Viral Accessory Protein Research Articles

Expression and purification of soluble HIV-2 viral protein R (Vpr) using a sandwich-fusion protein strategy

Viral accessory proteins of the human immunodeficiency virus (HIV), including virus protein R (Vpr), are crucial for the efficient replication of the virus in the host organism. While functional data are available for HIV-1 Vpr, there is a paucity of data describing the function and structure of HIV-2 Vpr. In this report, the construction of a His6-MBP-intein1-Vpr-intein2-Cyt b5-His6 fusion protein is presented. Unlike previous research efforts where only microgram quantities of HIV-1 Vpr could be produced, this construct enabled soluble milligram yields via an Escherichia coli over-expression system. Straightforward protein purification of HIV-2 Vpr was achieved by standard chromatography routines and autocatalytic intein cleavage. Preliminary structural studies by circular dichroism (CD) and NMR spectroscopy revealed that the protein is stable in the presence of micellar concentrations of the detergent DPC and adopts an α-helix secondary structure.

Structural basis of lentiviral subversion of a cellular protein degradation pathway

Lentiviruses contain accessory genes that have evolved to counteract the effects of host cellular defence proteins that inhibit productive infection. One such restriction factor, SAMHD1, inhibits HIV-1 infection of myeloid-lineage cells 1,2 as well as resting CD4+ T cells 3,4 by reducing the cellular dNTP concentration to a level where the viral reverse transcriptase cannot function 5,6. In other lentiviruses, including HIV-2 and related SIVs, SAMHD1 restriction is overcome by the action of viral accessory protein x (Vpx) or the related viral protein r (Vpr) that target and recruit SAMHD1 for proteasomal degradation 7,8. The molecular mechanism by which these viral proteins are able to usurp the host cell’s ubiquitination machinery to destroy the cell’s protection against these viruses has not been defined. We present here the crystal structure of a ternary complex of Vpx with the host cell’s E3 ligase substrate adaptor DCAF1 and the C-terminal region of SAMHD1. Vpx is made up of a three-helical bundle, stabilised by a zinc finger motif and wraps tightly around the disc-shaped DCAF1 molecule to present a new molecular surface. This adapted surface is then able to recruit SAMHD1 via its C-terminus making it a competent substrate for the E3 ligase to mark for proteasomal degradation. The structure provides the first description of how a lentiviral accessory protein is able to subvert the cell’s normal protein degradation pathway to inactivate the cellular viral defence system.

Antagonism of SAMHD1 is actively maintained in natural infections of simian immunodeficiency virus.

Restriction factors are effectors of the innate immune response to viral pathogens that inhibit viral replication by operating as molecular barriers to steps of the viral life cycle. The restriction factor SAMHD1 blocks lentiviral reverse transcription in myeloid cells and resting CD4+ T cells. Many lineages of lentiviruses, including HIV-2 and other simian immunodeficiency viruses, encode accessory genes that serve to counteract host SAMHD1 restriction by causing degradation of the antiviral factor. The viral accessory protein Vpr is responsible for SAMHD1 degradation in some lineages of lentiviruses, whereas in others the related protein Vpx assumes this task. However, HIV-1 has no SAMHD1 degradation capability, leading to questions about the selective advantage of this activity. We use an evolutionary approach to examine the importance of SAMHD1 antagonism for viral fitness by studying adaptation to host SAMHD1 in natural simian immunodeficiency virus infections of African Green Monkeys. We identified multiple SAMHD1 haplotypes in African Green Monkeys and find that the vpr gene from different strains of Simian Immunodeficiency Virus has adapted to the polymorphisms of the African Green Monkey population in which it is found. Such evidence of viral adaptation to host restriction indicates that SAMHD1 antagonism is actively maintained in natural infections and that this function must be advantageous to viral fitness, despite its absence in HIV-1.

Discovery of a diaminoquinoxaline benzenesulfonamide antagonist of HIV-1 Nef function using a yeast-based phenotypic screen

BackgroundHIV-1 Nef is a viral accessory protein critical for AIDS progression. Nef lacks intrinsic catalytic activity and binds multiple host cell signaling proteins, including Hck and other Src-family tyrosine kinases. Nef binding induces constitutive Hck activation that may contribute to HIV pathogenesis by promoting viral infectivity, replication and downregulation of cell-surface MHC-I molecules. In this study, we developed a yeast-based phenotypic screen to identify small molecules that inhibit the Nef-Hck complex.ResultsNef-Hck interaction was faithfully reconstituted in yeast cells, resulting in kinase activation and growth arrest. Yeast cells expressing the Nef-Hck complex were used to screen a library of small heterocyclic compounds for their ability to rescue growth inhibition. The screen identified a dihydrobenzo-1,4-dioxin-substituted analog of 2-quinoxalinyl-3-aminobenzene-sulfonamide (DQBS) as a potent inhibitor of Nef-dependent HIV-1 replication and MHC-I downregulation in T-cells. Docking studies predicted direct binding of DQBS to Nef which was confirmed in differential scanning fluorimetry assays with recombinant purified Nef protein. DQBS also potently inhibited the replication of HIV-1 NL4-3 chimeras expressing Nef alleles representative of all M-group HIV-1 clades.ConclusionsOur findings demonstrate the utility of a yeast-based growth reversion assay for the identification of small molecule Nef antagonists. Inhibitors of Nef function discovered with this assay, such as DQBS, may complement the activity of current antiretroviral therapies by enabling immune recognition of HIV-infected cells through the rescue of cell surface MHC-I.

The Innate Immune Factor Apolipoprotein L1 Restricts HIV-1 Infection

Apolipoprotein L1 (APOL1) is a major component of the human innate immune response against African trypanosomes. Although the mechanism of the trypanolytic activity of circulating APOL1 has been recently clarified, the intracellular function(s) of APOL1 in human cells remains poorly defined. Like that of many genes linked to host immunity, APOL1 expression is induced by proinflammatory cytokines gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α). Additionally, IFN-γ-polarized macrophages that potently restrict HIV-1 replication express APOL1, which suggests that APOL1 may contribute to HIV-1 suppression. Here, we report that APOL1 inhibits HIV-1 replication by multiple mechanisms. We found that APOL1 protein targeted HIV-1 Gag for degradation by the endolysosomal pathway. Interestingly, we found that APOL1 stimulated both endocytosis and lysosomal biogenesis by promoting nuclear localization of transcription factor EB (TFEB) and expression of TFEB target genes. Moreover, we demonstrated that APOL1 depletes cellular viral accessory protein Vif, which counteracts the host restriction factor APOBEC3G, via a pathway involving degradation of Vif in lysosomes and by secretion of Vif in microvesicles. As a result of Vif depletion by APOL1, APOBEC3G was not degraded and reduced infectivity of progeny virions. In support of this model, we also showed that endogenous expression of APOL1 in differentiated U937 monocytic cells stimulated with IFN-γ resulted in a reduced production of virus particles. This finding supports the hypothesis that induction of APOL1 contributes to HIV-1 suppression in differentiated monocytes. Deciphering the precise mechanism of APOL1-mediated HIV-1 restriction may facilitate the design of unique therapeutics to target HIV-1 replication.

Expression of HIV-1 Vpu Leads to Loss of the Viral Restriction Factor CD317/Tetherin from Lipid Rafts and Its Enhanced Lysosomal Degradation

CD317/tetherin (aka BST2 or HM1.24 antigen) is an interferon inducible membrane protein present in regions of the lipid bilayer enriched in sphingolipids and cholesterol (often termed lipid rafts). It has been implicated in an eclectic mix of cellular processes including, most notably, the retention of fully formed viral particles at the surface of cells infected with HIV and other enveloped viruses. Expression of the HIV viral accessory protein Vpu has been shown to lead to intracellular sequestration and degradation of tetherin, thereby counteracting the inhibition of viral release. There is evidence that tetherin interacts directly with Vpu, but it remains unclear where in the cell this interaction occurs or if Vpu expression affects the lipid raft localisation of tetherin. We have addressed these points using biochemical and cell imaging approaches focused on endogenous rather than ectopically over-expressed tetherin. We find i) no evidence for an interaction between Vpu and endogenous tetherin at the cell surface, ii) the vast majority of endogenous tetherin that is at the cell surface in control cells is in lipid rafts, iii) internalised tetherin is present in non-raft fractions, iv) expression of Vpu in cells expressing endogenous tetherin leads to the loss of tetherin from lipid rafts, v) internalised tetherin enters early endosomes, and late endosomes, in both control cells and cells expressing Vpu, but the proportion of tetherin molecules destined for degradation rather than recycling is increased in cells expressing Vpu vi) lysosomes are the primary site for degradation of endogenous tetherin in cells expressing Vpu. Our studies underlie the importance of studying endogenous tetherin and let us propose a model in which Vpu intercepts newly internalised tetherin and diverts it for lysosomal destruction rather than recycling to the cell surface.

Allosteric Inhibitors of the NS3 Protease from the Hepatitis C Virus

The nonstructural protein 3 (NS3) from the hepatitis C virus processes the non-structural region of the viral precursor polyprotein in infected hepatic cells. The NS3 protease activity has been considered a target for drug development since its identification two decades ago. Although specific inhibitors have been approved for clinical therapy very recently, resistance-associated mutations have already been reported for those drugs, compromising their long-term efficacy. Therefore, there is an urgent need for new anti-HCV agents with low susceptibility to resistance-associated mutations. Regarding NS3 protease, two strategies have been followed: competitive inhibitors blocking the active site and allosteric inhibitors blocking the binding of the accessory viral protein NS4A. In this work we exploit the intrinsic Zn+2-regulated plasticity of the protease to identify a new type of allosteric inhibitors. In the absence of Zn+2, the NS3 protease adopts a partially-folded inactive conformation. We found ligands binding to the Zn+2-free NS3 protease, trap the inactive protein, and block the viral life cycle. The efficacy of these compounds has been confirmed in replicon cell assays. Importantly, direct calorimetric assays reveal a low impact of known resistance-associated mutations, and enzymatic assays provide a direct evidence of their inhibitory activity. They constitute new low molecular-weight scaffolds for further optimization and provide several advantages: 1) new inhibition mechanism simultaneously blocking substrate and cofactor interactions in a non-competitive fashion, appropriate for combination therapy; 2) low impact of known resistance-associated mutations; 3) inhibition of NS4A binding, thus blocking its several effects on NS3 protease.

Evasion of Antiviral Innate Immunity by Theiler's Virus L* Protein through Direct Inhibition of RNase L

Theiler's virus is a neurotropic picornavirus responsible for chronic infections of the central nervous system. The establishment of a persistent infection and the subsequent demyelinating disease triggered by the virus depend on the expression of L*, a viral accessory protein encoded by an alternative open reading frame of the virus. We discovered that L* potently inhibits the interferon-inducible OAS/RNase L pathway. The antagonism of RNase L by L* was particularly prominent in macrophages where baseline oligoadenylate synthetase (OAS) and RNase L expression levels are elevated, but was detectable in fibroblasts after IFN pretreatment. L* mutations significantly affected Theiler's virus replication in primary macrophages derived from wild-type but not from RNase L-deficient mice. L* counteracted the OAS/RNase L pathway through direct interaction with the ankyrin domain of RNase L, resulting in the inhibition of this enzyme. Interestingly, RNase L inhibition was species-specific as Theiler's virus L* protein blocked murine RNase L but not human RNase L or RNase L of other mammals or birds. Direct RNase L inhibition by L* and species specificity were confirmed in an in vitro assay performed with purified proteins. These results demonstrate a novel viral mechanism to elude the antiviral OAS/RNase L pathway. By targeting the effector enzyme of this antiviral pathway, L* potently inhibits RNase L, underscoring the importance of this enzyme in innate immunity against Theiler's virus.

NS3 Protease from Hepatitis C Virus: Biophysical Studies on an Intrinsically Disordered Protein Domain

The nonstructural protein 3 (NS3) from the hepatitis C virus (HCV) is responsible for processing the non-structural region of the viral precursor polyprotein in infected hepatic cells. NS3 protease activity, located at the N-terminal domain, is a zinc-dependent serine protease. A zinc ion, required for the hydrolytic activity, has been considered as a structural metal ion essential for the structural integrity of the protein. In addition, NS3 interacts with another cofactor, NS4A, an accessory viral protein that induces a conformational change enhancing the hydrolytic activity. Biophysical studies on the isolated protease domain, whose behavior is similar to that of the full-length protein (e.g., catalytic activity, allosteric mechanism and susceptibility to inhibitors), suggest that a considerable global conformational change in the protein is coupled to zinc binding. Zinc binding to NS3 protease can be considered as a folding event, an extreme case of induced-fit binding. Therefore, NS3 protease is an intrinsically (partially) disordered protein with a complex conformational landscape due to its inherent plasticity and to the interaction with its different effectors. Here we summarize the results from a detailed biophysical characterization of this enzyme and present new experimental data.

Creation of a completely helper cell-dependent recombinant morbillivirus

We have created a completely helper cell-dependent morbillivirus by modifying the genome to remove the coding sequence of the phosphoprotein (P) and recovering the recombinant virus in a cell line constitutively expressing the P protein. The P protein-deleted virus (P−) grew very inefficiently unless both of the viral accessory proteins (V and C) were also expressed. Growth of the virus was restricted to the P-expressing cell line. The P− virus grew more slowly than the parental virus and expressed much less viral protein in infected cells. The technique could be used to create virus-like particles for use as a vaccine or as antigen in immunological or serological assays.

HIV-2 and SIVmac Accessory Virulence Factor Vpx Down-regulates SAMHD1 Enzyme Catalysis Prior to Proteasome-dependent Degradation

SAMHD1, a dGTP-regulated deoxyribonucleoside triphosphate (dNTP) triphosphohydrolase, down-regulates dNTP pools in terminally differentiated and quiescent cells, thereby inhibiting HIV-1 infection at the reverse transcription step. HIV-2 and simian immunodeficiency virus (SIV) counteract this restriction via a virion-associated virulence accessory factor, Vpx (Vpr in some SIVs), which loads SAMHD1 onto CRL4-DCAF1 E3 ubiquitin ligase for polyubiquitination, programming it for proteasome-dependent degradation. However, the detailed molecular mechanisms of SAMHD1 recruitment to the E3 ligase have not been defined. Further, whether divergent, orthologous Vpx proteins, encoded by distinct HIV/SIV strains, bind SAMHD1 in a similar manner, at a molecular level, is not known. We applied surface plasmon resonance analysis to assess the requirements for and kinetics of binding between various primate SAMHD1 proteins and Vpx proteins from SIV or HIV-2 strains. Our data indicate that Vpx proteins, bound to DCAF1, interface with the C terminus of primate SAMHD1 proteins with nanomolar affinity, manifested by rapid association and slow dissociation. Further, we provide evidence that Vpx binding to SAMHD1 inhibits its catalytic activity and induces disassembly of a dGTP-dependent oligomer. Our studies reveal a previously unrecognized biochemical mechanism of Vpx-mediated SAMHD1 inhibition: direct down-modulation of its catalytic activity, mediated by the same binding event that leads to SAMHD1 recruitment to the E3 ubiquitin ligase for proteasome-dependent degradation.

The progestin-only contraceptive medroxyprogesterone acetate, but not norethisterone acetate, enhances HIV-1 Vpr-mediated apoptosis in human CD4+ T cells through the glucocorticoid receptor.

The glucocorticoid receptor (GR) regulates several physiological functions, including immune function and apoptosis. The HIV-1 virus accessory protein, viral protein R (Vpr), can modulate the transcriptional response of the GR. Glucocorticoids (GCs) and Vpr have been reported to induce apoptosis in various cells, including T-cells. We have previously shown that the injectable contraceptive, medroxyprogesterone acetate (MPA) is a partial to full agonist for the GR, unlike norethisterone acetate (NET-A). We investigated the functional cross talk between the GR and Vpr in inducing apoptosis in CD4+ T-cells, in the absence and presence of GCs and these progestins, as well as progesterone. By using flow cytometry, we show that, in contrast to NET-A and progesterone, the synthetic GR ligand dexamethasone (Dex), cortisol and MPA induce apoptosis in primary CD4+ T-cells. Furthermore, the C-terminal part of the Vpr peptide, or HIV-1 pseudovirus, together with Dex or MPA further increased the apoptotic phenotype, unlike NET-A and progesterone. By a combination of Western blotting, PCR and the use of receptor- selective agonists, we provide evidence that the GR and the estrogen receptor are the only steroid receptors expressed in peripheral blood mononuclear cells. These results, together with the findings that RU486, a GR antagonist, prevents Dex-, MPA- and Vpr-mediated apoptosis, provide evidence for the first time that GR agonists or partial agonists increase apoptosis in primary CD4+ T-cells via the GR. We show that apoptotic induction involves differential expression of key apoptotic genes by both Vpr and GCs/MPA. This work suggests that contraceptive doses of MPA but not NET-A or physiological doses of progesterone could potentially accelerate depletion of CD4+ T-cells in a GR-dependent fashion in HIV-1 positive women, thereby contributing to immunodeficiency. The results imply that choice of progestin used in contraception may be critical to susceptibility and progression of diseases such as HIV-1.

Novel factors interfering with human immunodeficiency virus‐type 1 replication in vivo and in vitro

The strategy of all retroviral infections is based on establishing an equilibrium between virus replication and proviral latency in the infected host. The human immunodeficiency virus-type 1 (HIV-1), belonging to the subfamily of lentiviridae, adds an additional level of sophistication to this general rule by encoding two regulatory genes (tat and rev) and four accessory genes (nef, vif, vpr and vpu); HIV-2, structurally similar to HIV-1 but characterized by lower pathogenicity in vivo, encodes another accessory gene, vpx. The function of these accessory genes has become clear in recent years: they serve as countermeasures to host-cell restriction factors that prevent or curtail the capacity of HIV to productively infect its target cells (typically, CD4+ T lymphocytes, macrophages and dendritic cells). Some of the best characterized restriction factors for HIV-1 are Tripartite Motif-5α (TRIM5α), preventing infection of nonhuman primates, although not being effective in humans, and apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3G (APOBEC 3G), counteracted by the viral accessory protein Vif. In addition, several other molecules are under scrutiny for their mechanism of action and potential exploitation as novel anti-HIV agents. This review will summarize the recently emerging knowledge on these novel factors and their potential relevance for the discovery of new anti-HIV agents targeting not only the replicative, but also the latent state of HIV infection.

VprBP (DCAF1): a promiscuous substrate recognition subunit that incorporates into both RING-family CRL4 and HECT-family EDD/UBR5 E3 ubiquitin ligases

The terminal step in the ubiquitin modification system relies on an E3 ubiquitin ligase to facilitate transfer of ubiquitin to a protein substrate. The substrate recognition and ubiquitin transfer activities of the E3 ligase may be mediated by a single polypeptide or may rely on separate subunits. The latter organization is particularly prevalent among members of largest class of E3 ligases, the RING family, although examples of this type of arrangement have also been reported among members of the smaller HECT family of E3 ligases. This review describes recent discoveries that reveal the surprising and distinctive ability of VprBP (DCAF1) to serve as a substrate recognition subunit for a member of both major classes of E3 ligase, the RING-type CRL4 ligase and the HECT-type EDD/UBR5 ligase. The cellular processes normally regulated by VprBP-associated E3 ligases, and their targeting and subversion by viral accessory proteins are also discussed. Taken together, these studies provide important insights and raise interesting new questions regarding the mechanisms that regulate or subvert VprBP function in the context of both the CRL4 and EDD/UBR5 E3 ligases.

The Vpx Lentiviral Accessory Protein Targets SAMHD1 for Degradation in the Nucleus

Sterile alpha motif domain- and HD domain-containing protein 1 (SAMHD1) is a deoxynucleoside triphosphohydrolase that restricts the replication of lentiviruses in myeloid cells by hydrolyzing the cellular deoxynucleotide triphosphates to a level below that which is required for reverse transcription. Human immunodeficiency virus type 2 (HIV-2) and some simian immunodeficiency viruses (SIVs) encode the accessory protein viral protein X (Vpx) that counteracts SAMHD1. Vpx recruits SAMHD1 to a cullin4A-RING E3 ubiquitin ligase (CRL4), which targets the enzyme for proteasomal degradation. Vpx and SAMHD1 both localize to the nucleus of the cell. We identified the nuclear localization sequence (NLS) of SAMHD1 as a conserved KRPR sequence at amino acid residues 11 to 14. SAMHD1 lacking a functional NLS localized to the cytoplasm but retained its triphosphohydrolase and antiviral activities. However, cytoplasmic SAMHD1 was resistant to Vpx-induced degradation, and its antiviral activity was not counteracted by Vpx. Cytoplasmic SAMHD1 interacted with Vpx and retained it in the cytoplasm. The inhibition of nuclear export with leptomycin B did not impair the ability of Vpx to degrade SAMHD1. These findings suggest that SAMHD1 is targeted by Vpx for ubiquitination and degradation in the nucleus.

Role of SAMHD1 nuclear localization in restriction of HIV-1 and SIVmac

BackgroundSAMHD1 is a nuclear protein that blocks lentiviral infection before reverse transcription in macrophages and dendritic cells. The viral accessory protein Vpx overcomes the SAMHD1-mediated lentiviral block by inducing its proteasomal degradation.ResultsHere, we identified the nuclear localization signal (NLS) of SAMHD1, and studied its contribution to restriction of HIV-1 and SIVmac. By studying the cellular distribution of different SAMHD1 variants, we mapped the nuclear localization of SAMHD1 to residues 11KRPR14. Mutagenesis of these residues changed the cellular distribution of SAMHD1 from the nucleus to the cytoplasm. SAMHD1 mutants that lost nuclear localization restricted HIV-1 and SIV as potently as the wild type protein. Interestingly, SAMHD1 mutants that localized to the cytoplasm were not degraded by nuclear Vpx alleles. Therefore, nuclear Vpx alleles require nuclear localization of SAMHD1 in order to induce its degradation. In agreement, SIVmac viruses encoding Vpx did not overcome the restriction imposed by the cytoplasmic variants of SAMHD1.ConclusionsWe mapped the NLS of SAMHD1 to residues 11KRPR14 and studied the contribution of SAMHD1 nuclear localization to restriction of HIV-1 and SIV. These experiments demonstrate that cytoplasmic variants of SAMHD1 potently block lentiviral infection and are resistant to Vpx-mediated degradation. The nuclear Vpx alleles studied here are only capable of degrading a nuclearly localized SAMHD1 suggesting that Vpx-mediated degradation of SAMHD1 is initiated in the nucleus.

Sites of Action of HIV-1 Vpu in BST-2/Tetherin Downregulation

The interferon-inducible host restriction factor bone marrow stromal antigen 2 (BST-2/tetherin) blocks the release of human immunodeficiency virus type 1 (HIV-1) by directly cross-linking virions to the membrane of infected cells. This antiviral effect is counteracted by the HIV-1 accessory protein viral protein U (Vpu) through mechanisms that remain unclear. Accumulating evidence suggests that Vpu antagonizes BST-2 by removing it from the plasma membrane; however, neither the cellular sites of interaction nor the effector mechanisms that result in the downregulation of BST-2 cell-surface expression have been fully determined. Based on current evidence regarding the subcellular localization of Vpu and BST-2 and the latter's trafficking defects induced by their interaction, three models have been proposed. In the first, Vpu is hypothesized to block the traffic of newly synthesized BST-2 towards the cell surface by retaining it in the biosynthetic/secretory compartment. The second model suggests that Vpu sequesters BST-2 within intracellular compartments corresponding to recycling endosomes and the trans-Golgi network by blocking its recycling after endocytosis. In the third model, we and others have proposed that Vpu directly internalizes BST-2 from the plasma membrane and induces its enhanced endolysosomal trafficking and degradation. As for its intracellular fate, the viral antagonism of BST-2 is likely dependent on the intracellular sequestration, or the proteasomal/lysosomal degradation of the restriction factor. This review summarizes the current advances in our understanding of the cellular pathways and sites of action of Vpu in the downregulation of cell-surface BST-2.

HIV-1 Nef mobilizes lipid rafts in macrophages through a pathway that competes with ABCA1-dependent cholesterol efflux

HIV infection, through the actions of viral accessory protein Nef, impairs activity of cholesterol transporter ABCA1, inhibiting cholesterol efflux from macrophages and elevating the risk of atherosclerosis. Nef also induces lipid raft formation. In this study, we demonstrate that these activities are tightly linked and affect macrophage function and HIV replication. Nef stimulated lipid raft formation in macrophage cell line RAW 264.7, and lipid rafts were also mobilized in HIV-1-infected human monocyte-derived macrophages. Nef-mediated transfer of cholesterol to lipid rafts competed with the ABCA1-dependent pathway of cholesterol efflux, and pharmacological inhibition of ABCA1 functionality or suppression of ABCA1 expression by RNAi increased Nef-dependent delivery of cholesterol to lipid rafts. Nef reduced cell-surface accessibility of ABCA1 and induced ABCA1 catabolism via the lysosomal pathway. Despite increasing the abundance of lipid rafts, expression of Nef impaired phagocytic functions of macrophages. The infectivity of the virus produced in natural target cells of HIV-1 negatively correlated with the level of ABCA1. These findings demonstrate that Nef-dependent inhibition of ABCA1 is an essential component of the viral replication strategy and underscore the role of ABCA1 as an innate anti-HIV factor.

The HIV-1 Vpu Protein Induces Apoptosis in Drosophila via Activation of JNK Signaling

The genome of the human immunodeficiency virus type 1 (HIV-1) encodes the canonical retroviral proteins, as well as additional accessory proteins that enhance the expression of viral genes, the infectivity of the virus and the production of virions. The accessory Viral Protein U (Vpu), in particular, enhances viral particle production, while also promoting apoptosis of HIV-infected human T lymphocytes. Some Vpu effects rely on its interaction with the ubiquitin–proteasome protein degradation system, but the mechanisms responsible for its pro-apoptotic effects in vivo are complex and remain largely to be elucidated.We took advantage of the Drosophila model to study the effects of Vpu activity in vivo. Expression of Vpu in the developing Drosophila wing provoked tissue loss due to caspase-dependent apoptosis. Moreover, Vpu induced expression of the pro-apoptotic gene reaper, known to down-regulate Inhibitor of Apoptosis Proteins (IAPs) which are caspase-antagonizing E3 ubiquitin ligases. Indeed, Vpu also reduced accumulation of Drosophila IAP1 (DIAP1). Though our results demonstrate a physical interaction between Vpu and the proteasome-addressing SLIMB/β-TrCP protein, as in mammals, both SLIMB/βTrCP-dependent and -independent Vpu effects were observed in the Drosophila wing. Lastly, the pro-apoptotic effect of Vpu in this tissue was abrogated upon inactivation of the c-Jun N-terminal Kinase (JNK) pathway. Our results in the fly thus provide the first functional evidence linking Vpu pro-apoptotic effects to activation of the conserved JNK pathway.

APOBEC2 Is a Monomer in Solution: Implications for APOBEC3G Models

Although the physiological role of APOBEC2 is still largely unknown, a crystal structure of a truncated variant of this protein was determined several years ago [Prochnow, C. (2007) Nature445, 447-451]. This APOBEC2 structure had considerable impact in the HIV field because it was considered a good model for the structure of APOBEC3G, an important HIV restriction factor that abrogates HIV infectivity in the absence of the viral accessory protein Vif. The quaternary structure and the arrangement of the monomers of APOBEC2 in the crystal were taken as being representative for APOBEC3G and exploited in explaining its enzymatic and anti-HIV activity. Here we show, unambiguously, that in contrast to the findings for the crystal, APOBEC2 is monomeric in solution. The nuclear magnetic resonance solution structure of full-length APOBEC2 reveals that the N-terminal tail that was removed for crystallization resides close to strand β2, the dimer interface in the crystal structure, and shields this region of the protein from engaging in intermolecular contacts. In addition, the presence of the N-terminal region drastically alters the aggregation propensity of APOBEC2, rendering the full-length protein highly soluble and not prone to precipitation. In summary, our results cast doubt on all previous structure-function predictions for APOBEC3G that were based on the crystal structure of APOBEC2.