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.     

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 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.     

Simian and human immunodeficiency virus Nef proteins use different surfaces to downregulate class I major histocompatibility complex antigen expression

Simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 (HIV-1) Nef proteins are related regulatory proteins that share several functions, including the ability to downregulate class I major histocompatibility complex (MHC) and CD4 expression on the cell surface and to alter T-cell-receptor-initiated signal transduction in T cells. We compared the mechanisms used by SIV mac239 Nef and HIV-1 Nef to downregulate class I MHC and found that the ability of SIV Nef to downregulate class I MHC requires a unique C-terminal region of the SIV mac239 Nef molecule which is not found in HIV-1 Nef. Interestingly, mutation of the PxxP motif in SIV Nef, unlike in HIV-1 Nef, does not affect class I MHC downregulation. We also found that downregulation of class I MHC by SIV Nef requires a conserved tyrosine in the cytoplasmic domain of the class I MHC heavy chain and involves accelerated endocytosis of class I complexes, as previously found with HIV-1 Nef. Thus, while SIV and HIV-1 Nef proteins use a similar mechanism to downregulate class I MHC expression, they have evolved different surfaces for molecular interactions with cell factors that regulate class I MHC traffic. Mutations in the C-terminal domain of SIV mac239 Nef selectively disrupt class I MHC downregulation, having no detectable effect on other functions of Nef, such as the downregulation of CD4 and CD3 surface expression, the stimulation of SIV virion infectivity, and the induction of SIV replication from T cells infected in the absence of stimulation. The resulting mutants will be useful reagents for studying the importance of class I MHC downregulation for SIV replication and AIDS pathogenesis in infected rhesus macaques.     

Down-modulation of CD8αβ is a fundamental activity of primate lentiviral Nef proteins

It is well established that the Nef proteins of human and simian immunodeficiency viruses (HIV and SIV) modulate major histocompatibility complex class I (MHC-I) cell surface expression to protect infected cells against lysis by cytotoxic T lymphocytes (CTLs). Recent data supported the observation that Nef also manipulates CTLs directly by down-modulating CD8αβ (J. A. Leonard, T. Filzen, C. C. Carter, M. Schaefer, and K. L. Collins, J. Virol. 85:6867-6881, 2011), but it remained unknown whether this Nef activity is conserved between different lineages of HIV and SIV. In this study, we examined a total of 42 nef alleles from 16 different primate lentiviruses representing most major lineages of primate lentiviruses, as well as nonpandemic HIV-1 strains and the direct precursors of HIV-1 (SIVcpz and SIVgor). We found that the vast majority of these nef alleles strongly down-modulate CD8β in human T cells. Primate lentiviral Nefs generally interacted specifically with the cytoplasmic tail of CD8β, and down-modulation of this receptor was dependent on the conserved dileucine-based motif and two adjacent acidic residues (DD/E) in the C-terminal flexible loop of SIV Nef proteins. Both of these motifs are known to be important for the interaction of HIV-1 Nef with AP-2, and they were also shown to be critical for down-modulation of CD4 and CD28, but not MHC-I, by SIV Nefs. Our results show that down-modulation of CD4, CD8β, and CD28 involves largely overlapping (but not identical) domains and is most likely dependent on conserved interactions of primate lentiviral Nefs with cellular adaptor proteins. Furthermore, our data demonstrate that Nef-mediated down-modulation of CD8αβ is a fundamental property of primate lentiviruses and suggest that direct manipulation of CD8+ T cells plays a relevant role in viral immune evasion.     

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.     

Efficient class I major histocompatibility complex down-regulation by simian immunodeficiency virus Nef is associated with a strong selective advantage in infected rhesus macaques

Substitution of Y223F disrupts the ability of simian immunodeficiency virus (SIV) Nef to down-modulate major histocompatibility complex (MHC) class I from the cell surface but has no effect on other Nef functions, such as down-regulation of CD4, CD28, and CD3 cell surface expression or stimulation of viral replication and enhancement of virion infectivity. Inoculation of three rhesus macaques with the SIVmac239 Y223F-Nef variant revealed that this point mutation consistently reverts and that Nef activity in MHC class I down-modulation is fully restored within 4 weeks after infection. Our results demonstrate a strong selective pressure for a tyrosine at amino acid position 223 in SIV Nef, and they constitute evidence that Nef-mediated MHC class I down-regulation provides a selective advantage for viral replication in vivo.     

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.     

Cutting edge: SIV Nef protein utilizes both leucine- and tyrosine-based protein sorting pathways for down-regulation of CD4.

The Nef protein is unique to primate lentiviruses and is closely linked to accelerated pathogenesis in both human and monkey hosts. Nef acts to down-regulate CD4 and MHC class I, two receptors important for immune function. A recent report demonstrated the presence of two tyrosine motifs in SIV Nef that contribute to its ability to down-regulate CD4 and to associate with clathrin adaptors. These tyrosine motifs are not present in HIV-1 Nef, which instead utilizes a leucine-based motif for its down-regulation of CD4. We now report that SIV Nef also contains a conserved leucine-based motif that contributes to CD4 down-regulation, functions to stimulate internalization, and contributes to the association of SIV Nef with clathrin adaptors AP-1 and AP-2. These results demonstrate that SIV Nef differs from HIV-1 Nef by its ability to use two parallel pathways of the protein-sorting machinery based on either tyrosine or leucine motifs.     

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.     

Genetic and functional diversity of human immunodeficiency virus type 1 subtype B Nef primary isolates

We have characterized the functional integrity of seven primary Nef isolates: five from a long-term nonprogressing human immunodeficiency virus (HIV)-infected individual and one each from two patients with AIDS. One of the seven Nefs was defective for CD4 downregulation, two others were defective for PAK-2 activation, and one Nef was defective for PAK-2 activation and major histocompatibility complex (MHC) class I downregulation. Five of the Nefs were tested and found to be functional for the enhancement of virus particle infectivity. The structural basis for each of the functional defects has been analyzed by constructing a consensus nef, followed by mutational analysis of the variant amino acid residues. Mutations A29V and F193I were deleterious to CD4 downregulation and PAK-2 activation, respectively, while S189R rendered Nef defective for both MHC class I downregulation and PAK-2 activation. A search of the literature identified HIVs from five patients with Nefs predominantly mutated at F193 and from one patient with Nefs predominantly mutated at A29. A29 is highly conserved in all HIV subtypes except for subtype E. F193 is conserved in subtype B (and possibly in the closely related subtype D), but none of the other HIV group M subtypes. Our results suggest that functional distinctions may exist between HIV subtypes.     

The SH3 domain-binding surface and an acidic motif in HIV-1 Nef regulate trafficking of class I MHC complexes.

Nef, a regulatory protein of human and simian immunodeficiency viruses, downregulates cell surface expression of both class I MHC and CD4 molecules in T cells by accelerating their endocytosis. Fibroblasts were used to study alterations in the traffic of class I MHC complexes induced by Nef. We found that Nef downregulates class I MHC complexes by a novel mechanism involving the accumulation of endocytosed class I MHC in the trans-Golgi, where it colocalizes with the adaptor protein-1 complex (AP-1). This effect of Nef on class I MHC traffic requires the SH3 domain-binding surface and a cluster of acidic amino acid residues in Nef, both of which are also required for Nef to downregulate class I MHC surface expression and to alter signal transduction in T cells. Downregulation of class I MHC complexes from the surface of T cells also requires a tyrosine residue in the cytoplasmic domain of the class I MHC heavy chain molecule. The requirement of the same surfaces of the Nef molecule for downregulation of surface class I MHC complexes in T cells and for their accumulation in the trans-Golgi of fibroblasts indicates that the two effects of Nef involve similar interactions with the host cell machinery and involve a molecular mechanism regulating class I MHC traffic that is common for both of these cell types. Interestingly, the downregulation of class I MHC does not require the ability of Nef to colocalize with the adaptor protein-2 complex (AP-2). We showed previously that the ability of Nef to colocalize with AP-2 correlates with the ability of Nef to downregulate CD4 expression. Our observations indicate that Nef downregulates class I MHC and CD4 surface expression via different interactions with the protein sorting machinery, and link the sorting and signal transduction machineries in the regulation of class I MHC surface expression by Nef.     

Two sorting motifs, a ubiquitination motif and a tyrosine motif, are involved in HIV-1 and simian immunodeficiency virus Nef-mediated receptor endocytosis

HIV-1 and SIV Nef proteins downregulate cell surface CD4 and MHC class I (MHC-I) molecules of infected cells, which are necessary for efficient viral replication and pathogenicity. We previously reported that K144 in HIV-1 Nef is di-ubiquitinated, and K144R substitution impairs Nef-mediated CD4 downregulation. In this report, we extend the role of ubiquitination at this lysine residue from Nef-mediated CD4 downregulation to Nef-mediated MHC-I downregulation and from HIV Nef to SIV Nef. All HIV-1 Nef mutants that contain K144R substitution are inactive in MHC-I downregulation. Tested MHC-I alleles include HLA-ABC endogenously expressed and HLA-A2 exogenously expressed in Jurkat T cells. CD4 downregulation by SIV Nef involves K176 that aligns with K144 in HIV-1 Nef, as well as an N-terminal tyrosine motif Y28Y39 not present in HIV-1 Nef. Dual mutation at K176 and Y28Y39 completely impaired SIV Nef-mediated CD4 and MHC-I downregulation, whereas a single mutation at K176 or Y28Y39 did not. The involvement of tyrosine motif in SIV Nef-mediated CD4 and MHC-I downregulation prompted us to investigate a putative tyrosine motif (Y202Y/F203) in HIV-1 Nef that is conserved among HIV-1 species. Single mutation at the tyrosine motif Y202F203 in HIV-1 Nef (NA7) greatly impaired Nef-mediated CD4 downregulation, which is similar to what we observed previously with the single mutation at lysine K144. Thus, our study demonstrated that Nef-mediated receptor endocytosis involves the ubiquitination motif and tyrosine motif.     

Disrupting surfaces of nef required for downregulation of CD4 and for enhancement of virion infectivity attenuates simian immunodeficiency virus replication in vivo

The multifunctional simian and human immunodeficiency virus (SIV and HIV) Nef proteins are important for virulence. We studied the importance of selected Nef functions using an SIV Nef with mutations in two regions that are required for CD4 downregulation. This Nef mutant is defective for downregulating CD4 and, in addition, for enhancing SIV infectivity and induction of SIV replication from infected quiescent peripheral blood mononuclear cells, but not for other known functions, including downregulation of class I major histocompatibility complex (MHC) cell surface expression. Replication of SIV containing this Nef variant in rhesus monkeys was attenuated early during infection. Subsequent increases in viral load coincided with selection of reversions and second-site compensatory changes in Nef. Our results indicate that the surfaces of Nef that mediate CD4 downregulation and the enhancement of virion infectivity are critical for SIV replication in vivo. Furthermore, these findings indicate that class I MHC downregulation by Nef is not sufficient for SIV virulence early in infection.     

Negative factor from SIV binds to the catalytic subunit of the V-ATPase to internalize CD4 and to increase viral infectivity

The accessory protein negative factor (Nef) from human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) is required for optimal viral infectivity and the progression to acquired immunodeficiency syndrome (AIDS). Nef interacts with the endocytic machinery, resulting in the down-regulation of cluster of differentiation antigen 4 (CD4) and major histocompatibility complex class I (MHCI) molecules on the surface of infected cells. Mutations in the C-terminal flexible loop of Nef result in a lower rate of internalization by this viral protein. However, no loop-dependent binding of Nef to adaptor protein-2 (AP-2), which is the adaptor protein complex that is required for the internalization of proteins from the plasma membrane, could be demonstrated. In this study we investigated the relevance of different motifs in Nef from SIV(mac239) for its internalization, CD4 down-regulation, binding to components of the trafficking machinery, and viral infectivity. Our data suggest that the binding of Nef to the catalytic subunit H of the vacuolar membrane ATPase (V-ATPase) facilitates its internalization. This binding depends on the integrity of the whole flexible loop. Subsequent studies on Nef mutant viruses revealed that the flexible loop is essential for optimal viral infectivity. Therefore, our data demonstrate how Nef contacts the endocytic machinery in the absence of its direct binding to AP-2 and suggest an important role for subunit H of the V-ATPase in viral infectivity.     

Primary sooty mangabey simian immunodeficiency virus and human immunodeficiency virus type 2 nef alleles modulate cell surface expression of various human receptors and enhance viral infectivity and replication

The nef gene of the pathogenic simian immunodeficiency virus (SIV) mac239 clone has been well characterized. Little is known, however, about the function of nef alleles derived from naturally SIVsm-infected sooty mangabeys (Cercocebus atys) and from human immunodeficiency virus type 2 (HIV-2)-infected individuals. Addressing this, we demonstrate that, similarly to the SIVmac239 nef, primary SIVsm and HIV-2 nef alleles down-modulate cell surface expression of human CD4, CD28, CD3, and class I or II major histocompatibility complex (MHC-I or MHC-II, respectively) molecules, up-regulate surface expression of the invariant chain (Ii) associated with immature MHC-II, inhibit early T-cell activation events, and enhance virion infectivity. Both also stimulate viral replication, although HIV-2 nef alleles were less active in this assay than SIVsm nef alleles. Mutational analysis showed that a dileucine-based sorting motif in the C-proximal loop of SIV or HIV-2 Nef is critical for its effects on CD4, CD28, and Ii but dispensable for down-regulation of CD3, MHC-I, and MHC-II. The C terminus of SIV and HIV-2 Nef was exclusively required for down-modulation of MHC-I, further demonstrating that analogous functions are mediated by different domains in Nef proteins derived from different groups of primate lentiviruses. Our results demonstrate that none of the eight Nef functions investigated had been newly acquired after cross-species transmission of SIVsm from naturally infected mangabeys to humans or macaques. Notably, HIV-2 and SIVsm nef alleles efficiently down-modulate CD3 and C28 surface expression and inhibit T-cell activation more efficiently than HIV-1 nef alleles. These differences in Nef function might contribute to the relatively low levels of immune activation observed in HIV-2-infected human individuals.     

In vitro treatment of human monocytes/macrophages with myristoylated recombinant Nef of human immunodeficiency virus type 1 leads to the activation of mitogen-activated protein kinases, IkappaB kinases, and interferon regulatory factor 3 and to the release of beta interferon

The viral protein Nef is a virulence factor that plays multiple roles during the early and late phases of human immunodeficiency virus (HIV) replication. Nef regulates the cell surface expression of critical proteins (including down-regulation of CD4 and major histocompatibility complex class I), T-cell receptor signaling, and apoptosis, inducing proapoptotic effects in uninfected bystander cells and antiapoptotic effects in infected cells. It has been proposed that Nef intersects the CD40 ligand signaling pathway in macrophages, leading to modification in the pattern of secreted factors that appear able to recruit and activate T lymphocytes, rendering them susceptible to HIV infection. There is also increasing evidence that in vitro cell treatment with Nef induces signaling effects. Exogenous Nef treatment is able to induce apoptosis in uninfected T cells, maturation in dendritic cells, and suppression of CD40-dependent immunoglobulin class switching in B cells. Previously, we reported that Nef treatment of primary human monocyte-derived macrophages (MDMs) induces a cycloheximide-independent activation of NF-kappaB and the synthesis and secretion of a set of chemokines/cytokines that activate STAT1 and STAT3. Here, we show that Nef treatment is capable of hijacking cellular signaling pathways, inducing a very rapid regulatory response in MDMs that is characterized by the rapid and transient phosphorylation of the alpha and beta subunits of the IkappaB kinase complex and of JNK, ERK1/2, and p38 mitogen-activated protein kinase family members. In addition, we have observed the activation of interferon regulatory factor 3, leading to the synthesis of beta interferon mRNA and protein, which in turn induces STAT2 phosphorylation. All of these effects require Nef myristoylation.     

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.     

Simian immunodeficiency virus Nef protein delays the progression of CD4+ T cells through G1/S phase of the cell cycle

Human and simian immunodeficiency virus (HIV and SIV, respectively) infections are characterized by gradual depletion of CD4+ T cells. The underlying mechanisms of CD4+ T-cell depletion and HIV and SIV persistence are not fully determined. The Nef protein is expressed early in infection and is necessary for pathogenesis. Nef can cause T-cell activation and downmodulates cell surface signaling molecules. However, the effect of Nef on the cell cycle has not been well characterized. To determine the role of Nef in the cell cycle, we investigated whether the SIV Nef protein can modulate cell proliferation and apoptosis in CD4+ Jurkat T cells. We developed a CD4+ Jurkat T-cell line that stably expresses SIV Nef under the control of an inducible promoter. Alterations in cell proliferation were determined by flow cytometry using stable intracytoplasmic fluorescent dye 5- and 6-carboxyfluorescein diacetate succinimidyl ester and bromodeoxyuridine incorporation. Apoptotic cell death was measured by annexin V and propidium iodide staining. Our results demonstrated that SIV Nef inhibited Fas-induced apoptosis in these cells and that the mechanism involved upregulation of the Bcl-2 protein. SIV Nef suppressed CD4+ T-cell proliferation by inhibiting the progression of cells into S phase of the cell cycle. Suppression involved an upregulation of cyclin-dependent kinase inhibitors p21 and p27 and the downregulation of cyclin D1 and cyclin A. In summary, inhibition of apoptosis by Nef can lead to persistence of infected cells and can support viral replication. In addition, a Nef-mediated delay in cell cycle progression may contribute to CD4+ T-cell anergy/depletion seen in HIV and SIV disease.