Comparative immunofluorescence of murine leukemia virus-derived membrane-associated antigens.

We recently reported that the distribution and location of Moloney murine leukemia virus-derived membrane-associated gp70 and p30 antigens on the surface of 3.7% formaldehyde-fixed, chronically infected mouse fibroblasts were completely distinct, as judged by immunofluorescent light microscopy (M. Satake, P. N. McMillan, and R. B. Luftig (1981), Proc. Nat. Acad. Sci. USA 78, 6266). gp70, one of the two env gene products, exhibited a multiple-dot fluorescent pattern on the external surface of infected cells, while p30, one of the gag gene products, exhibited a diffuse fluorescence pattern which was apparently derived from Pr65gag molecules associated with the cytoplasmic face of the cell membrane. We have now examined the membrane fluorescence patterns of p15E, the other env gene product, as well as p15, p12, and p10, the other gag gene products. In these studies, both multivalent and monoclonal antibodies as well as fluorescein- and rhodamine-conjugated probes were used. We found that: (i) each of the env gene products, gp70 and p15E, exhibited characteristic and distinctive multiple-dot staining patterns. Further, each protein was labeled on intact cells with 125I-protein A plus homologous antiserum, confirming that both gp70 and p15E had externally exposed antigenic determinants. (ii) Among the gag gene products, p15 exhibited a different membrane fluorescence pattern than the diffuse pattern seen with p30, p12, and p10. The p15 pattern had an additional multiple-dot component. (iii) By double immunofluorescence we observed that the p15E and p15 multiple-dot patterns were superimposable at the same loci on infected cells. These three results suggest, first, that the cleavage of gp70 and p15E occurs prior to the arrival of the env polyprotein precursor at the cell surface and, second, there is an association between p15E and p15 antigenic determinants at the cell membrane. This latter association between an env and a gag gene product may be important for viral assembly.