Overexpression of tetraspanins decreases the infectivity of cell-free virus. (A)HeLa cells were co-transfected with pNL4-3 and the indicated plasmids. Released virus was pelleted through a 20% sucrose cushion, immunoprecipitated using HIVIG and protein A sepharose beads (to purify the virus from tetraspanin-bearing exosomes or microvesicles), then eluted and analyzed for p24 content by ELISA. Equal amounts of p24 were loaded for each sample and analyzed for tetraspanin incorporation by Western blot. (B) HeLa cells were co-transfected with pNL4-3 and the indicated tetraspanin expression plasmids as in (A). Supernatants were collected, normalized for p24 content, and the infectivity assay was carried out as described in Methods. Data shown represent the mean of 5 independent experiments (normalized to vector control) +/- SEM. * denotes a statistically significant difference from vector control (p < 0.001).