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Background:  To characterize signals that control the routing of the structural components of HIV-1, we are analyzing their trafficking in intact cells and we are investigating whether and how they interact with elements of the cellular protein sorting machinery in vitro. We have previously shown that the HIV-1 structural protein Gag interacts with the cellular adaptor protein AP-2 in vitro. We have begun to study the significance of HIV-1 Gag-AP-2 interactions by looking at particle release, viral infectivity, and Gag distribution in cells expressing wild type or mutant AP-2. In those studies we found an enhancement in viral release from AP-2-mutant cells, but a decrease in the infectivity of those released virions. We also observed an altered distribution of Gag in AP-2-mutant cells. Based on these data, it is apparent that AP-2, and thus the early endosomal pathway, plays a role in the trafficking of Gag during viral assembly and release. It is known that HIV-1 and other primate lentiviruses selectively incorporate cellular membrane proteins that normally shuttle between endosomes and the plasma membrane. Also, recent biochemical and genetic studies implicate several components of the mammalian vacuolar protein sorting pathway, part of the endosomal compartment, in retroviral budding. Proteins such as VPS4, TSG101, and AIP1/Alix have been found to interact with HIV-1 Gag and to affect viral budding. Together, these observations suggest that HIV-1 morphogenesis is rooted in the endosomal system.

Methods:  To determine the role of AP-2 in viral assembly and release we have begun to look into interactions between AP-2 and proteins of the late endosomal pathway.

Results:  Utilizing imuunofluoresecence microscopy we found that significant fractions of AP-2 and AIP1/Alix co-localize. Further, these components of the cellular endocytic machinery and the mammalian vacuolar sorting pathway were found to interact in an in vitro binding assay.

Conclusions:  We hypothesize that AP-2 serves as a docking point at the plasma membrane for both Gag and AIP1/Alix during HIV-1 assembly and release.

Keywords: HIV-1 release; AIP1/Alix; adaptor protein, AP-2