Degradation Of CD4 Research Articles

Structural similarity-based prediction of host factors associated with SARS-CoV-2 infection and pathogenesis

The current pandemic resulted from SARS-CoV-2 still remains as the major public health concern globally. The precise mechanism of viral pathogenesis is not fully understood, which remains a major hurdle for medical intervention. Here we generated an interactome profile of protein-protein interactions based on host and viral protein structural similarities information. Further computational biological study combined with Gene enrichment analysis predicted key enriched pathways associated with viral pathogenesis. The results show that axon guidance, membrane trafficking, vesicle-mediated transport, apoptosis, clathrin-mediated endocytosis, Vpu mediated degradation of CD4 T cell, and interferon-gamma signaling are key events associated in SARS-CoV-2 life cycle. Further, degree centrality analysis reveals that IRF1/9/7, TP53, and CASP3, UBA52, and UBC are vital proteins for IFN-γ-mediated signaling, apoptosis, and proteasomal degradation of CD4, respectively. We crafted chronological events of the virus life cycle. The SARS-CoV-2 enters through clathrin-mediated endocytosis, and the genome is trafficked to the early endosomes in a RAB5-dependent manner. It is predicted to replicate in a double-membrane vesicle (DMV) composed of the endoplasmic reticulum, autophagosome, and ERAD machinery. The SARS-CoV-2 down-regulates host translational machinery by interacting with protein kinase R, PKR-like endoplasmic reticulum kinase, and heme-regulated inhibitor and can phosphorylate eIF2a. The virion assembly occurs in the ER-Golgi intermediate compartment (ERGIC) organized by the spike and matrix protein. Collectively, we have established a spatial link between viral entry, RNA synthesis, assembly, pathogenesis, and their associated diverse host factors, those could pave the way for therapeutic intervention.

Conserved residues within the HIV-1 Vpu transmembrane-proximal hinge region modulate BST2 binding and antagonism

BackgroundBST2 inhibits HIV-1 release by tethering nascent virions to the surface of infected cells. HIV-1 Vpu overcomes this restriction by removing BST2 from viral budding sites via BST2 intracellular trapping and sequestration, surface downregulation and/or displacement mechanisms. Vpu is composed of a short luminal tail, a transmembrane domain (TMD) and a cytoplasmic hinge region that is followed by two helices. BST2 counteraction relies on the ability of Vpu to physically bind BST2 through TMD interactions and recruit the clathrin-dependent trafficking machinery via a canonical acidic di-leucine signalling motif within the helix-2 of Vpu. The highly conserved Vpu transmembrane-proximal hinge region encompasses residues that resemble an acidic leucine-based trafficking motif, whose functional roles are currently ill-defined. In this study, we investigated the contribution of these residues towards Vpu-mediated BST2 antagonism.ResultsWe show that while these conserved residues have no intrinsic activity on the cellular distribution of Vpu in the absence of BST2, they regulate the ability of Vpu to bind to BST2 and, consequently, govern both BST2-dependent trafficking properties of the protein as well as its co-localization with BST2. Moreover, these residues, particularly a glutamic acid residue positioned immediately following the TMD, are a determinant not only for efficient targeting of BST2, but also binding and degradation of CD4, another host membrane protein targeted by Vpu. Mechanistically, our data are consistent with a role of these residues in the maintenance of the Vpu TMD conformational configuration such that interactions with membrane-associated host targets are favoured.ConclusionsAltogether, this work demonstrates an important regulatory role of the transmembrane-proximal Vpu hinge region residues towards enabling the protein to efficiently engage its target host proteins. Thus, this highly conserved, cytosolic Vpu hinge region may represent an attractive target for the development of anti-Vpu inhibitors.

INTERLEUKIN-10 PLASMA DAN LIMFOSIT-T CD4+ PENDERITA TERINFEKSI HIV

HIV & AIDS have become a global problem throughout the world including Indonesia. The incidence is rapidly increasing. Various treatment and efforts have been carried out but until now have not yet been succeeded, the mortality remains high. Examination of CD4+ lymphocyte-T which is carried out to determine the immune status and monitoring the treatment has some limitation. Besides the degradation of CD4+ lymphocyte-T, the increase of the disease progression is also followed by an increase of interleukin-10 as well. The determination of interleukin-10 is expected to be use as an alternative examination if CD4+ lymphocyte-T can not be performed. To analyze the correlation between the levels of plasma interleukin-10 and amount of CD4+ lymphocyte-T in the asymptomatic HIV infected patients. A cross sectional, observational analytical study of 41 patients with HIV infection stage I, has been conducted from March–April 2009, at the Intermediate Infectious Disease Care Unit Dr. Soetomo General Hospital, Surabaya. The diagnosis of HIV patients was based on positive result of HIV test using three (3) different methods. Eight mL venous blood were taken from each patient, 6 mL is put into a heparin tube for the examination of IL-10 plasma with ELISA method and 2 mL is put into a K3EDTA tube for examining the CD4+ lymphocyte-T using flowcytometry. The results showed of plasma IL-10 level and the amount of CD4+ lymphocyte-T, which were analyzed by Pearson correlation test to determine the correlation between the two (2) variabels. The IL-10 levels in HIV patients were 3.80–44.50 pg/mL (mean 18.09 pg/mL, SD 8.84 pg/mL). The amount of absolute CD4+ lymphocyte-T was 5–846 cells/µL (mean 322.07 cells/µL and SD 221.89 cells/µL), while the amount of percentage CD4+ lymphocyte-T was 0.41–29.48% (mean 13.99%, SD 7.62%). Statistical analyzes show a significant negative correlation either between plasma IL-10 level with absolute CD4+lymphocyte-T and plasma IL-10 level with percentage of CD4+ lymphocyte-T, e ach level of r was –0.652 and –0,683. A significant negative correlation was also formed between plasma IL-10 level and the amount of CD4+ lymphocyte-T in the HIV infected patients. In the HIV infected patients, the increase of plasma IL-10 level was followed by a decrease of CD4+ lymphocyte-T.

Vpu-Mediated Counteraction of Tetherin Is a Major Determinant of HIV-1 Interferon Resistance

ABSTRACTHuman immunodeficiency virus type 1 (HIV-1) groups M, N, O, and P are the result of independent zoonotic transmissions of simian immunodeficiency viruses (SIVs) infecting great apes in Africa. Among these, only Vpu proteins of pandemic HIV-1 group M strains evolved potent activity against the restriction factor tetherin, which inhibits virus release from infected cells. Thus, effective Vpu-mediated tetherin antagonism may have been a prerequisite for the global spread of HIV-1. To determine whether this particular function enhances primary HIV-1 replication and interferon resistance, we introduced mutations into the vpu genes of HIV-1 group M and N strains to specifically disrupt their ability to antagonize tetherin, but not other Vpu functions, such as degradation of CD4, down-modulation of CD1d and NTB-A, and suppression of NF-κB activity. Lack of particular human-specific adaptations reduced the ability of HIV-1 group M Vpu proteins to enhance virus production and release from primary CD4+ T cells at high levels of type I interferon (IFN) from about 5-fold to 2-fold. Interestingly, transmitted founder HIV-1 strains exhibited higher virion release capacity than chronic control HIV-1 strains irrespective of Vpu function, and group M viruses produced higher levels of cell-free virions than an N group HIV-1 strain. Thus, efficient virus release from infected cells seems to play an important role in the spread of HIV-1 in the human population and requires a fully functional Vpu protein that counteracts human tetherin.

CD4 downregulation by the HIV-1 protein Nef reveals distinct roles for the γ1 and γ2 subunits of the AP-1 complex in protein trafficking.

The HIV accessory protein Nef is a major determinant of viral pathogenesis that facilitates viral particle release, prevents viral antigen presentation and increases infectivity of new virus particles. These functions of Nef involve its ability to remove specific host proteins from the surface of infected cells, including the CD4 receptor. Nef binds to the adaptor protein 2 (AP-2) and CD4 in clathrin-coated pits, forcing CD4 internalization and its subsequent targeting to lysosomes. Herein, we report that this lysosomal targeting requires a variant of AP-1 containing isoform 2 of γ-adaptin (AP1G2, hereafter γ2). Depletion of the γ2 or μ1A (AP1M1) subunits of AP-1, but not of γ1 (AP1G1), precludes Nef-mediated lysosomal degradation of CD4. In γ2-depleted cells, CD4 internalized by Nef accumulates in early endosomes and this alleviates CD4 removal from the cell surface. Depletion of γ2 also hinders EGFR-EGF-complex targeting to lysosomes, an effect that is not observed upon γ1 depletion. Taken together, our data provide evidence that the presence of γ1 or γ2 subunits delineates two distinct variants of AP-1 complexes, with different functions in protein sorting.

Pharmacologic Inhibition of Nedd8 Activation Enzyme Exposes CD4-Induced Epitopes within Env on Cells Expressing HIV-1.

HIV-1 Vpu decreases the exposure of epitopes within the viral envelope glycoprotein (Env) on the surface of infected cells by downregulating both BST2 and CD4. To test the hypothesis that inhibiting Vpu activity would increase the exposure of these epitopes and sensitize infected cells to antibody-dependent cellular cytotoxicity (ADCC), we treated cells with the Nedd8 activation enzyme (NAE) inhibitor MLN4924, which inhibits the cullin1-based ubiquitin ligase complex coopted by Vpu to degrade cellular targets. Treatment of HeLa cells with MLN4924 or expression of a dominant negative mutant of cullin1 inhibited the Vpu-mediated downregulation of CD4 but not the downregulation of BST2. NAE inhibition also increased the surface exposure of CD4-induced epitopes within Env on HEK293 cells containing an inducible HIV genome, on infected CEM T cells, and on infected primary T cells. In contrast, the Vpu-mediated downregulation of BST2 was substantially inhibited by MLN4924 only when T cells were treated with alpha interferon (IFN-α) to induce high levels of BST2 expression. As reported previously, the absence of vpu or nef and even more so the combined absence of these two genes sensitized infected cells to ADCC. However, NAE inhibition affected ADCC minimally. Paradoxically, even in infected, IFN-treated cells in which NAE inhibition substantially rescued the surface level of BST2, the surface level of Env detected with an antibody recognizing a CD4-independent epitope (2G12) was minimally increased. Mutation of the C-terminal Vpu residue W76, which supports the ability of Vpu to stimulate virion release by displacing BST2 from assembly sites on the plasma membrane by a cullin1-independent mechanism, increased the exposure of Env detected by 2G12 on infected T cells. Thus, inhibiting the displacement function of Vpu together with its ability to degrade CD4 and BST2 may be required to sensitize infected cells to ADCC. Pathogenic viruses encode gene products that enable evasion of host immune surveillance mechanisms. One such mechanism is antibody-dependent cellular cytotoxicity (ADCC), whereby host antibodies bind envelope glycoproteins of the virus that are inserted into the cellular membrane and direct the destruction of infected cells. Targeting pharmacologically the activity of HIV-1 Vpu, which contributes to evasion of ADCC, could potentially sensitize infected cells to this immune surveillance mechanism, an outcome that would have therapeutic implications with respect to the goal of curing HIV-1 infection. The Nedd8 activation enzyme inhibitor MLN4924 blocks the activity of the host ubiquitin ligase that Vpu coopts to direct the degradation of CD4 and BST2. We observed that while MLN4924 partially reverses the activity of Vpu and could become part of a therapeutic approach by virtue of CD4-induced epitope exposure, sufficient Vpu activity as an antagonist of BST2 persists despite this drug to allow escape from ADCC.

Mutational analysis of HIV-1 viral protein U at Ser52 and Ser56 among the HIV-1 infected patients of Manipur

BackgroundPhosphorylation by Casein kinase 2 at two conserved seryl residues, Ser52 and Ser56 at the cytoplasmic domain of HIV-1 Viral protein U (Vpu) are essential for inducing CD4 degradation. This study is conducted to investigate the variability of HIV-1 viral protein unique particularly at amino-acid position Ser52 and Ser56 among the HIV infected individuals in the northeastern region of India. Materials and methodsBlood samples from 90 HIV-1 infected patients of Manipur have been studied by viral amplification and sequencing at vpu gene of HIV-1. For analysis of amino acid variation at Ser52 and Ser56, nucleic acid sequences were translated into amino acid and aligned with reference strain of HIV-1. Phylogenetic tree was also inferred among the studied samples. ResultsThe results revealed that 93% of HIV-1 infected individuals harbored virus with conserved serine at both amino acid positions 52 and 56 of vpu which were known to have the ability to induce CD4 degradation while 1% harbored viral mutation at both Ser52 and Ser56, replaced by asparagine which has been predicted to may have lost the ability to induce CD4 degradation. Moreover, 5% and 1% of HIV-1 infected individuals were found to be infected with mutated virus at ser52 and ser56 of vpu respectively. ConclusionThe overall finding of this study reveals that seryl residue of vpu at Ser52 and Ser56 was highly conserved among the HIV-1 infected individuals of the study population.

HIV-1 Vpu utilizes both cullin-RING ligase (CRL) dependent and independent mechanisms to downmodulate host proteins.

BackgroundHijacking of the cullin-RING E3 ubiquitin ligase (CRL) machinery is a common mechanism employed by diverse groups of viruses for the efficient counteraction and degradation of host proteins. In particular, HIV-1 Vpu usurps the SCFβ-TrCP E3 ubiquitin ligase complex to mark CD4 for degradation by the 26S proteasome. Vpu also interacts with and downmodulates a number of other host proteins, including the restriction factor BST-2. However, whether Vpu primarily relies on a cullin-dependent or -independent mechanism to antagonize its cellular targets has not been fully elucidated.ResultsWe utilized a sulphamate AMP analog, MLN4924, to effectively block the activation of CRLs within infected primary CD4+ T cells. MLN4924 treatment, in a dose dependent manner, efficiently relieved surface downmodulation and degradation of CD4 by NL4-3 Vpu. MLN4924 inhibition was highly specific, as this inhibitor had no effect on Nef’s ability to downregulate CD4, which is accomplished by a CRL-independent mechanism. In contrast, NL4-3 Vpu’s capacity to downregulate BST-2, NTB-A and CCR7 was not inhibited by the drug. Vpu’s from both a transmitted founder (T/F) and chronic carrier (CC) virus preserved the ability to downregulate BST-2 in the presence of MLN4924. Finally, depletion of cellular pools of cullin 1 attenuated Vpu’s ability to decrease CD4 but not BST-2 surface levels.ConclusionsWe conclude that Vpu employs both CRL-dependent and CRL-independent modes of action against host proteins. Notably, we also establish that Vpu-mediated reduction of BST-2 from the cell surface is independent of β-TrCP and the CRL- machinery and this function is conserved by Vpu’s from primary isolates. Therefore, potential therapies aimed at antagonizing the activities of Vpu may need to address these distinct mechanisms of action in order to achieve a maximal effect.

Bacteria-based analysis of HIV-1 Vpu channel activity.

HIV-1 Vpu is a small, single-span membrane protein with two attributed functions that increase the virus' pathogenicity: degradation of CD4 and inactivation of BST-2. Vpu has also been shown to posses ion channel activity, yet no correlation has been found between this attribute and Vpu's role in viral release. In order to gain further insight into the channel activity of Vpu we devised two bacteria-based assays that can examine this function in detail. In the first assay Vpu was over-expressed, such that it was deleterious to bacterial growth due to membrane permeabilization. In the second and more sensitive assay, the channel was expressed at low levels in K+ transport deficient bacteria. Consequently, Vpu expression enabled the bacteria to grow at otherwise non permissive low K+ concentrations. Hence, Vpu had the opposite impact on bacterial growth in the two assays: detrimental in the former and beneficial in the latter. Furthermore, we show that channel blockers also behave reciprocally in the two assays, promoting growth in the first assay and hindering it in the second assay. Taken together, we investigated Vpu's channel activity in a rapid and quantitative approach that is amenable to high-throughput screening, in search of novel blockers.

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

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

HIV-1 Nef Employs Cellular Autophagy Machinery to Downregulate CD4

During HIV infection, expression of the viral protein, HIV-1 negative regulatory factor (Nef) leads to the lysosomal degradation of CD4 and other surface molecules. In addition, lysosome degradation through autophagy is upregulated. Here, we investigated whether autophagosomes participate in directing CD4 to lysosomes in Nef-expressing cells. Autophagosomes are membrane-bounded structures formed in response to specific stimuli (including starvation and buildup of ubiquitinated protein aggregates). They engulf various cytoplasmic components (i.e., damaged organelles, misfolded proteins and intracellular pathogens), and then fuse with lysosomes for degradation. We found that co-expression of Nef and CD4 in HeLa cells caused significant accumulation of the autophagosomal marker, microtubule-associated protein 1 light chain 3 (LC3) on highly motile subcellular vesicles. This occurred within 2-3 days of Nef expression and could be monitored by imaging of single cells or by flow cytometry in large cell populations. In the absence of Nef, CD4-expressing cells showed very few autophagosomes. LC3-containing vesicles resided in close proximity or overlapped with Nef-containing vesicles in cells co-expressing CD4 and Nef. Since Nef is known to trigger CD4 downregulation, we next looked to see if CD4 was associated with vesicles positive for LC3 and Nef. For this experiment, we inhibited autophagosome fusion with lysosomes using the drugs bafliomycin or chloroquine since this increases autophagosome number within cells. Both drug treatments led to extensive co-localization of CD4 in LC3-positive vesicles. When Nef-expressing cells were treated with 3-methyl adenine to inhibit autophagy, CD4 was still internalized from the plasma membrane but was retained in static endocytic structures rather than being transferred to highly dynamic structures as occurred without the inhibitor. These results suggest that cellular autophagy machinery is involved in transferring CD4 from the endosomal system to lysosomes during Nef-induced downregulation of this protein.

Evidence that Vpu modulates HIV-1 Gag-envelope interaction towards envelope incorporation and infectivity in a cell type dependent manner.

The HIV-1 Vpu is required for efficient virus particle release from the plasma membrane and intracellular CD4 degradation in infected cells. In the present study, we found that the loss of virus infectivity as a result of envelope (Env) incorporation defect caused by a Gag matrix (MA) mutation (L30E) was significantly alleviated by introducing a start codon mutation in vpu. Inactivation of Vpu partially restored the Env incorporation defect imposed by L30E substitution in MA. This effect was found to be comparable in cell types such as 293T, HeLa, NP2 and GHOST as well as in peripheral blood mononuclear cells (PBMC) and monocyte-derived macrophages (MDM). However, in HeLa cells BST-2 knockdown was found to further alleviate the effect of Vpu inactivation on infectivity of L30E mutant. Our data demonstrated that the impaired infectivity of virus particles due to Env incorporation defect caused by MA mutation was modulated by start codon mutation in Vpu.

The transmembrane domain of HIV-1 Vpu is sufficient to confer anti-tetherin activity to SIVcpz and SIVgor Vpu proteins: cytoplasmic determinants of Vpu function

BackgroundThe acquisition of effective Vpu-mediated anti-tetherin activity to promote virion release following transmission of SIVcpzPtt from central chimpanzees (Pan troglodytes troglodytes) to humans distinguishes pandemic HIV-1 group M strains from non-pandemic group N, O and P viruses and may have been a prerequisite for their global spread. Some functional motifs in the cytoplasmic region of HIV-1 M Vpus proposed to be important for anti-tetherin activity are more frequently found in the Vpu proteins of SIVcpzPtt than in those of SIVcpzPts infecting eastern chimpanzees (P. t. schweinfurthii), that have not been detected in humans, and SIVgor from gorillas, which is closely related to HIV-1 O and P. Thus, SIVcpzPtt strains may require fewer adaptive changes in Vpu than SIVcpzPts or SIVgor strains to counteract human tetherin.ResultsTo examine whether SIVcpzPtt may only need changes in the transmembrane domain (TMD) of Vpu to acquire anti-tetherin activity, whereas SIVcpzPts and SIVgor may also require changes in the cytoplasmic region, we analyzed chimeras between the TMD of an HIV-1 M Vpu and the cytoplasmic domains of SIVcpzPtt (n = 2), SIVcpzPts (n = 2) and SIVgor (n = 2) Vpu proteins. Unexpectedly, all of these chimeras were capable of counteracting human tetherin to enhance virion release, irrespective of the presence or absence of the putative adaptor protein binding sites and the DSGxxS β-TrCP binding motif reported to be critical for effective anti-tetherin activity of M Vpus. It was also surprising that in three of the six chimeras the gain of anti-tetherin function was associated with a loss of the CD4 degradation activity since this function was conserved among all parental HIV-1, SIVcpz and SIVgor Vpu proteins.ConclusionsOur results show that changes in the TMD of SIVcpzPtt, SIVcpzPts and SIVgor Vpus are sufficient to render them active against human tetherin. Thus, several previously described domains in the extracellular region of Vpu are not absolutely essential for tetherin antagonism but may be required for other Vpu functions.

Mapping the interaction between the cytoplasmic domains of HIV‐1 viral protein U and human CD4 with NMR spectroscopy

Viral protein U (VpU) of HIV-1 plays an important role in downregulation of the main HIV-1 receptor CD4 from the surface of infected cells. Physical binding of VpU to newly synthesized CD4 in the endoplasmic reticulum is an early step in a pathway leading to proteasomal degradation of CD4. In this study, regions in the cytoplasmic domain of VpU involved in CD4 binding were identified by NMR spectroscopy. Amino acids in both helices found in the cytoplasmic region of VpU in membrane-mimicking detergent micelles experience chemical shift perturbations upon binding to CD4, whereas amino acids between the two helices and at the C-terminus of VpU show no or only small changes, respectively. The topology of the complex was further studied with paramagnetic relaxation enhancement. Paramagnetic spin labels were attached at three sequence positions of a CD4 peptide comprising the transmembrane and cytosolic domains of the receptor. VpU binds to a membrane-proximal region in the cytoplasmic domain of CD4.

Minor contribution of HLA class I-associated selective pressure to the variability of HIV-1 accessory protein Vpu

Host HLA class I (HLA-I) allele-associated immune responses are major forces driving the evolution of HIV-1 proteins such as Gag and Nef. The viral protein U (Vpu) is an HIV-1 accessory protein responsible for CD4 degradation and enhancement of virion release by antagonizing tetherin/CD317. Although Vpu represents one of the most variable proteins in the HIV-1 proteome, it is still not clear to what extent HLA-I influence its evolution. To examine this issue, we enrolled 240 HLA-I-typed, treatment naïve, chronically HIV-infected subjects in Japan, and analyzed plasma HIV RNA nucleotide sequences of the vpu region. Using a phylogenetically-informed method incorporating corrections for HIV codon covariation and linkage disequilibrium among HLA alleles, we investigated HLA-associated amino acid mutations in the Vpu protein as well as in the translational products encoded by alternative reading frames. Despite substantial amino acid variability in Vpu, we identified only 4 HLA-associations in all possible translational products encoded in this region, suggesting that HLA-associated immune responses had minor effects on Vpu variability in this cohort. Rather, despite its size (81 amino acids), Vpu showed 103 codon–codon covariation associations, suggesting that Vpu conformation and function are preserved through many possible combinations of primary and secondary polymorphisms. Taken together, our study suggests that Vpu has been comparably less influenced by HLA-I-associated immune-driven evolution at the population level compared to other highly variable HIV-1 accessory proteins.

Ion channel activity of HIV-1 Vpu is dispensable for counteraction of CD317

While the C-terminal domain of HIV-1 Vpu is critical for CD4 degradation, the transmembrane domain (TM) mediates ion channel activity, enhances virus release and is essential for counteracting CD317/Bst-2/Tetherin. Here we analyzed whether the ion channel activity of Vpu is required to antagonize CD317-mediated restriction of virion release. We examined TM-mutants of three conserved residues: the S23A mutation, which was previously shown to abrogate ion channel function, did not affect Vpu mediated augmentation of virus release. In contrast, the A14N and A18N mutation did not affect ion channel activity of Vpu, but substantially reduced its ability to support virus release and to down-regulate CD317 from the cell surface. Altogether, our data suggest that not the ion channel activity of Vpu, but its ability to remove CD317 from the cell surface is required to augment HIV-1 release.

Proteomic-Based Identification of CD4-Interacting Proteins in Human Primary Macrophages

BackgroundHuman macrophages (Mφ) express low levels of CD4 glycoprotein, which is constitutively recycled, and 40–50% of its localization is intracellular at steady-state. Although CD4-interacting proteins in lymphoid cells are well characterised, little is known about the CD4 protein interaction-network in human Mφ, which notably lack LCK, a Src family protein tyrosine kinase believed to stabilise CD4 at the surface of T cells. As CD4 is the main cellular receptor used by HIV-1, knowledge of its molecular interactions is important for the understanding of viral infection strategies.Methodology/Principal FindingsWe performed large-scale anti-CD4 immunoprecipitations in human primary Mφ followed by high-resolution mass spectrometry analysis to elucidate the protein interaction-network involved in induced CD4 internalization and degradation. Proteomic analysis of CD4 co-immunoisolates in resting Mφ showed CD4 association with a range of proteins found in the cellular cortex, membrane rafts and components of clathrin-adaptor proteins, whereas in induced internalization and degradation CD4 is associated with components of specific signal transduction, transport and the proteasome.Conclusions/SignificanceThis is the first time that the anti-CD4 co-immunoprecipitation sub-proteome has been analysed in human primary Mφ. Our data have identified important Mφ cell surface CD4-interacting proteins, as well as regulatory proteins involved in internalization and degradation. The data give valuable insights into the molecular pathways involved in the regulation of CD4 expression in Mφ and provide candidates/targets for further biochemical studies.

β-TrCP is dispensable for Vpu's ability to overcome the CD317/Tetherin-imposed restriction to HIV-1 release

BackgroundThe cellular transmembrane protein CD317/BST-2/HM1.24/Tetherin restricts HIV-1 infection by physically tethering mature virions to the surface of infected cells. HIV-1 counteracts this restriction by expressing the accessory protein Vpu, yet the mechanism of this antagonism is incompletely understood. β-TrCP is the substrate recognition domain of an E3 ubiquitin ligase complex that interacts with the di-serine motif S52/S56 in the cytoplasmic tail of Vpu to target the CD4 receptor for proteasomal degradation. Recently, it has been suggested that β-TrCP is also critically involved in Vpu's ability to overcome the CD317-mediated virion release block.ResultsTo test this model, we analyzed the consequences of several experimental strategies to interfere with the Vpu-β-TrCP protein-protein interaction. Under these conditions, we studied effects of Vpu on expression and localization of CD317 and CD4, as well as on its ability to promote HIV-1 release. Our results demonstrate a strict requirement for Vpu's di-serine motif for degradation of CD4 and also CD317, reduction of cell surface exposure of CD317, and HIV-1 release enhancement. We further show a critical role of β-TrCP2, but not of the structurally related β-TrCP1 isoform, for Vpu-mediated degradation of both receptors. Most importantly, Vpu remained active in downregulating CD317 from the cell surface and in overcoming the HIV-1 release restriction in β-TrCP-depleted cells.ConclusionsThese results demonstrate that β-TrCP is not strictly required for Vpu's ability to counteract the CD317-imposed virion release block and support the relevance of cell surface down-modulation of the restriction factor as a central mechanism of Vpu antagonism. Moreover, we propose the existence of a critical, yet to be identified cellular factor that interacts with Vpu via its di-serine motif to alter the trafficking of the restriction factor.

Characterizing the Interaction of Human CD4 and the HIV-1 accessory Protein VpU using Liquid State NMR

Human CD4 is a 433 residue transmembrane protein involved in the body's adaptive immune response. The extracellular domain of CD4 serves as primary receptor of the human immunodeficiency virus (HIV-1) and binds the viral glycoprotein gp120. The cytoplasmic domain of the 81 residue Virus protein U (VpU) plays an important role in downregulation of CD4. It directly binds the cytoplasmic domain of CD4 in the endoplasmic reticulum, which initiates a series of events resulting in CD4 degradation in the proteasome. We characterized the CD4 - VpU interaction by liquid state NMR spectroscopy utilizing paramagnetic relaxation enhancement (PRE). The PRE effect is a distance-dependent enhancement of the spin relaxation in magnetically active nuclei in the vicinity of a paramagnetic centre. PRE leads to line broadening or complete quenching of NMR signals and indicates spatial proximity to the probe. We recorded NMR spectra of the 15N-labelled cytoplasmic domain of VpU in the presence of varying amounts of a single-cysteine-mutant of CD4(372-433), which was labelled with the active PRE-probe, methanethiosulfonate (MTSL). This CD4 polypeptide contains the single transmembrane and the C-terminal cytoplasmic domain of CD4. Experiments were conducted in membrane-mimicking dodecyl phosphocholine (DPC) micelles. Spectra recorded in the presence of paramagnetic MTSL show reduced NMR signal intensities of certain amino acids of 15N-labeled VpU. Additionally, we studied chemical shift perturbations (CSP's) of VpU resonances observed on titrating increasing concentrations of unlabelled CD4. Such chemical shift changes are expected for residues in the binding interface but may also occur at remote sites due to allosteric effects. Our data reveal VpU residues involved in CD4 binding, provide insight into the exchange regime, and yield an estimate of the binding affinity.

HIV-1 Vpu Disarms Natural Killer Cells

Natural killer (NK)-cell killing of virus-infected cells is regulated in part by the engagement of activation and coactivation receptors. In this issue of Cell Host & Microbe, Shah etal. (2010) demonstrate that HIV-1 protects infected cells from NK-cell-mediated killing by hindering NK-cell degranulation through downmodulation of NTB-A coactivation receptor ligands by the Vpu accessory protein.