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Comparative immunofluorescence of murine leukemia virus-derived membrane-associated antigens
Abstract:
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.
Insights
Moloney murine leukemia virus proteins gp70 and p15E are located externally on infected cells, while gag proteins p30, p12, and p10 are internal. Protein p15 shows both internal and external patterns, suggesting viral assembly associations.
Area of Science:
- Virology
- Cell Biology
- Molecular Biology
Background:
- Moloney murine leukemia virus (MMLV) infection alters cell surface protein expression.
- Previous studies identified distinct surface distributions for MMLV gp70 (env gene product) and p30 (gag gene product).
Purpose of the Study:
- To investigate the membrane localization of other MMLV env (p15E) and gag (p15, p12, p10) gene products.
- To determine potential associations between env and gag gene products at the cell membrane.
Main Methods:
- Immunofluorescence microscopy using multivalent and monoclonal antibodies.
- Fluorescein and rhodamine conjugated probes for labeling.
- 125I-protein A labeling to confirm external antigenic determinants.
- Double immunofluorescence to compare protein localization.
Main Results:
- Both MMLV env proteins, gp70 and p15E, exhibited distinct, externally located multiple-dot staining patterns.
- MMLV gag proteins p30, p12, and p10 showed diffuse fluorescence, indicating internal localization.
- MMLV gag protein p15 displayed a unique pattern with both diffuse and multiple-dot components.
- p15E and p15 multiple-dot patterns were superimposed, suggesting an association at the cell membrane.
Conclusions:
- Cleavage of MMLV env polyprotein precursors (gp70, p15E) occurs before cell surface expression.
- An association exists between MMLV env (p15E) and gag (p15) gene products at the cell membrane.
- This env-gag association may play a role in viral assembly.