human immunodeficiency virus type 1 (HIV-1) Nef interacts with the clathrin-associated AP-1 and AP-3 adaptor complexes, stabilizing their association with endosomal membranes. These findings led us to hypothesize a general impact of this viral protein on the endosomal system. Here, we have shown that Nef specifically disturbs the morphology of the early/recycling compartment, inducing a redistribution of early endosomal markers and a shortening of the tubular recycling endosomal structures. Furthermore, Nef modulates the trafficking of the transferrin receptor (TfR), the prototypical recycling surface protein, indicating that it also disturbs the function of this compartment. Nef reduces the rate of recycling of TfR to the plasma membrane, causing TfR to accumulate in early endosomes and reducing its expression at the cell surface. These effects depend on the leucine-based motif of Nef, which is required for the membrane stabilization of AP-1 and AP-3 complexes. Since we show that this motif is also required for the full infectivity of HIV-1 virions, these results indicate that the positive influence of Nef on viral infectivity may be related to its general effects on early/recycling endosomal compartments.Trafficking of membrane proteins is governed by a regulated machinery that involves the vesicular transport of proteins throughout different intracellular compartments. One major regulatory mechanism is related to the function of the adaptor protein (AP) 1 complexes that assemble on donor membranes of the endocytic pathway to form transport vesicles (for review, see Ref. 1). The sorting of transmembrane proteins into these vesicles requires the recognition by the AP complexes of specific tyrosine-or leucine-based motifs contained within the cytoplasmic domains of cargo proteins (2). Four different types of heterotetrameric AP complexes (AP-1-AP-4) have been identified (3). AP-2 is specifically involved in the formation of clathrincoated vesicles at the plasma membrane, whereas AP-1 and AP-3 mediate the formation of clathrin-coated vesicles at the levels of the trans-Golgi network (TGN) and endosomes. The function of AP-4 is less well documented, but it regulates formation of non-clathrin-coated vesicles at the TGN. The association of the AP-1, AP-3, and AP-4 complexes with TGN and endosomal membranes is regulated by ADP-ribosylation factor 1 (ARF1). The Nef protein of HIV-1 is a 27-kDa protein that associates with the cell membranes through N-terminal myristoylation and is abundantly produced shortly after virus infection (for review, see Refs. 4 and 5). Nef is an essential factor in vivo for efficient viral replication and pathogenesis. In vitro, Nef also facilitates virus replication and enhances the infectivity of virions. Although the positive influence of Nef on viral replication and infectivity may be multifactorial, genetic evidence suggests a relationship between these virological effects and the ability of Nef to modulate the cell surface expression of multiple membrane-associated proteins. In additio...

The maximal virulence of HIV-1 requires Nef, a virally encoded peripheral membrane protein. Nef binds to the adaptor protein (AP) complexes of coated vesicles, inducing an expansion of the endosomal compartment and altering the surface expression of cellular proteins including CD4 and class I major histocompatibility complex. Here, we show that Nef stabilizes the association of AP-1 and AP-3 with membranes. These complexes remained with Nef on juxtanuclear membranes despite the treatment of cells with brefeldin A, which induced the release of ADP-ribosylation factor 1 (ARF1) from these membranes to the cytosol. Nef also induced a persistent association of AP-1 and AP-3 with membranes despite the expression of dominant-negative ARF1 or the overexpression of an ARF1-GTPase activating protein. Mutational analysis indicated that the direct binding of Nef to the AP complexes is essential for this stabilization. The leucine residues of the EXXXLL motif found in Nef were required for binding to AP-1 and AP-3 in vitro and for the stabilization of these complexes on membranes in vivo, whereas the glutamic acid residue of this motif was required specifically for the binding and stabilization of AP-3. These data indicate that Nef mediates the persistent attachment of AP-1 and AP-3 to membranes by an ARF1-independent mechanism. The stabilization of these complexes on membranes may underlie the pleiotropic effects of Nef on protein trafficking within the endosomal system.

The human immunodeficiency virus type 1 virulence protein Nef interacts with the endosomal sorting machinery via a leucine-based motif. Similar sequences within the cytoplasmic domains of cellular transmembrane proteins bind to the adaptor protein (AP) complexes of coated vesicles to modulate protein traffic, but the molecular basis of the interactions between these motifs and the heterotetrameric complexes is controversial. To identify the target of the Nef leucine motif, the native sequence was replaced with either leucine-or tyrosine-based AP-binding sequences from cellular proteins, and the interactions with AP subunits were correlated with function. Tyrosine motifs predictably modulated the interactions between Nef and the subunits of AP-1, AP-2, and AP-3; heterologous leucine motifs caused little change in these interactions. Conversely, leucine motifs mediated a ternary interaction between Nef and hemicomplexes containing the 1 plus ␥ subunits of AP-1 or the 3 plus ␦ subunits of AP-3, whereas tyrosine motifs did not. Similarly, only leucine motifs supported the Nef-mediated association of AP-1 and AP-3 with endosomal membranes in cells treated with brefeldin A. Functionally, Nef proteins containing leucine motifs down-regulated CD4 from the cell surface and enhanced viral replication, whereas those containing tyrosine motifs were inactive. Apparently, the interaction of Nef with the subunits of AP complexes is insufficient for function. A leucine-specific mode of interaction that likely involves AP hemicomplexes is further required for Nef activity. The and hemicomplex interactions may cooperate to yield high avidity binding of AP complexes to Nef. This binding likely underlies the unusual ability of Nef to induce the stabilization of these complexes on endosomal membranes, an activity that correlates with enhancement of viral replication.

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HIV-1 Nef affects the trafficking of numerous cellular proteins to optimize viral replication and evade host defenses. The adaptor protein (AP) complexes, which form part of the cytoplasmic coat of endosomal vesicles, are key cellular co-factors for Nef. Nef binds these complexes and alters their physiologic cycle of attachment and release from membranes. Specifically, while AP-1 normally becomes cytosolic when attachment events are blocked by inhibition of the GTPase cycle of ADP-ribosylation factor-1 (ARF1), the complex remains membrane-associated in Nef-expressing cells. To investigate the mechanism of this effect, we used a permeabilized cell system to detect the de novo attachment of exogenous AP-1 to endosomal membranes. Nef did not mediate de novo attachment independently of ARF1, despite its ability to maintain the association of AP-1 with endosomal membranes when the activity of ARF1 was blocked. We conclude that Nef stabilizes AP complexes on endosomal membranes after ARF1-dependent attachment. This stabilization may facilitate coat formation and stimulate the trafficking of multiple cellular proteins.