Interactions of HIV-1 Nef with the mu subunits of adaptor protein complexes 1, 2, and 3: Role of the dileucine-based sorting motif

HIV-1 Nef interacts with cellular adaptor protein (AP) complexes and their medium (mu) subunits. However, the role of the dileucine-based sorting moti f within Nef in these interactions has been incompletely characterized. Her e, yeast two-hybrid assays indicated that HIV-1 Nef interacted not only wit h the mu subunits of AP-1 and AP-2, but also with that of AP-3. The interac tions with mu 1 and mu 3 were markedly stronger than the interaction with m u 2. Leucine residues of the sorting motif were required for the interactio ns with mu 3 and mu 2 and contributed to the interaction with mu 1. Confoca l immunofluorescence microscopy indicated that Nef, AP-I, and AP-3 (but not AP-2) were concentrated in a juxtanuclear region near the cell center, pot entially facilitating interaction between Nei and the mu 1 and mu 3 subunit s. However, leucine residues of the sorting motif were not required for thi s subcellular localization of Nef. These data suggest that the dileucine mo tif, required for optimal viral replication, functions through interactions with a variety of AP complexes, including AP-3, potentially by recruiting adaptor complexes to subcellular locations specified by additional determin ants in the Nef protein. (C) 2000 Academic Press.