesis, we have suggested that this results from the inhibition of the splicing of genome size RNA to form env mRNA, perhaps owing to the absence of specific methylations of the viral RNA (6).We have previously reported decreases in the rate of infectious virion production after a 12-to-24-h ex...