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 type 1 Nef-induced CD4 cell surface downregulation is inhibited by ikarugamycin

One well-characterized in vitro function of Nef is its ability to remove CD4, the human immunodeficiency virus (HIV) receptor, from the cell surface. Nef accomplishes this by accelerating the internalization and degradation of CD4. Current models propose that Nef promotes CD4 internalization via an increased association of CD4 with clathrin-coated pits (CCP). Here, we investigated the effect of a naturally occurring antiprotozoan antibiotic, ikarugamycin (IKA), on CD4 cell surface expression in human monocytic cells stably expressing HIV type 1 SF2 Nef. IKA was able to efficiently restore CD4 cell surface expression in Nef-expressing cells without affecting either CD4 synthesis or Nef expression. In addition, we demonstrate that IKA is also capable of efficiently blocking CD4 down-modulation in response to phorbol myristate acetate. Our data suggest that IKA may be an efficient and useful inhibitor of CCP-dependent endocytosis.     

Dynamic interaction of HIV-1 Nef with the clathrin-mediated endocytic pathway at the plasma membrane

The HIV-1 Nef protein perturbs the trafficking of membrane proteins such as CD4 by interacting with clathrin-adaptor complexes. We previously reported that Nef alters early/recycling endosomes, but its role at the plasma membrane is poorly documented. Here, we used total internal reflection fluorescence microscopy, which restricts the analysis to a approximately 100 nm region of the adherent surface of the cells, to focus on the dynamic of Nef at the plasma membrane relative to that of clathrin. Nef colocalized both with clathrin spots (CS) that remained static at the cell surface, corresponding to clathrin-coated pits (CCPs), and with approximately 50% of CS that disappeared from the cell surface, corresponding to forming clathrin-coated vesicles (CCVs). The colocalization of Nef with clathrin required the di-leucine motif essential for Nef binding to AP complexes and was independent of CD4 expression. Furthermore, analysis of Nef mutants showed that the capacity of Nef to induce internalization and downregulation of CD4 in T lymphocytes correlated with its localization into CCPs. In conclusion, this analysis shows that Nef is recruited into CCPs and into forming CCVs at the plasma membrane, in agreement with a model in which Nef uses the clathrin-mediated endocytic pathway to induce internalization of some membrane proteins from the surface of HIV-1-infected T cells.     

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.     

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.     

Mechanisms and functions of AT(1) angiotensin receptor internalization

The type 1 (AT(1)) angiotensin receptor, which mediates the known physiological and pharmacological actions of angiotensin II, activates numerous intracellular signaling pathways and undergoes rapid internalization upon agonist binding. Morphological and biochemical studies have shown that agonist-induced endocytosis of the AT(1) receptor occurs via clathrin-coated pits, and is dependent on two regions in the cytoplasmic tail of the receptor. However, it is independent of G protein activation and signaling, and does not require the conserved NPXXY motif in the seventh transmembrane helix. The dependence of internalization of the AT(1) receptor on a cytoplasmic serine-threonine-rich region that is phosphorylated during agonist stimulation suggests that endocytosis is regulated by phosphorylation of the AT(1) receptor tail. beta-Arrestins have been implicated in the desensitization and endocytosis of several G protein-coupled receptors, but the exact nature of the adaptor protein required for association of the AT(1) receptor with clathrin-coated pits, and the role of dynamin in the internalization process, are still controversial. There is increasing evidence for a role of internalization in sustained signal generation from the AT(1) receptor. Several aspects of the mechanisms and specific function of AT(1) receptor internalization, including its precise mode and route of endocytosis, and the potential roles of cytoplasmic and nuclear receptors, remain to be elucidated.     

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.     

Ligand-induced internalization of TNF receptor 2 mediated by a di-leucin motif is dispensable for activation of the NFκB pathway

Endocytosis is an important mechanism to regulate tumor necrosis factor (TNF) signaling. In contrast to TNF receptor 1 (TNFR1; CD120a), the relevance of receptor internalization for signaling as well as the fate and route of internalized TNF receptor 2 (TNFR2; CD120b) is poorly understood. To analyze the dynamics of TNFR2 signaling and turnover at the plasma membrane we established a human TNFR2 expressing mouse embryonic fibroblast cell line in a TNFR1^−/−/TNFR2^−/− background. TNF stimulation resulted in a decrease of constitutive TNFR2 ectodomain shedding. We hypothesized that reduced ectodomain release is a result of TNF/TNFR2 complex internalization. Indeed, we could demonstrate that TNFR2 was internalized together with its ligand and cytoplasmic binding partners. Upon endocytosis the TNFR2 signaling complex colocalized with late endosome/lysosome marker Rab7 and entered the lysosomal degradation pathway. Furthermore, we identified a di-leucin motif in the cytoplasmic part of TNFR2 suggesting clathrin-dependent internalization of TNFR2. Internalization defective TNFR2 mutants are capable to signal, i.e. activate NFκB, demonstrating that the di-leucin motif dependent internalization is dispensable for this response. We therefore propose that receptor internalization primarily serves as a negative feed-back to limit TNF responses via TNFR2.     

Nef-Induced CD4 and Major Histocompatibility Complex Class I (MHC-I) Down-Regulation Are Governed by Distinct Determinants: N-Terminal Alpha Helix and Proline Repeat of Nef Selectively Regulate MHC-I Trafficking

The Nef protein of primate lentiviruses triggers the accelerated endocytosis of CD4 and of class I major histocompatibility complex (MHC-I), thereby down-modulating the cell surface expression of these receptors. Nef acts as a connector between the CD4 cytoplasmic tail and intracellular sorting pathways both in the Golgi and at the plasma membrane, triggering the de novo formation of CD4-specific clathrin-coated pits (CCP). The downstream partners of Nef in this event are the adapter protein complex (AP) of CCP and possibly a subunit of the vacuolar ATPase. Whether Nef-induced MHC-I down-regulation stems from a similar mechanism is unknown. By comparing human immunodeficiency virus type 1 (HIV-1) Nef mutants for their ability to affect either CD4 or MHC-I expression, both in transient-transfection assays and in the context of HIV-1 infection, it was determined that Nef-induced CD4 and MHC-I down-regulation constitute genetically and functionally separate properties. Mutations affecting only CD4 regulation mapped to residues previously shown to mediate the binding of Nef to this receptor, such as W57 and L58, as well as to an AP-recruiting dileucine motif and to an acidic dipeptide in the C-terminal region of the protein. In contrast, mutation of residues in an alpha-helical region in the proximal portion of Nef and amino acid substitutions in a proline-based SH3 domain-binding motif selectively affected MHC-I down-modulation. Although both the N-terminal alpha-helix and the proline-rich region of Nef have been implicated in recruiting Src family protein kinases, the inhibitor herbimycin A did not block MHC-I down-regulation, suggesting that the latter process is not mediated through an activation of this family of tyrosine kinases.The Nef protein of primate lentiviruses plays a multifaceted role in the life cycle of these pathogens (reviewed in reference 17). Produced in abundance from the earliest stage of viral gene expression, Nef associates with the membranes of infected cells by virtue of its N-terminal myristoylation (21, 36, 46), and it accomplishes several distinct functions. First, it down-regulates the cell surface expression of class I major histocompatibility complex (MHC-I), preventing the recognition and lysis of infected cells by cytotoxic lymphocytes (14, 48, 50, 66). Second, it decreases the surface expression of CD4, the primary viral receptor (1, 25, 36). Third, it stimulates virion infectivity by as yet ill-defined influences exerted during viral particle formation (3, 13, 54, 72, 73). Finally, it alters T-cell activation pathways, an effect that can be observed both in tissue culture and in transgenic mice (7, 9, 37, 51, 71).Several lines of evidence indicate that Nef down-modulates CD4 by acting as a receptor-specific sorting adapter. The Nef effect is exerted at a posttranslational level and, unlike phorbol myristate acetate-induced down-regulation, does not require phosphorylation of the CD4 cytoplasmic tail (25). The membrane-proximal 20 amino acids of CD4, including an essential dileucine motif, are necessary for Nef-mediated down-modulation and can transfer Nef sensitivity to another integral membrane protein (1). Although difficult to detect in mammalian cells, an interaction between Nef and CD4 could be demonstrated in insect cells infected with baculovirus vectors, in the yeast two-hybrid system, and in vitro with recombinant Nef protein and CD4-derived peptide (35, 39, 64). In these last two settings, the importance of the CD4 dileucine motif for association with Nef was confirmed (35, 64). Nuclear magnetic resonance (NMR) analyses further defined the CD4 binding site of Nef (33, 35). A pocket formed of the noncontiguous amino acids WLE₅₉, GGL₉₇, R₁₀₆, and L₁₁₀ bound a peptide corresponding to the CD4 tail, albeit with a low affinity. Supporting the functional relevance of these data, a mutation targeting WL₅₈ abrogated Nef-induced CD4 down-regulation (42). Additionally, human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus (SIV) Nef proteins require slightly different sequences in the CD4 cytoplasmic tail for efficient down-modulation, arguing against the existence of a cellular intermediate bridging Nef with CD4 (43).While it now appears well established that Nef binds CD4, overwhelming evidence also indicates that the viral protein interacts with the endocytic machinery. HIV-1 Nef can trigger the de novo formation of clathrin-coated pits (CCP) that preferentially incorporate CD4 (20). Furthermore, a chimeric integral membrane protein composed of the extracellular and transmembrane domains of CD4 or CD8 with Nef as its cytoplasmic tail undergoes rapid internalization and causes an increase in the clathrin lattice on the inner side of the cell membrane (20, 53). Not strictly a cell surface phenomenon, Nef-induced CD4 down-regulation additionally reflects some degree of intracellular retention and rerouting from the Golgi to the endosomal compartment (53).The model in which Nef acts as a connector between CD4 and CCP implies that the viral protein recognizes some component of the internalization machinery. Two such downstream partners have been recently proposed: the μ chain of the so-called adapter protein complexes (AP) (48, 60), and a subunit of the vacuolar ATPase, NBP1 (52). APs are heterotetrameric complexes which normally associate with receptor cytoplasmic tails containing tyrosine-based (8, 27, 56) and perhaps dileucine-based (40) signals and which recruit clathrin to induce the formation of CCP (24, 28, 69). AP-1 is present in the Golgi, and AP-2 is found at the plasma membrane (62). A third class of AP, AP-3, was recently identified and might be involved in lysosomal targeting (15, 18, 70). Nef proteins from HIV-1, HIV-2, and SIV were found to associate with the μ chain of both the Golgi (μ1) and plasma membrane (μ2) APs (48, 60). Mutating tyrosine residues at the N terminus of SIV Nef abrogated the Nef-μ interaction and prevented Nef-mediated CD4 down-regulation (60). In HIV-1 Nef, where these tyrosine-based motifs are absent, mutating a dileucine motif in a C-terminal disordered loop of the protein abrogated CD4 down-modulation (16). Furthermore, a 10- to 11-amino-acid sequence including this Nef-derived dileucine motif induced the accelerated internalization of a chimeric integral membrane protein (10, 16). Finally, the dileucine-dependent binding of HIV-1 Nef to APs could be demonstrated both in vitro and in tissue culture (16, 30). In another study, direct interactions between HIV-1 Nef and NBP1, the catalytic subunit of the vacuolar ATPase (V-ATPase), correlated with CD4 down-regulation (52). However, loss of interaction with NBP1 led to only a partial loss of the effect of Nef on CD4.Although less information is available about the mechanisms of Nef-induced MHC-I down-regulation, this receptor also exhibits increased rates of internalization and lysosomal degradation in the presence of the viral protein (66). Furthermore, HLA-A and HLA-B accumulate in the Golgi and colocalize with clathrin-coated vesicles in this setting (31, 48). Whether the parallel between CD4 and MHC-I down-modulation can be extended further is, however, unknown.To address this question, we analyzed the ability of a series of HIV-1 Nef mutants to down-regulate CD4 and MHC-I and to trigger in cis the accelerated endocytosis of a chimeric integral membrane protein. The results of our experiments support a model in which Nef uses distinct domains for connecting CD4 with cellular mediators of protein sorting and for down-modulating MHC-I. Additionally, we identify an N-terminal domain of Nef, shown by NMR to be an alpha-helix (5), as being crucial for MHC-I down-regulation.     

HIV-1 Nef Targets MHC-I and CD4 for Degradation Via a Final Common β-COP–Dependent Pathway in T Cells

To facilitate viral infection and spread, HIV-1 Nef disrupts the surface expression of the viral receptor (CD4) and molecules capable of presenting HIV antigens to the immune system (MHC-I). To accomplish this, Nef binds to the cytoplasmic tails of both molecules and then, by mechanisms that are not well understood, disrupts the trafficking of each molecule in different ways. Specifically, Nef promotes CD4 internalization after it has been transported to the cell surface, whereas Nef uses the clathrin adaptor, AP-1, to disrupt normal transport of MHC-I from the TGN to the cell surface. Despite these differences in initial intracellular trafficking, we demonstrate that MHC-I and CD4 are ultimately found in the same Rab7⁺ vesicles and are both targeted for degradation via the activity of the Nef-interacting protein, β-COP. Moreover, we demonstrate that Nef contains two separable β-COP binding sites. One site, an arginine (RXR) motif in the N-terminal α helical domain of Nef, is necessary for maximal MHC-I degradation. The second site, composed of a di-acidic motif located in the C-terminal loop domain of Nef, is needed for efficient CD4 degradation. The requirement for redundant motifs with distinct roles supports a model in which Nef exists in multiple conformational states that allow access to different motifs, depending upon which cellular target is bound by Nef.     

Loss of Myosin VI No Insert Isoform (NoI) Induces a Defect in Clathrin-mediated Endocytosis and Leads to Caveolar Endocytosis of Transferrin Receptor

Myosin VI is a motor protein that moves toward the minus end of actin filaments. It is involved in clathrin-mediated endocytosis and associates with clathrin-coated pits/vesicles at the plasma membrane. In this article the effect of the loss of myosin VI no insert isoform (NoI) on endocytosis in nonpolarized cells was examined. The absence of myosin VI in fibroblasts derived from the Snell''s waltzer mouse (myosin VI knock-out) gives rise to defective clathrin-mediated endocytosis with shallow clathrin-coated pits and a strong reduction in the internalization of clathrin-coated vesicles. To compensate for this defect in clathrin-mediated endocytosis, plasma membrane receptors such as the transferrin receptor (TfR) are internalized by a caveola-dependent pathway. Moreover the clathrin adaptor protein, AP-2, necessary for TfR internalization, follows the receptor and relocalizes in caveolae in Snell''s waltzer fibroblasts.     

Endocytosis of the ASGP receptor H1 is reduced by mutation of tyrosine- 5 but still occurs via coated pits

The clustering of plasma membrane receptors in clathrin-coated pits depends on determinants within their cytoplasmic domains. In several cases, individual tyrosine residues were shown to be necessary for rapid internalization. We have mutated the single tyrosine at position 5 in the cytoplasmic domain of the major subunit H1 of the asialoglycoprotein receptor to alanine. Expressed in fibroblasts cells, the mutant protein was accumulated in the plasma membrane, and its rate of internalization was reduced by a factor of four. The residual rate of endocytosis, however, was still significantly higher than that of resident plasma membrane proteins. Upon acidification of the cytoplasm, which specifically inhibits the formation of clathrin-coated vesicles but not uptake of the fluid phase marker Lucifer yellow, residual endocytosis was blocked. By immunoelectron microscopy mutant H1 could be directly demonstrated in coated pits. The fraction of wild-type and mutant H1 present in coated pits as determined by immunogold localization correlated well with the respective rates of internalization. Thus, mutation of tyrosine-5 only partially inactivates recognition of H1 for incorporation into coated pits.     

Mediation of Clathrin-Dependent Trafficking during Cytokinesis and Cell Expansion by Arabidopsis STOMATAL CYTOKINESIS DEFECTIVE Proteins

STOMATAL CYTOKINESIS DEFECTIVE1 (SCD1) encodes a putative Rab guanine nucleotide exchange factor that functions in membrane trafficking and is required for cytokinesis and cell expansion in Arabidopsis thaliana. Here, we show that the loss of SCD2 function disrupts cytokinesis and cell expansion and impairs fertility, phenotypes similar to those observed for scd1 mutants. Genetic and biochemical analyses showed that SCD1 function is dependent upon SCD2 and that together these proteins are required for plasma membrane internalization. Further specifying the role of these proteins in membrane trafficking, SCD1 and SCD2 proteins were found to be associated with isolated clathrin-coated vesicles and to colocalize with clathrin light chain at putative sites of endocytosis at the plasma membrane. Together, these data suggest that SCD1 and SCD2 function in clathrin-mediated membrane transport, including plasma membrane endocytosis, required for cytokinesis and cell expansion.     

Internalization of proprotein convertase PC7 from plasma membrane is mediated by a novel motif

Proprotein convertase 7 (PC7) is a member of the subtilisin-like proprotein convertase family, which is involved in the endoproteolysis of a variety of precursor proteins. Under steady state conditions, PC7 is mainly localized in the trans-Golgi network, but a small fraction is found at the cell surface. So far, no sorting signals for membrane trafficking have been identified in PC7. In this study, we have examined the internalization of PC7 from the plasma membrane. Our results show that internalization of PC7 is mediated by clathrin-coated vesicles. After inhibition of clathrin-mediated endocytosis using hypertonic conditions or the small molecule inhibitor, Pitstop 2, PC7 accumulated at the plasma membrane. Furthermore, PC7 was present in isolated clathrin-coated vesicles. To determine the internalization motif, constructs were generated in which parts of the N and C terminus of the cytoplasmic tail of PC7 were deleted, and chimeric proteins were constructed consisting of the luminal and transmembrane domains of Tac (CD25) and parts of the cytoplasmic domain of PC7. Antibody uptake experiments as well as surface biotinylation experiments demonstrated that the region between Ala(713) and Cys(726) in the cytoplasmic domain of PC7 is essential and sufficient for the internalization of PC7 but not for trans-Golgi network localization. Individual amino acids in this region were substituted with alanine, which identified Pro, Leu, and Cys as the essential amino acids. In conclusion, internalization of PC7 depends on a short transferable sequence in the cytoplasmic tail, which contains the three crucial amino acids PLC.     

Nef-induced CD4 degradation: a diacidic-based motif in Nef functions as a lysosomal targeting signal through the binding of beta-COP in endosomes

The Nef protein of primate lentiviruses downregulates the cell surface expression of CD4 through a two-step process. First, Nef connects the cytoplasmic tail of CD4 with adaptor protein complexes (AP), thereby inducing the formation of CD4-specific clathrin-coated pits that rapidly endocytose the viral receptor. Second, Nef targets internalized CD4 molecules for degradation. Here we show that Nef accomplishes this second task by acting as a connector between CD4 and the beta subunit of COPI coatomers in endosomes. A sequence encompassing a critical acidic dipeptide, located nearby but distinct from the AP-binding determinant of HIV-1 Nef, is responsible for beta-COP recruitment and for routing to lysosomes. A novel class of endosomal sorting motif, based on acidic residues, is thus revealed, and beta-COP is identified as its downstream partner.     

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.     

Lentiviral Nef suppresses iron uptake in a strain specific manner through inhibition of Transferrin endocytosis

Background

Increased cellular iron levels are associated with high mortality in HIV-1 infection. Moreover iron is an important cofactor for viral replication, raising the question whether highly divergent lentiviruses actively modulate iron homeostasis. Here, we evaluated the effect on cellular iron uptake upon expression of the accessory protein Nef from different lentiviral strains.

Results

Surface Transferrin receptor (TfR) levels are unaffected by Nef proteins of HIV-1 and its simian precursors but elevated in cells expressing Nefs from most other primate lentiviruses due to reduced TfR internalization. The SIV Nef-mediated reduction of TfR endocytosis is dependent on an N-terminal AP2 binding motif that is not required for downmodulation of CD4, CD28, CD3 or MHCI. Importantly, SIV Nef-induced inhibition of TfR endocytosis leads to the reduction of Transferrin uptake and intracellular iron concentration and is accompanied by attenuated lentiviral replication in macrophages.

Conclusion

Inhibition of Transferrin and thereby iron uptake by SIV Nef might limit viral replication in myeloid cells. Furthermore, this new SIV Nef function could represent a virus-host adaptation that evolved in natural SIV-infected monkeys.     

Human immunodeficiency virus type 1 envelope protein endocytosis mediated by a highly conserved intrinsic internalization signal in the cytoplasmic domain of gp41 is suppressed in the presence of the Pr55gag precursor protein.

The mechanisms involved in the incorporation of viral glycoproteins into virions are incompletely understood. For retroviruses, incorporation may involve interactions between the Gag proteins of these viruses and the cytoplasmic domains of the relevant envelope (Env) glycoproteins. Recent studies have identified within the cytoplasmic tail of the human immunodeficiency virus type 1 (HIV-1) Env protein a tyrosine-containing internalization motif similar to those found in the cytoplasmic domains of certain cell surface proteins that undergo rapid constitutive endocytosis in clathrin-coated pits. Given that surface expression of the HIV-1 Env protein is essential for the production of infectious virus, the presence of this internalization motif is surprising. We show here that in contrast to the rapid rate of Env protein internalization observed in cells expressing the Env protein in the absence of other HIV-1 proteins, the rate of internalization of Env protein from the surfaces of HIV-1-infected cells is extremely slow. The presence of the Pr55gag precursor protein is necessary and sufficient for inhibition of Env protein internalization, while a mutant Pr55-gag that is incapable of mediating Env incorporation into virions is also unable to inhibit endocytosis of the Env protein. The failure of the Env protein to undergo endocytosis from the surface of an HIV-1-infected cell may reflect the fact that the proposed interaction of the matrix domain of the Gag protein with Env during assembly prevents the interaction of Env with host adaptin molecules that recruit plasma membrane molecules such as the transferrin receptor into clathrin-coated pits. When the normal ratio of Gag and Env proteins in the infected cells is altered by overexpression of Env protein, this mechanism allows removal of excess Env protein from the cell surface. Taken together, these results suggest that a highly conserved system to reduce surface levels of the Env protein functions to remove Env protein that is not associated with Gag and that is therefore not destined for incorporation into virions. This mechanism for the regulation of surface levels of Env protein may protect infected cells from Env-dependent cytopathic effects or Env-specific immune responses.     

Basolateral sorting in MDCK cells requires a distinct cytoplasmic domain determinant

In MDCK cells, Golgi to basolateral transport of several membrane proteins has been found to involve a cytoplasmic domain determinant. In some cases (Fc receptor, lysosomal glycoprotein Igp120), the determinant appears similar to that required for endocytosis via clathrin-coated pits; for Igp120, elimination of a single cytoplasmic domain tyrosine both blocks internalization and results in apical transport. In other cases (LDL receptor), the determinant does not involve the cytoplasmic domain tyrosine required for endocytosis. Thus, contrary to current models, basolateral transport in MCDK cells occurs not by default but depends on one or more cytoplasmic domain determinants, the precise nature of which is unknown. For some proteins, it is closely related to coated pit determinants. The fact that many membrane proteins can reach the apical surface in the absence of this determinant suggests that signals for apical transport are widely distributed.     

CD4 down-regulation by HIV-1 and simian immunodeficiency virus (SIV) Nef proteins involves both internalization and intracellular retention mechanisms

Among the pleiotropic effects of Nef proteins of HIV and simian immunodeficiency virus (SIV), down-modulation of cell surface expression of CD4 is a prominent phenotype. It has been presumed that Nef proteins accelerate endocytosis of CD4 by linking the receptor to the AP-2 clathrin adaptor. However, the related AP-1 and AP-3 adaptors have also been shown to interact with Nef, hinting at role(s) for these complexes in the intracellular retention of CD4. By using genetic inhibitors of endocytosis and small interfering RNA-induced knockdown of AP-2, we show that accelerated CD4 endocytosis is not a dominant mechanism of HIV-1 (NL4-3 strain) Nef in epithelial cells, T lymphocyte cell lines, or peripheral blood lymphocytes. Furthermore, we show that both the CD4 recycling from the plasma membrane and the nascent CD4 in transit to the plasma membrane are susceptible to intracellular retention in HIV-1 Nef-expressing cells. In contrast, AP-2-mediated enhanced endocytosis constitutes the predominant mechanism for SIV (MAC-239 strain) Nef-induced down-regulation of human CD4 in human cells.