The HIV-1 p6/EIAV p9 docking site in Alix is autoinhibited as revealed by a conformation-sensitive anti-Alix monoclonal antibody

Alix [ALG-2 (apoptosis-linked gene 2)-interacting protein X], a component of the endosomal sorting machinery, contains a three-dimensional docking site for HIV-1 p6^Gag or EIAV (equine infectious anaemia virus) p9^Gag, and binding of the viral protein to this docking site allows the virus to hijack the host endosomal sorting machinery for budding from the plasma membrane. In the present study, we identified a monoclonal antibody that specifically recognizes the docking site for p6^Gag/p9^Ga^g and we used this antibody to probe the accessibility of the docking site in Alix. Our results show that the docking site is not available in cytosolic or recombinant Alix under native conditions and becomes available upon addition of the detergent Nonidet P40 or SDS. In HEK (human embryonic kidney)-293 cell lysates, an active P6^Gag/p9^Gag docking site is specifically available in Alix from the membrane fraction. The findings of the present study demonstrate that formation or exposure of the p6^Gag/p9^Gag docking site in Alix is a regulated event and that Alix association with the membrane may play a positive role in this process.