SUMMARYThe initial env gene polyproteins present in Moloney murine leukaemia virus (M-MuLV)-infected NIH/3T3 cells were examined to determine their relationship to each other as well as their role in generating env gene products gp70, pl5(E) and p l2(E). Steady-state labelling with [3H]glucosamine revealed anti-gp69/71 immunoprecipitable proteins of mol. wt. 93000 (gPr93env), 83000 (gPr83 env) and 70000 (gp70), whereas similar labelling with [3H]fucose showed only two bands of anti-gp69/71 immunoprecipitable radioactivity migrating in SDS-polyacrylamide gels with gPr93 env and gp70. Pulse-chase experiments employing [3H]leucine labelling instead of labelled sugars failed to detect gPr93 e"v using similar techniques. The gPr83 env was the only polypeptide detected in 15 min [3H]leucine pulselabeUings, whereas gPr83 env, gp70, pl5(E) and pl2(E) were detected in chase experiments using appropriate antisera in immunoprecipitation experiments. Pretreatment of infected cells with tunicamycin, an inhibitor of glycosylation, allowed the synthesis of a major band at mol. wt. 62000 (Pr62 env) and a minor band of 73 000 mol. wt. at the expense of gPr83 env. In pulse-chase experiments conducted in the presence of tunicamycin, Pr62 e"v increased during the early chase period but disappeared during the later stages of the chase. No product of Pr62 env was detected. Cation-exchange chromatography of tryptic digests of radioactive tyrosine-labelled gPr83 env, Pr62 env and gp70 showed sequence relationships among the three proteins.Comparison of the two-dimensional fingerprints of [3H]leucine-labelled gPr83 env and the mature proteins gp70 and pl2(E) support their precursor-product relationship. Of interest is the observation that gp70 and pl2(E) seemed to share a few leucine-containing tryptic peptides. These results provide strong evidence that gPr83 ear is the primary product of the env gene which, upon tunicamycin treatment, is synthesized as a subglycosylated protein, Pr62 e"v. It appears that gPr83 env undergoes further modification of its core oligosaccharide structure as detected by fucosylation to yield gPr93 env. Our inability to detect gPr93 e"v by [3H]leucine labeUings suggests a close chronological relationship between fucosylation and cleavage of the precursor polyprotein, suggesting that cleavage of gPr93 env yields gp70 and pl5(E). The latter is further cleaved to yield pl2(E) plus a polypeptide containing the C-terminal end of p 15 (E).