Interactions of HIV-1 Nef with the μ Subunits of Adaptor Protein Complexes 1, 2, and 3: Role of the Dileucine-Based Sorting Motif

Abstract

HIV-1 Nef interacts with cellular adaptor protein (AP) complexes and their medium (μ) subunits. However, the role of the dileucine-based sorting motif within Nef in these interactions has been incompletely characterized. Here, yeast two-hybrid assays indicated that HIV-1 Nef interacted not only with the μ subunits of AP-1 and AP-2, but also with that of AP-3. The interactions with μ1 and μ3 were markedly stronger than the interaction with μ2. Leucine residues of the sorting motif were required for the interactions with μ3 and μ2 and contributed to the interaction with μ1. Confocal immunofluorescence microscopy indicated that Nef, AP-1, and AP-3 (but not AP-2) were concentrated in a juxtanuclear region near the cell center, potentially facilitating interaction between Nef and the μ1 and μ3 subunits. However, leucine residues of the sorting motif were not required for this subcellular localization of Nef. These data suggest that the dileucine motif, 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 determinants in the Nef protein.