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 (mu) 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 mu subunits of AP-1 and AP-2, but also with that of AP-3. The interactions with mu1 and mu3 were markedly stronger than the interaction with mu2. Leucine residues of the sorting motif were required for the interactions with mu3 and mu2 and contributed to the interaction with mu1. 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 mu1 and mu3 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.

Nef Stimulates Human Immunodeficiency Virus Type 1 Replication in Primary T Cells by Enhancing Virion-Associated gp120 Levels: Coreceptor-Dependent Requirement for Nef in Viral Replication

“…Nef, in contrast, is expressed immediately following infection and stimulates the internalization and degradation of CD4 molecules already present on the surface of the infected cell prior to Env and Vpu expression (2,12,59). Nef interacts simultaneously with the cytoplasmic tail of CD4 and members of the cellular clathrin endocytic machinery, such as adaptor proteins (9,19,27,30,41), ␤-cop (5,56), and/or vacuolar ATPase (42,44). Nef thus directs infected cell surface CD4 to cellular endocytic pathways and to its subsequent degradation in lysosomes.…”

Nef Is Physically Recruited into the Immunological Synapse and Potentiates T Cell Activation Early after TCR Engagement

“…Of note, Nef-GFP appears as effective as wild-type Nef for all its functions studied to date (35,36). To label the lipid rafts, which are enriched with the ganglioside GM1, a sphingolipid specifically recognized by CT-B, we incubated the cells with recombinant CT-B conjugated to Alexa Fluo 594.…”

HIV Type 1 Nef Is Released from Infected Cells in CD45+Microvesicles and Is Present in the Plasma of HIV-Infected Individuals

“…For example, interactions between Nef and b-Cop target CD4 to late acidic endosomes whereas interactions with AP-2 target CD4 to clathrin-coated pits (early endosomes). [32][33][34] Studies have shown that Nef expression leads to changes within the endosomal compartment and increases endosome, lysosome, and late endosome/multivesicular body (MVB) production in hematopoietic cells. [35][36][37] MVBs contain smaller membrane-bound vesicles that fuse with plasma membranes and are released via exocytosis into the extracellular space as exosomes ranging in size from 30 to 200 nm in diameter.…”