Particles of Reduced Infectivity and Deficient in Envelope Glycoproteins Are Produced in Cycloleucine-Treated B77 Avian Sarcoma Virus-Infected Chicken Embryo Fibroblasts

Abstract

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...