Partial inhibition of HIV-1 release using anti-tetraspanin antibodies. (A) MOLT/HIV-1 cells were incubated for one hour with anti-tetraspanin antibodies, or with anti-VSVg, anti-CD45 or anti-Lamp2; the antibodies were then removed, and virus release was measured in the supernatant 3 hours post incubation with the antibodies. The results of two independent experiments are presented on the chart. The percentage of virus release is evaluated by RT assay in comparison to the release in the absence of antibodies, normalized to 100%. (B) Cell surface tetraspanin inaccessibility after treatment of MOLT/HIV-1 cells with the anti-tetraspanin antibodies was evaluated by FACS analysis. The histograms present the surface staining of untreated cells and cells treated with anti-CD81 (first panel) and anti-CD45 (last panel), as indicated, at 3 hours post-viral release. When the proteins are expressed at the cell surface (i.e. CD81 or CD45), the antibody treatment leads to a decrease of the Mean Fluorescent Intensity measured. (C) Infectivity of the released virus after the treatment with anti-tetraspanin antibodies. The same amount of virus was inoculated on SupT1 cells, and the resulting RT activity from de novo produced virions was detected (See Materials and Methods). The infectivity obtained from the "No Ab" control virus was referred as 100%. (D) Virus maturation (and/or retention) as well as virus release at 3 hours after anti-tetraspanin treatment in MOLT/HIV-1 cells were evaluated by immunoblotting using an anti-CAp24 monoclonal antibody. Cells were lysed and 50 μg of proteins were deposited on a gel. On the upper panel ("cell lysate") two expositions of the film are presented: at 15 seconds, only the viral capsid could be detected; at 1 hour, all maturation products appeared. Partial inhibition of virus release could be observed on the lower panel ("virus release") which is consistent with that observed by the RT assay on Fig. 4A.