Interaction of HIV-1 Nef Protein with the Host Protein Alix Promotes Lysosomal Targeting of CD4 Receptor

Nef is an accessory protein of human immunodeficiency viruses that promotes viral replication and progression to AIDS through interference with various host trafficking and signaling pathways. A key function of Nef is the down-regulation of the coreceptor CD4 from the surface of the host cells. Nef-induced CD4 down-regulation involves at least two independent steps as follows: acceleration of CD4 endocytosis by a clathrin/AP-2-dependent pathway and targeting of internalized CD4 to multivesicular bodies (MVBs) for eventual degradation in lysosomes. In a previous work, we found that CD4 targeting to the MVB pathway was independent of CD4 ubiquitination. Here, we report that this targeting depends on a direct interaction of Nef with Alix/AIP1, a protein associated with the endosomal sorting complexes required for transport (ESCRT) machinery that assists with cargo recruitment and intraluminal vesicle formation in MVBs. We show that Nef interacts with both the Bro1 and V domains of Alix. Depletion of Alix or overexpression of the Alix V domain impairs lysosomal degradation of CD4 induced by Nef. In contrast, the V domain overexpression does not prevent cell surface removal of CD4 by Nef or protein targeting to the canonical ubiquitination-dependent MVB pathway. We also show that the Nef-Alix interaction occurs in late endosomes that are enriched in internalized CD4. Together, our results indicate that Alix functions as an adaptor for the ESCRT-dependent, ubiquitin-independent targeting of CD4 to the MVB pathway induced by Nef.     

Two elements target SIV Nef to the AP-2 clathrin adaptor complex, but only one is required for the induction of CD4 endocytosis.

The simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 (HIV-1) Nef proteins induce the endocytosis of CD4 and class I MHC molecules. Here we show that SIV Nef interacts with the AP-2 adaptor complex via two elements located in the N-terminal region of the Nef molecule, but only the N-distal element is required to induce CD4 endocytosis. This N-distal AP-2 targeting element contains no canonical endocytic signals and probably contacts the AP-2 complex via a novel interaction surface. The data support a model where SIV Nef induces CD4 endocytosis by promoting the normal interactions between the di-leucine sorting signal in the CD4 cytoplasmic domain and AP-2, but does not substitute for the CD4-AP-2 adaptor interaction. Neither element is important for the induction of class I MHC endocytosis, thus indicating that different mechanisms underlie the induction of class I MHC and CD4 endocytosis by Nef. In contrast to SIV Nef, HIV-1 Nef interacts with AP-2 via a surface containing a di-leucine endocytosis signal in the C-terminal disordered loop of Nef. The fact that genetic selection maintains similar molecular interactions via different surfaces in SIV and HIV-1 Nef proteins indicates that these interactions have critical roles for the viral life cycle in vivo.     

HIV Nef-mediated CD4 down-regulation is adaptor protein complex 2 dependent

Nef is a crucial viral protein for HIV to replicate at high titers and in the development of AIDS. One Nef function is down-regulating CD4 from the cell surface, which correlates with Nef-enhanced viral pathogenicity. Nef down-regulates CD4 by linking CD4 to clathrin-coated pits. However, the mechanistic connection between the C-terminal dileucine motif of Nef and the component(s) of the clathrin-coated pits has not been pinpointed. In this report we used two AP-2 complex-specific inhibitors: a dominant negative mutant of Eps15 (Eps15DIII) that binds to the alpha subunit of AP-2 complex and a small interference RNA that is specific for the mu2 subunit of AP-2 complex. We show that both HIV Nef- and SIV Nef-mediated CD4 down-regulations were profoundly blocked by the synergistic effect of Eps15DIII and RNA interference of AP-2 expression. The results demonstrate that HIV/SIV Nef-mediated CD4 down-regulation is AP-2 dependent. We also show that the PMA-induced CD4 down-regulation was blocked by these two inhibitors. Therefore, PMA-induced CD4 down-regulation is also AP-2 dependent. The results demonstrate that, like the tyrosine sorting motif-dependent endocytosis (for which the transferrin receptor and the epidermal growth factor receptor are the two prototypes), dileucine sorting motif-dependent endocytosis of Nef and CD4 are also AP-2 dependent.     

Human immunodeficiency virus type 1 Nef mediates sustained membrane expression of tumor necrosis factor and the related cytokine LIGHT on activated T cells

Human immunodeficiency virus (HIV) Nef downregulates the antigen recognition molecules major histocompatibility complex class I and CD4. Downregulation of surface CD4 by Nef relies on the ability of this viral protein to redirect the endocytic machinery to CD4. However, by redirecting the endocytic machinery, Nef may affect the internalization rates of other proteins. Here we show that Nef simultaneously enhances surface expression of the effector cytokines tumor necrosis factor (TNF) and LIGHT, leading to enhanced cytokine activity. A dileucine motif in Nef, which is essential for CD4 downregulation and is involved in the recruitment of adapter protein complexes by Nef, was required to increase surface levels of both cytokines. The physiological impact of the Nef-mediated interference with endocytosis was demonstrated by the fact that a TNF-responsive T-cell line chronically infected with HIV produced higher levels of p24 viral protein following expression of a Nef-green fluorescent protein (GFP) fusion protein. This enhancement was dependent on the levels of membrane-bound TNF, since it was abrogated by a recombinant soluble TNF receptor. Expression of Nef-GFP in human 293T cells reduced the endocytosis of LIGHT, whereas at the same time CD4 internalization was accelerated. Taken together, these results suggest that in infected cells Nef interferes with the internalization of these effector cytokines. By increasing TNF expression, Nef could accelerate disease progression in infected individuals. These findings may help explain the pleiotropic functions that Nef plays during infection and disease.     

Cooperative interactions of simian immunodeficiency virus Nef,AP-2, and CD3-zeta mediate the selective induction of T-cell receptor-CD3 endocytosis

The Nef proteins of human immunodeficiency virus and simian immunodeficiency virus (SIV) bind the AP-1 and AP-2 clathrin adaptors to downmodulate the expression of CD4 and CD28 by recruiting them to sites of AP-2 clathrin-dependent endocytosis. Additionally, SIV Nef directly binds the CD3-zeta subunit of the CD3 complex and downmodulates the T-cell receptor (TCR)-CD3 complex. We report here that SIV mac239 Nef induces the endocytosis of TCR-CD3 in Jurkat T cells. SIV Nef also induces the endocytosis of a chimeric CD8-CD3-zeta protein containing only the CD3-zeta cytoplasmic domain (8-zeta), in the absence of other CD3 subunits. Thus, the interaction of SIV Nef with CD3-zeta likely mediates the induction of TCR-CD3 endocytosis. In cells expressing SIV Nef and 8-zeta, both proteins colocalize with AP-2, indicating that Nef induces 8-zeta internalization via this pathway. Surprisingly, deletion of constitutively strong AP-2 binding determinants (CAIDs) in SIV Nef had little effect on its ability to induce TCR-CD3, or 8-zeta endocytosis, even though these determinants are required for the induction of CD4 and CD28 endocytosis via this pathway. Fluorescent microscopic analyses revealed that while neither the mutant SIV Nef protein nor 8-zeta colocalized with AP-2 when expressed independently, both proteins colocalized with AP-2 when coexpressed. In vitro binding studies using recombinant SIV Nef proteins lacking CAIDs and recombinant CD3-zeta cytoplasmic domain demonstrated that SIV Nef and CD3-zeta cooperate to bind AP-2 via a novel interaction. The fact that Nef uses distinct AP-2 interaction surfaces to recruit specific membrane receptors demonstrates how Nef independently selects distinct types of target receptors and recruits them to AP-2 for endocytosis.     

Downregulation of CD4 by human immunodeficiency virus type 1 Nef is dependent on clathrin and involves direct interaction of Nef with the AP2 clathrin adaptor

Nef, an accessory protein of human and simian immunodeficiency viruses, is a critical determinant of pathogenesis that promotes the progression from infection to AIDS. The pathogenic effects of Nef are in large part dependent on its ability to downregulate the macrophage and T-cell coreceptor, CD4. It has been proposed that Nef induces downregulation by linking the cytosolic tail of CD4 to components of the host-cell protein trafficking machinery. To identify these components, we developed a novel Nef-CD4 downregulation system in Drosophila melanogaster S2 cells. We found that human immunodeficiency virus type 1 (HIV-1) Nef downregulates human CD4 in S2 cells and that this process is subject to the same sequence requirements as in human cells. An RNA interference screen targeting protein trafficking genes in S2 cells revealed a requirement for clathrin and the clathrin-associated, plasma membrane-localized AP2 complex in the downregulation of CD4. The requirement for AP2 was confirmed in the human cell line HeLa. We also used a yeast three-hybrid system and glutathione S-transferase pull-down analyses to demonstrate a robust, direct interaction between HIV-1 Nef and AP2. This interaction requires a dileucine motif in Nef that is also essential for downregulation of CD4. Together, these results support a model in which HIV-1 Nef downregulates CD4 by promoting its accelerated endocytosis by a clathrin/AP2 pathway.     

Co-localization of HIV-1 Nef with the AP-2 adaptor protein complex correlates with Nef-induced CD4 down-regulation.

The nef gene of human and simian immunodeficiency viruses is critical for AIDS pathogenesis. Its function in vivo is unknown, but in vitro natural isolates of Nef down-regulate expression of the cell surface CD4 molecule, a component of the T cell antigen receptor and the viral receptor, by accelerating its endocytosis. We have used chimeric proteins comprised of the natural HIV-1 NA7 Nef fused to a strongly fluorescing mutant of green fluorescent protein (GFP) to correlate Nef function with intracellular localization in human CD4-positive Jurkat T cells. The NA7-GFP fusion protein co-localizes with components of the clathrin coat, including clathrin and the beta-subunit of the AP-2 adaptor protein complex, at discrete locations that are consistent with the normal cellular distribution of clathrin coats at the plasma membrane. The NA7-GFP protein is also found in the perinuclear region of the cell, which is likely to reflect the Golgi apparatus. Evidence from a CD4-negative fibroblast cell line indicates that co-localization of NA7-GFP with components of the clathrin coat does not require expression of the CD4 molecule. Analysis of a large panel of chimeric molecules containing mutant Nef moieties demonstrated that the N-terminal membrane targeting signal cooperates with additional element(s) in the disordered loops in the Nef molecule to co-localize the Nef protein with AP-2 adaptor complexes at the cell margin. This localization of NA7-GFP correlates with, but is not sufficient for, down-regulation of surface CD4 and at least one additional function of Nef is required. In T cells co-expressing CD4 and NA7-GFP, CD4 at the cell surface is redistributed into a discrete pattern that co-localizes with that of NA7-GFP. Our observations place NA7-GFP in physical proximity to AP-2-containing clathrin coat at the plasma membrane and imply that Nef interacts, either directly or indirectly, with a component of the AP-2-containing coat at this location. This evidence supports a model whereby Nef recruits CD4 to the endocytic machinery via AP-2-containing clathrin coats at the plasma membrane.     

HIV-Nef and AIDS pathogenesis: are we barking up the wrong tree?

After two decades of research the Nef protein of human immunodeficiency virus (HIV) remains a mysterious protein with an indisputable role in HIV pathogenesis. The ability to downregulate CD4 and major histocompatibility complex class I (MHC-I) was the first ascribed function of Nef and, whereas the number of downmodulated receptors by Nef is rising, so are the explanations for how their downregulation could contribute to HIV pathogenesis. At the same time there is increasing evidence that Nef not only induces endocytosis but also exocytosis, namely of cytokines and microvesicles that contain Nef itself. Because endocytosis and exocytosis are connected events, this is not surprising - and raises the intriguing possibility that HIV aims at secretion rather than ingestion. Have we therefore barked up the wrong tree over the past two decades? In this opinion article I argue that Nef-induced secretion is most probably the pathogenesis-relevant function behind this elusive viral effector.     

Mechanism of Nef-induced CD4 endocytosis: Nef connects CD4 with the mu chain of adaptor complexes.

The Nef protein of primate lentiviruses down-regulates the cell surface expression of CD4 and probably MHC I by connecting these receptors with the endocytic machinery. Here, we reveal that Nef interacts with the mu chains of adaptor complexes, key components of clathrin-coated pits. For human immunodeficiency virus type 2 (HIV-2) and simian immunodeficiency virus (SIV) Nef, this interaction occurs via tyrosine-based motifs reminiscent of endocytosis signals. Mutating these motifs prevents the binding of SIV Nef to the mu chain of plasma membrane adaptor complexes, abrogates its ability to induce CD4 internalization, suppresses the accelerated endocytosis of a chimeric integral membrane protein harboring Nef as its cytoplasmic domain and confers a dominant-negative phenotype to the viral protein. Taken together, these data identify mu adaptins as downstream mediators of the down-modulation of CD4, and possibly MHC I, by Nef.     

Human immunodeficiency virus Nef induces rapid internalization of the T-cell coreceptor CD8alphabeta

Human immunodeficiency virus (HIV) Nef is a membrane-associated protein decreasing surface expression of CD4, CD28, and major histocompatibility complex class I on infected cells. We report that Nef strongly down-modulates surface expression of the beta-chain of the CD8alphabeta receptor by accelerated endocytosis, while CD8 alpha-chain expression is less affected. By mutational analysis of the cytoplasmic tail of the CD8 beta-chain, an FMK amino acid motif was shown to be critical for Nef-induced endocytosis. Although independent of CD4, endocytosis of the CD8 beta-chain was abrogated by the same mutations in Nef that affect CD4 down-regulation, suggesting common molecular interactions. The ability to down-regulate the human CD8 beta-chain was conserved in HIV-1, HIV-2, and simian immunodeficiency virus SIVmac239 Nef and required an intact AP-2 complex. The Nef-mediated internalization of receptors, such as CD4, major histocompatibility complex class I, CD28, and CD8alphabeta, may contribute to the subversion of the host immune system and progression towards AIDS.     

HIV-1 Nef stabilizes the association of adaptor protein complexes with membranes

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.     

Nef proteins from diverse groups of primate lentiviruses downmodulate CXCR4 to inhibit migration to the chemokine stromal derived factor 1

Nef proteins of primate lentiviruses promote viral replication, virion infectivity, and evasion of antiviral immune responses by modulating signal transduction pathways and downregulating expression of receptors at the cell surface that are important for efficient antigen-specific responses, such as CD4, CD28, T-cell antigen receptor, and class I and class II major histocompatibility complex. Here we show that Nef proteins from diverse groups of primate lentiviruses which do not require the chemokine receptor CXCR4 for entry into target cells strongly downmodulate the cell surface expression of CXCR4. In contrast, all human immunodeficiency virus type 1 (HIV-1) and the majority of HIV-2 Nef proteins tested did not have such strong effects. SIVmac239 Nef strongly inhibited lymphocyte migration to CXCR4 ligand, the chemokine stromal derived factor 1 (SDF-1). SIVmac239 Nef downregulated CXCR4 by accelerating the rate of its endocytosis. Downmodulation of CXCR4 was abolished by mutations that disrupt the constitutively strong AP-2 clathrin adaptor binding element located in the N-terminal region of the Nef molecule, suggesting that Nef accelerates CXCR4 endocytosis via an AP-2-dependent pathway. Together, these results point to CXCR4 as playing an important role in simian immunodeficiency virus and possibly also HIV-2 persistence in vivo that is unrelated to viral entry into target cells. We speculate that Nef targets CXCR4 to disrupt ordered trafficking of infected leukocytes between local microenvironments in order to facilitate their dissemination and/or impair the antiviral immune response.     

Molecular design, functional characterization and structural basis of a protein inhibitor against the HIV-1 pathogenicity factor Nef

Increased spread of HIV-1 and rapid emergence of drug resistance warrants development of novel antiviral strategies. Nef, a critical viral pathogenicity factor that interacts with host cell factors but lacks enzymatic activity, is not targeted by current antiviral measures. Here we inhibit Nef function by simultaneously blocking several highly conserved protein interaction surfaces. This strategy, referred to as "wrapping Nef", is based on structure-function analyses that led to the identification of four target sites: (i) SH3 domain interaction, (ii) interference with protein transport processes, (iii) CD4 binding and (iv) targeting to lipid membranes. Screening combinations of Nef-interacting domains, we developed a series of small Nef interacting proteins (NIs) composed of an SH3 domain optimized for binding to Nef, fused to a sequence motif of the CD4 cytoplasmic tail and combined with a prenylation signal for membrane association. NIs bind to Nef in the low nM affinity range, associate with Nef in human cells and specifically interfere with key biological activities of Nef. Structure determination of the Nef-inhibitor complex reveals the molecular basis for binding specificity. These results establish Nef-NI interfaces as promising leads for the development of potent Nef inhibitors.     

Mechanism for down-regulation of CD28 by Nef

SIV and HIV Nef proteins disrupt T-cell receptor machinery by down-modulating cell surface expression of CD4 and expression or signaling of CD3-TCR. Nef also down-modulates class I major histocompatibility complex (MHC) surface expression. We show that SIV and HIV-1 Nefs down-modulate CD28, a major co-stimulatory receptor that mediates effective T-cell activation, by accelerating CD28 endocytosis. The effects of Nef on CD28, CD4, CD3 and class I MHC expression are all genetically separable, indicating that all are selected independently. In cells expressing a Nef-green fluorescent protein (GFP) fusion, CD28 co-localizes with the AP-2 clathrin adaptor and Nef-GFP. Mutations that disrupt Nef interaction with AP-2 disrupt CD28 down-regulation. Furthermore, HIV and SIV Nefs use overlapping but distinct target sites in the membrane-proximal region of the CD28 cytoplasmic domain. Thus, Nef probably induces CD28 endocytosis via the AP-2 pathway, and this involves a ternary complex containing Nef, AP-2 and CD28. The likely consequence of the concerted down-regulation of CD28, CD4 and/or CD3 by Nef is disruption of antigen-specific signaling machineries in infected T cells following a productive antigen recognition event.     

Human immunodeficiency virus type 1 Nef induces accumulation of CD4 in early endosomes.

We have studied the fate of CD4 in CEM T cells expressing a human immunodeficiency virus type 1 HIV-1 Nef protein. Nef triggered a rapid endocytosis and a degradation of CD4, while most of the p56lck was upheld at the cell membrane. In the presence of Nef, CD4 accumulated in acidic intracellular vesicles that were not stained by antibodies against rab6, a marker of the Golgi apparatus complex. Detection of transferrin in CD4-containing vesicles showed that CD4 was trapped in early endosomes, without significant accumulation of CD4 in late endocytic compartments. Internalization pathways taken by CD4 in Nef+ cells may therefore be different from those observed after treatment with phorbol esters.     

Mutation of a conserved residue (D123) required for oligomerization of human immunodeficiency virus type 1 Nef protein abolishes interaction with human thioesterase and results in impairment of Nef biological functions

Nef is a myristoylated protein of 27 to 35 kDa that is conserved in primate lentiviruses. In vivo, Nef is required for high viral load and full pathological effects. In vitro, Nef has at least four activities: induction of CD4 and major histocompatibility complex (MHC) class I downregulation, enhancement of viral infectivity, and alteration of T-cell activation pathways. We previously reported that the Nef protein from human immunodeficiency virus type 1 interacts with a novel human thioesterase (hTE). In the present study, by mutational analysis, we identified a region of the Nef core, extending from the residues D108 to W124, that is involved both in Nef-hTE interaction and in Nef-induced CD4 downregulation. This region of Nef is located on the oligomer interface and is in close proximity to the putative CD4 binding site. One of the mutants carrying a mutation in this region, targeted to the conserved residue D123, was also found to be defective in two other functions of Nef, MHC class I downmodulation and enhancement of viral infectivity. Furthermore, mutation of this residue affected the ability of Nef to form dimers, suggesting that the oligomerization of Nef may be critical for its multiple functions.     

Human immunodeficiency virus type 1 nef expression prevents AP-2-mediated internalization of the major histocompatibility complex class II-associated invariant chain

The lentiviral Nef protein has been studied extensively for its ability to induce the downregulation of several immunoreceptors on the surfaces of infected cells. However, Nef expression is unique in inducing highly effective upregulation of the major histocompatibility complex class II-associated chaperone invariant (Ii) chain complexes in different cell types. Under normal conditions, endocytosis of the Ii chain and other molecules, like the transferrin receptor and CD4, is rapid and AP-2 dependent. Human immunodeficiency virus type 1 (HIV-1) Nef expression strongly reduces the internalization of the Ii chain, enhances that of CD4, and does not modify transferrin uptake. The mutation of AP-2 binding motifs LL164 and DD174 in Nef leads to the inhibition of Ii chain upregulation. In AP-2-depleted cells, surface levels of the Ii chain are high and remain unmodified by Nef expression, further indicating that Nef regulates Ii chain internalization via the AP-2 pathway. Immunoprecipitation experiments revealed that the Ii chain can interact with Nef in a dileucine-dependent manner. Importantly, we have shown that Nef-induced CD4 downregulation and Ii chain upregulation are genetically distinguishable. We have identified natural nef alleles that have lost one of the two functions but not the other one. Moreover, we have characterized Nef mutant forms possessing a similar phenotype in the context of HIV-1 infection. Therefore, the Nef-induced accumulation of Ii chain complexes at the cell surface probably results from a complex mechanism leading to the impairment of AP-2-mediated endocytosis rather than from direct competition between Nef and the Ii chain for binding AP-2.     

Structural basis for the interaction between focal adhesion kinase and CD4

Focal adhesion kinase (FAK) and CD4 fulfil vital functions in cellular signal transduction: FAK is a central component in integrin signalling, whereas CD4 plays essential roles in the immune defence. In T lymphocytes, FAK and CD4 localise to the same signalling complexes after stimulation by either the human immunodeficiency virus (HIV) gp120 glycoprotein or an antigen, suggesting the concerted action of FAK and CD4 in these cells. Using crystallography and microcalorimetry, we here show that the focal adhesion targeting (FAT) domain of FAK binds specifically to the CD4 endocytosis motif in vitro. This FAT-CD4 complex is structurally and thermodynamically similar to the one FAT forms with paxillin LD motifs. The CD4 binding site on FAT presents the same features as the established CD4 binding site on the HIV-1 Nef protein. The binding of FAT to CD4 is incompatible with the binding of Lck to CD4. We further show that HIV-1 gp120 triggers the association of CD4 with FAK in T cells, under conditions that are known to dissociate Lck from CD4. Our results suggest that the FAK-CD4 complex represents an alternative route for eliciting T-cell-specific signals and that it links gp120 engagement to distinctive T-cell signalling during HIV infection. In infected cells, HIV-1 Nef may displace FAK from CD4 to protect the cells from apoptosis.     

Lysine 144, a ubiquitin attachment site in HIV-1 Nef, is required for Nef-mediated CD4 down-regulation

Nef is a HIV-1 accessory protein critical for the replication of the virus and the development of AIDS. The major pathological activity of Nef is the down-regulation of CD4, the primary receptor of HIV-1 infection. The mechanism underlying Nef-mediated CD4 endocytosis and degradation remains incompletely understood. Since protein ubiquitination is the predominant sorting signal in receptor endocytosis, we investigated whether Nef is ubiquitinated. The in vivo ubiquitination assay showed that both HIV-1 and SIV Nef proteins expressed in Jurkat T cells and 293T cells were multiple ubiquitinated by ubiquitin-His. The lysine-free HIV-1 Nef mutant (Delta10K) generated by replacing all 10 lysines with arginines was not ubiquitinated and the major ubiquitin-His attachment sites in HIV-1 Nef were determined to be lysine 144 (di-ubiquitinated) and lysine 204 (mono-ubiquitinated). Lysine-free HIV-1 Nef was completely inactive in Nef-mediated CD4 down-regulation, so was the Nef mutant with a single arginine substitution at K144 but not at K204. A mutant HIV-1 provirion NL4-3 with a single arginine substitution in Nef at K144 was also inactive in Nef-mediated CD4 down-regulation. Lysine-free Nef mutant reintroduced with lysine 144 (DeltaK10 + K144) was shown active in CD4 down-regulation. These data suggest that ubiquitination of Nef, particularly diubiquitination of the lysine 144, is necessary for Nef-mediated CD4 down-regulation.