IDENTIFICATION OF RETROVIRUS MATRIX PROTEINS BY LIPID-PROTEIN CROSS-LINKING

Dimethyl suberimidate and its analogs are symmetrical bifunctional reagents that form amidine linkages with primary amino groups. These reagents were used previously to study nearest neighbor relationships of proteins in viruses and in complex structures such as ribosomes. Dimethyl suberimidate also reacts with phosphatidylethanolamine, which can be radioactively labeled specifically with [14C]ethanolamine. When enveloped viruses containing radioactive phosphatidylethanolamine are exposed to the diimido ester, a fraction of the radioactivity becomes linked to viral structural proteins. Upon separation by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, the lipid-protein complexes can be visualized on fluorograms of the gels. The cross-linking of lipids to proteins is specific, since it requires the viral structure to be intact, and since only certain proteins become chemically linked to phosphatidylethanolamine even though all the proteins react with dimethyl suberimidate. In vesicular stomatitis virus, the structure of which was well characterized, only the glycoprotein and the matrix protein become linked to lipid. This is consistent with their known locations protruding outwards and inwards from the virus membrane, respectively. Thus, the cross-linking technique can be used to identify proteins in close proximity to the lipid bilayer. In the avian leukemia and sarcoma viruses the protein designated p19, and in the murine leukemia viruses, p15, become linked to radioactive lipid. Since avian p19 and murine p15 are internal structural proteins, apparently they are equivalent to the matrix protein defined for other enveloped viruses.