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 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.

The nef gene contributes to the replication of primate lentiviruses by altering the trafficking of cellular proteins involved in adaptive immunity (class I and II major histocompatibility complex [MHC]) and viral transmission (CD4 and DC-SIGN). A conserved acidic leucine-based sequence (E 160 xxxLL) within human immunodeficiency virus type 1 (HIV-1) Nef binds to the cellular adaptor protein (AP) complexes, which mediate protein sorting into endosomal vesicles. The leucine residues in this motif are required for the down-regulation of CD4 and for the up-regulation of DC-SIGN and the invariant chain of MHC class II, but the role of the acidic residue is unclear. Here, substitution of E160 with uncharged residues impaired the ability of Nef to up-regulate the expression of the invariant chain and DC-SIGN at the cell surface, whereas substitution with a basic residue was required for a similar effect on the down-regulation of CD4. All substitutions of E160 relieved the Nef-mediated block to transferrin uptake. The nef gene of primate lentiviruses is required for high-level viremia and the efficient pathogenesis of AIDS (12,25,44). These effects are at least partly due to the effect of Nef on the cellular protein trafficking environment. Nef alters the subcellular localization of a number of proteins, including CD4, DC-SIGN, transferrin receptor, tumor necrosis factor, LIGHT, CD28, class I major histocompatibility complex (MHC), and both mature and immature class II MHC (1,27,39,40,42,43). These effects likely influence the efficiency of viral replication. For example, the down-regulation of the cell surface level of CD4 by Nef prevents the binding of the viral envelope glycoprotein (gp120) to CD4 on the surface of the virus-producing cell, preserving the infectivity of newly formed virions and potentially enhancing their release (26, 37). In contrast to CD4, Nef up-regulates the surface level of DC-SIGN, a C-type lectin expressed on dendritic cells that both allows the uptake of mannosylated antigens and serves as an adhesion molecule, facilitating the interaction of dendritic cells with T cells during antigen presentation (17, 40). Although DC-SIGN binds gp120, the human immunodeficiency virus virions internalized into dendritic cells remain infectious and are subsequently transmitted to T cells, a process that Nef may facilitate. Finally, Nef disrupts the presentation of viral antigens by down-regulating class I MHC and mature class II MHC from the cell surface, while up-regulating the surface expression of the invariant chain, which normally chaperones the immature class II complex to an endosomal compartment in which antigens derived from the extracellular space are processed (42).The down-regulation of CD4, CD28, and transferrin receptor as well as the up-regulation of tumor necrosis factor, LIGHT, invariant chain, and DC-SIGN require two leucine residues within a C-terminal, solvent-exposed loop of the Nef protein (4,9,18,27,40,42,43). The leucine codons are conserved among human immunodeficiency virus type 1 ...

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.

HIV-1 Nef is a peripheral membrane protein that affects both signal transduction and membrane trafficking in infected cells. Alterations in these cellular processes enhance the efficiency of viral replication and the pathogenesis of AIDS in vivo. The precise mechanisms by which Nef functions are not fully elucidated. Nef is not an enzyme but appears to act as a linker molecule, mediating a variety of protein-protein interactions. Structural, biochemical and mutational data have allowed tentative identification of the key interactive surfaces on Nef, their cellular partners and their roles in Nef activity. Nef contains an SH3-binding surface through which it can interact with cellular Src-family tyrosine kinases and/or activator molecules for small GTPases involved in signal transduction. This SH3-binding surface is important for the ability of Nef to facilitate the activation of host T-lymphocytes, a process which renders the cells more permissive for viral replication. Nef also contains two relatively unstructured, solvent-exposed loops, through which it interacts with the cellular proteins that coat vesicles involved in membrane trafficking. These surfaces are important for Nef-mediated alterations in the subcellular distribution of transmembrane proteins, a process which causes diverse effects, including the assembly of maximally infectious viral particles and viral evasion of the host immune system. These data provide precise molecular targets within the Nef protein. Molecules that bind these interactive surfaces are predicted to inhibit Nef activity and provide the basis for novel chemotherapeutic agents for the treatment of HIV-infection.