Two-dimensional analysis of murine leukemia virus gag-gene polyproteins.

The processing of gag translational products in a Gross Murine Leukemia virus (MuLV)-induced leukemia (E λ G2) was studied with two-dimensional gel electrophoresis, combining separation based upon charge in the first dimension and separation based upon size in the second dimension. In most experiments, the gag species were compared to the env species; gag species were precipitated from labeled cells or virus with antisera to the virion gag proteins p30 or p10, whereas env species were precipitated from labeled cells or virus with anti-gp70 serum. Three viral proteins were detected on the surface of E λ G2 cells with ¹²⁵I] lactoperoxidase labelings: these included gp70 and two glycosylated gag gene species (gpP95^gag and gpP85^gag). Neuraminidase treatment of ¹²⁵I] lactoperoxidase-labeled cells did not affect the antigenicity of gp70, gpP95^gag, or gpP85^gag. However, the neuraminidase treatment caused gp70, gpP95^gag, and gpP85^gag to migrate as more basic species, indicating that all three glycoproteins contain terminal sialic acid. The cytoplasmic gag-gene products were studied with ³⁵S]methionine labelings of E λ G2 cells; seven relatively stable gag species were identified. In general, none of the gag intermediates were single proteins; rather, each of the species exhibited multiple, specific modifications that resulted in complex yet reproducible patterns in the two-dimensional gel system. The core polyproteins Pr75^gag and Pr65^gag were formed rapidly after pulse-labelings, with Pr65^gag being processed into Pr55^gag involving cleavage of p10. The smaller gag species (Pr45^gag and p30) also appeared to result from processing of Pr65^gag. In contrast, Pr75^gag was directly processed to form gpP95^gag. A protein of approximately 58,000 daltons, designated P58^gag, qualified as a gag species since it was specifically precipitated by anti-p30 serum. However, P58^gag did not appear to be a precursor of p30 since it was long-lived in the cytoplasm. Multiple forms of p30 were precipitated from the cytoplasm and from the virion, with unique forms of p30 present in both the cytoplasm and the virion. Comparisons of the gag species from several AKR leukemias indicated that similar, but not identical gag gene products were present in the various leukemias.