Figure 4

The tetraspanins CD63, CD81 and CD82 are associated with purified HIV-1 virions. (A). Cell lysate from MOLT/HIV-1 cells was run on SDS-PAGE and probed with antibodies against CD45, CD9, CD63, CD81, CD82, and Lamp2 as indicated ("cells"). Purified viral pellet from MOLT/HIV-1 was immunoblotted with the same antibodies ("virus"). (B). Purified virions produced by MOLT/HIV-1 cells (left panel) were loaded on 20–70% sucrose density gradient. After ultracentrifugation at equilibrium, the gradient was fractionated and the density (g/ml) of each fraction was determined, as indicated. Immunoblots of all fractions were performed using antibodies against Gag and Env, the tetraspanins CD63, CD81, CD82, or CD9, and CD45 or Lamp2 as controls. HIV-1 virions, as seen by the CAp24 and TMgp41, appeared in fractions with a density between 1.15–1.17 g/ml. In the same viral fractions, signals were obtained for the tetraspanins CD63, CD81 and CD82. Control gradient from uninfected MOLT cells is presented on the right panel. (C). Purified HIV-1 virions from MOLT/HIV-1 were submitted to immunoprecipitation with CD45, CD9, CD63, CD81 and CD82 antibodies (lane 4 to 7), or with HIV-1 serum and Env gp120 antibody as positive controls (lane 1 and 2) or without antibody as a negative control (No Ab – lane 3). Immunoprecipitated virions were run on SDS-PAGE gels and revealed with an anti-CAp24 antibody.