Accessory Protein Vpu Research Articles

HIV-1 Vpu induces neurotoxicity by promoting Caspase 3-dependent cleavage of TDP-43

Despite the efficacy of highly active antiretroviral therapy in controlling the incidence and mortality of AIDS, effective interventions for HIV-1-induced neurological damage and cognitive impairment remain elusive. In this study, we found that HIV-1 infection can induce proteolytic cleavage and aberrant aggregation of TAR DNA-binding protein 43 (TDP-43), a pathological protein associated with various severe neurological disorders. The HIV-1 accessory protein Vpu was found to be responsible for the cleavage of TDP-43, as ectopic expression of Vpu alone was sufficient to induce TDP-43 cleavage, whereas HIV-1 lacking Vpu failed to cleave TDP-43. Mechanistically, the cleavage of TDP-43 at Asp89 by HIV-1 relies on Vpu-mediated activation of Caspase 3, and pharmacological inhibition of Caspase 3 activity effectively suppressed the HIV-1-induced aggregation and neurotoxicity of TDP-43. Overall, these results suggest that TDP-43 is a conserved host target of HIV-1 Vpu and provide evidence for the involvement of TDP-43 dysregulation in the neural pathogenesis of HIV-1.

NTB-A and 2B4 Natural Killer Cell Receptors Modulate the Capacity of a Cocktail of Non-Neutralizing Antibodies and a Small CD4-Mimetic to Eliminate HIV-1-Infected Cells by Antibody-Dependent Cellular Cytotoxicity.

Natural Killer (NK) cells have the potential to eliminate HIV-1-infected cells by antibody-dependent cellular cytotoxicity (ADCC). NK cell activation is tightly regulated by the engagement of its inhibitory and activating receptors. The activating receptor CD16 drives ADCC upon binding to the Fc portion of antibodies; NK cell activation is further sustained by the co-engagement of activating receptors NTB-A and 2B4. During HIV-1 infection, Nef and Vpu accessory proteins contribute to ADCC escape by downregulating the ligands of NTB-A and 2B4. HIV-1 also evades ADCC by keeping its envelope glycoproteins (Env) in a "closed" conformation which effectively masks epitopes recognized by non-neutralizing antibodies (nnAbs) which are abundant in the plasma of people living with HIV. To achieve this, the virus uses its accessory proteins Nef and Vpu to downregulate the CD4 receptor, which otherwise interacts with Env and exposes the epitopes recognized by nnAbs. Small CD4-mimetic compounds (CD4mc) have the capacity to expose these epitopes, thus sensitizing infected cells to ADCC. Given the central role of NK cell co-activating receptors NTB-A and 2B4 in Fc-effector functions, we studied their contribution to CD4mc-mediated ADCC. Despite the fact that their ligands are partially downregulated by HIV-1, we found that both co-activating receptors significantly contribute to CD4mc sensitization of HIV-1-infected cells to ADCC.

Antiviral factors and their counteraction by HIV-1: many uncovered and more to be discovered.

Extensive studies on HIV-1 have led to the discovery of a variety of structurally and functionally diverse innate defense factors that target various steps of the retroviral replication cycle. Some of them, such as APOBEC3, tetherin, and SERINC5, are well established. Their importance is evident from the fact that HIV-1 uses its accessory proteins Vif, Vpu, and Nef to counteract them. However, the list of antiviral factors is constantly increasing, and accumulating evidence suggests that innate defense mechanisms, which restrict HIV-1 and/or are counteracted by viral proteins, remain to be discovered. These antiviral factors are relevant to diseases other than HIV/AIDS, since they are commonly active against various viral pathogens. In this review, we provide an overview of recently reported antiretroviral factors and viral countermeasures, present the evidence suggesting that more innate defense mechanisms remain to be discovered, and discuss why this is a challenging but rewarding task.

MicroRNA-25/93 induction by Vpu as a mechanism for counteracting MARCH1-restriction on HIV-1 infectivity in macrophages.

In order to efficiently produce infectious viral particles, HIV must counter several restrictions exerted by host cell antiviral proteins. MARCH1 is a member of the MARCH protein family that restricts HIV infection by limiting the incorporation of viral envelope glycoproteins into nascent virions. Here, we identified two regulatory RNAs, microRNAs-25 and -93, induced by the HIV-1 accessory protein Vpu, that downregulate MARCH1 mRNA. We also show that Vpu induces these cellular microRNAs in macrophages by hijacking the cellular β-catenin pathway. The notion that HIV-1 has evolved a mechanism to counteract MARCH1 restriction on viral infectivity underlines the importance of MARCH1 in the host antiviral response.

Small but Highly Versatile: The Viral Accessory Protein Vpu.

Human and simian immunodeficiency viruses (HIVs and SIVs, respectively) encode several small proteins (Vif, Vpr, Nef, Vpu, and Vpx) that are called accessory because they are not generally required for viral replication in cell culture. However, they play complex and important roles for viral immune evasion and spread in vivo. Here, we discuss the diverse functions and the relevance of the viral protein U (Vpu) that is expressed from a bicistronic RNA during the late stage of the viral replication cycle and found only in HIV-1 and closely related SIVs. It is well established that Vpu counteracts the restriction factor tetherin, mediates degradation of the primary viral CD4 receptors, and inhibits activation of the transcription factor nuclear factor kappa B. Recent studies identified additional activities and provided new insights into the sophisticated mechanisms by which Vpu enhances and prolongs the release of fully infectious viral particles. In addition, it has been shown that Vpu prevents superinfection not only by degrading CD4 but also by modulating DNA repair mechanisms to promote degradation of nuclear viral complementary DNA in cells that are already productively infected.

Piperidine CD4-Mimetic Compounds Expose Vulnerable Env Epitopes Sensitizing HIV-1-Infected Cells to ADCC.

The ability of the HIV-1 accessory proteins Nef and Vpu to decrease CD4 levels contributes to the protection of infected cells from antibody-dependent cellular cytotoxicity (ADCC) by preventing the exposure of Env vulnerable epitopes. Small-molecule CD4 mimetics (CD4mc) based on the indane and piperidine scaffolds such as (+)-BNM-III-170 and (S)-MCG-IV-210 sensitize HIV-1-infected cells to ADCC by exposing CD4-induced (CD4i) epitopes recognized by non-neutralizing antibodies that are abundantly present in plasma from people living with HIV. Here, we characterize a new family of CD4mc, (S)-MCG-IV-210 derivatives, based on the piperidine scaffold which engages the gp120 within the Phe43 cavity by targeting the highly conserved Asp368 Env residue. We utilized structure-based approaches and developed a series of piperidine analogs with improved activity to inhibit the infection of difficult-to-neutralize tier-2 viruses and sensitize infected cells to ADCC mediated by HIV+ plasma. Moreover, the new analogs formed an H-bond with the α-carboxylic acid group of Asp368, opening a new avenue to enlarge the breadth of this family of anti-Env small molecules. Overall, the new structural and biological attributes of these molecules make them good candidates for strategies aimed at the elimination of HIV-1-infected cells.

The Feline Immunodeficiency Virus Envelope Signal Peptide Is a Tetherin Antagonizing Protein.

Signal peptides are N-terminal peptides, generally less than 30 amino acids in length, that direct translocation of proteins into the endoplasmic reticulum and secretory pathway. The envelope glycoprotein (Env) of the nonprimate lentivirus feline immunodeficiency virus (FIV) contains the longest signal peptide of all eukaryotic, prokaryotic, and viral proteins (175 amino acids), yet the reason is unknown. Tetherin is a dual membrane-anchored host protein that inhibits the release of enveloped viruses from cells. Primate lentiviruses have evolved three antagonists: the small accessory proteins Vpu and Nef, and in the case of HIV-2, Env. Here, we identify the FIV Env signal peptide (Fsp) as the FIV tetherin antagonist. A short deletion in the central portion of Fsp had no effect on viral replication in the absence of tetherin, but severely impaired virion budding in its presence. Fsp is necessary and sufficient, acting as an autonomous accessory protein with the rest of Env dispensable. In contrast to primate lentivirus tetherin antagonists, its mechanism is to stringently block the incorporation of this restriction factor into viral particles rather than by degrading it or downregulating it from the plasma membrane. IMPORTANCE The study of species- and virus-specific differences in restriction factors and their antagonists has been central to deciphering the nature of these key host defenses. FIV is an AIDS-causing lentivirus that has achieved pandemic spread in the domestic cat. We now identify its tetherin antagonist as the signal sequence of the Envelope glycoprotein, thus identifying the fourth lentiviral anti-tetherin protein and the first new lentiviral accessory protein in decades. Fsp is necessary and sufficient and functions by stringently blocking particle incorporation of tetherin, which differs from the degradation or surface downregulation mechanisms used by primate lentiviruses. Fsp also is a novel example of signal peptide dual function, being both a restriction factor antagonist and a mediator of protein translocation into the endoplasmic reticulum.

HIV-induced bystander cell death in astrocytes requires cell-to-cell viral transmission.

Human immunodeficiency virus (HIV) neuroinvasion occurs early after infection through the trafficking of virus-infected immune cells into the central nervous system (CNS) and viral dissemination into the brain. There, it can infect resident brain cells including astrocytes, the most abundant cell type that is crucial to brain homeostasis. In this report, we examined the HIV-related mechanism able to induce bystander cell death in astrocytes mediated by cell-to-cell contact with productively infected (PI) ones. We first demonstrate that HIV-induced bystander cell death involves mitochondrial dysfunction that promotes exacerbated reactive oxygen species production. Such a phenomenon is a contagious cell death that requires contact with HIV-PI astrocytes that trigger caspase-dependent (apoptosis and pyroptosis) and caspase-independent cell death pathways. The HIV accessory proteins Nef, Vpu, and Vpr counteract astrocyte death among PI cells but, in contrast, participate to promote contagious bystander cell death by inducing mitochondrial reactive oxygen species production. Our findings indicate that astrocytes PI by HIV became capable to counteract infection-derived death signals, surviving, and spreading the bystander cell death into neighboring uninfected cells by a cell-to-cell contact-dependent mechanism. Considering that astrocytes have been proposed as a long-term HIV reservoir in the CNS, ascertaining the mechanism of survival and contagious bystander death will afford clear targets in the current goal to achieve a functional cure.

Attenuated HIV-1 Nef But Not Vpu Function in a Cohort of Rwandan Long-Term Survivors.

HIV-1 accessory proteins Nef and Vpu enhance viral pathogenesis through partially overlapping immune evasion activities. Attenuated Nef or Vpu functions have been reported in individuals who display slower disease progression, but few studies have assessed the relative impact of these proteins in non-B HIV-1 subtypes or examined paired proteins from the same individuals. Here, we examined the sequence and function of matched Nef and Vpu clones isolated from 29 long-term survivors (LTS) from Rwanda living with HIV-1 subtype A and compared our results to those of 104 Nef and 62 Vpu clones isolated from individuals living with chronic untreated HIV-1 subtype A from the same geographic area. Nef and vpu coding regions were amplified from plasma HIV RNA and cloned. The function of one intact, phylogenetically-validated Nef and Vpu clone per individual was then quantified by flow cytometry following transient expression in an immortalized CD4+ T-cell line. We measured the ability of each Nef clone to downregulate CD4 and HLA class I, and of each Vpu clone to downregulate CD4 and Tetherin, from the cell surface. Results were normalized to reference clones (Nef-SF2 and Vpu-NL4.3). We observed that Nef-mediated CD4 and HLA downregulation functions were lower in LTS compared to the control cohort (Mann-Whitney p=0.03 and p<0.0001, respectively). Moreover, we found a positive correlation between Nef-mediated CD4 downregulation function and plasma viral load in LTS and controls (Spearman ρ= 0.59, p=0.03 and ρ=0.30, p=0.005, respectively). In contrast, Vpu-mediated functions were similar between groups and did not correlate with clinical markers. Further analyses identified polymorphisms at Nef codon 184 and Vpu codons 60-62 that were associated with function, which were confirmed through mutagenesis. Overall, our results support attenuated function of Nef, but not Vpu, as a contributor to slower disease progression in this cohort of long-term survivors with HIV-1 subtype A.

Detection of the HIV-1 Accessory Proteins Nef and Vpu by Flow Cytometry Represents a New Tool to Study Their Functional Interplay within a Single Infected CD4+ T Cell.

The HIV-1 Nef and Vpu accessory proteins are known to protect infected cells from antibody-dependent cellular cytotoxicity (ADCC) responses by limiting exposure of CD4-induced (CD4i) envelope (Env) epitopes at the cell surface. Although both proteins target the host receptor CD4 for degradation, the extent of their functional redundancy is unknown. Here, we developed an intracellular staining technique that permits the intracellular detection of both Nef and Vpu in primary CD4+ T cells by flow cytometry. Using this method, we show that the combined expression of Nef and Vpu predicts the susceptibility of HIV-1-infected primary CD4+ T cells to ADCC by HIV+ plasma. We also show that Vpu cannot compensate for the absence of Nef, thus providing an explanation for why some infectious molecular clones that carry a LucR reporter gene upstream of Nef render infected cells more susceptible to ADCC responses. Our method thus represents a new tool to dissect the biological activity of Nef and Vpu in the context of other host and viral proteins within single infected CD4+ T cells. IMPORTANCE HIV-1 Nef and Vpu exert several biological functions that are important for viral immune evasion, release, and replication. Here, we developed a new method allowing simultaneous detection of these accessory proteins in their native form together with some of their cellular substrates. This allowed us to show that Vpu cannot compensate for the lack of a functional Nef, which has implications for studies that use Nef-defective viruses to study ADCC responses.

A combined EM and proteomic analysis places HIV-1 Vpu at the crossroads of retromer and ESCRT complexes: PTPN23 is a Vpu-cofactor.

The HIV-1 accessory protein Vpu modulates membrane protein trafficking and degradation to provide evasion of immune surveillance. Targets of Vpu include CD4, HLAs, and BST-2. Several cellular pathways co-opted by Vpu have been identified, but the picture of Vpu’s itinerary and activities within membrane systems remains incomplete. Here, we used fusion proteins of Vpu and the enzyme ascorbate peroxidase (APEX2) to compare the ultrastructural locations and the proximal proteomes of wild type Vpu and Vpu-mutants. The proximity-omes of the proteins correlated with their ultrastructural locations and placed wild type Vpu near both retromer and ESCRT-0 complexes. Hierarchical clustering of protein abundances across the mutants was essential to interpreting the data and identified Vpu degradation-targets including CD4, HLA-C, and SEC12 as well as Vpu-cofactors including HGS, STAM, clathrin, and PTPN23, an ALIX-like protein. The Vpu-directed degradation of BST-2 was supported by STAM and PTPN23 and to a much lesser extent by the retromer subunits Vps35 and SNX3. PTPN23 also supported the Vpu-directed decrease in CD4 at the cell surface. These data suggest that Vpu directs targets from sorting endosomes to degradation at multi-vesicular bodies via ESCRT-0 and PTPN23.

Vpr counteracts the restriction of LAPTM5 to promote HIV-1 infection in macrophages

The HIV-1 accessory proteins Vif, Vpu, and Nef can promote infection by overcoming the inhibitory effects of the host cell restriction factors APOBEC3G, Tetherin, and SERINC5, respectively. However, how the HIV-1 accessory protein Vpr enhances infection in macrophages but not in CD4+ T cells remains elusive. Here, we report that Vpr counteracts lysosomal-associated transmembrane protein 5 (LAPTM5), a potent inhibitor of HIV-1 particle infectivity, to enhance HIV-1 infection in macrophages. LAPTM5 transports HIV-1 envelope glycoproteins to lysosomes for degradation, thereby inhibiting virion infectivity. Vpr counteracts the restrictive effects of LAPTM5 by triggering its degradation via DCAF1. In the absence of Vpr, the silencing of LAPTM5 precisely phenocopied the effect of Vpr on HIV-1 infection. In contrast, Vpr did not enhance HIV-1 infection in the absence of LAPTM5. Moreover, LAPTM5 was highly expressed in macrophages but not in CD4+ T lymphocytes. Re-expressing LAPTM5 reconstituted the Vpr-dependent promotion of HIV-1 infection in primary CD4+ T cells, as observed in macrophages. Herein, we demonstrate the molecular mechanism used by Vpr to overcome LAPTM5 restriction in macrophages, providing a potential strategy for anti-HIV/AIDS therapeutics.

Vpu modulates DNA repair to suppress innate sensing and hyper-integration of HIV-1.

To avoid innate sensing and immune control, human immunodeficiency virus type 1 (HIV-1) has to prevent the accumulation of viral complementary DNA species. Here, we show that the late HIV-1 accessory protein Vpu hijacks DNA repair mechanisms to promote degradation of nuclear viral cDNA in cells that are already productively infected. Vpu achieves this by interacting with RanBP2-RanGAP1*SUMO1-Ubc9 SUMO E3-ligase complexes at the nuclear pore to reprogramme promyelocytic leukaemia protein nuclear bodies and reduce SUMOylation of Bloom syndrome protein, unleashing end degradation of viral cDNA. Concomitantly, Vpu inhibits RAD52-mediated homologous repair of viral cDNA, preventing the generation of dead-end circular forms of single copies of the long terminal repeat and permitting sustained nucleolytic attack. Our results identify Vpu as a key modulator of the DNA repair machinery. We show that Bloom syndrome protein eliminates nuclear HIV-1 cDNA and thereby suppresses immune sensing and proviral hyper-integration. Therapeutic targeting of DNA repair may facilitate the induction of antiviral immunity and suppress proviral integration replenishing latent HIV reservoirs.

Convergent Evolution of HLA-C Downmodulation in HIV-1 and HIV-2.

HLA-C-mediated antigen presentation induces the killing of human immunodeficiency virus (HIV)-infected CD4+ T cells by cytotoxic T lymphocytes (CTLs). To evade killing, many HIV-1 group M strains decrease HLA-C surface levels using their accessory protein Vpu. However, some HIV-1 group M isolates lack this activity, possibly to prevent the activation of natural killer (NK) cells. Analyzing diverse primate lentiviruses, we found that Vpu-mediated HLA-C downregulation is not limited to pandemic group M but is also found in HIV-1 groups O and P as well as several simian immunodeficiency viruses (SIVs). We show that Vpu targets HLA-C primarily at the protein level, independently of its ability to suppress NF-κB-driven gene expression, and that in some viral lineages, HLA-C downregulation may come at the cost of efficient counteraction of the restriction factor tetherin. Remarkably, HIV-2, which does not carry a vpu gene, uses its accessory protein Vif to decrease HLA-C surface expression. This Vif activity requires intact binding sites for the Cullin5/Elongin ubiquitin ligase complex but is separable from its ability to counteract APOBEC3G. Similar to HIV-1 Vpu, the degree of HIV-2 Vif-mediated HLA-C downregulation varies considerably among different virus isolates. In agreement with opposing selection pressures in vivo, we show that the reduction of HLA-C surface levels by HIV-2 Vif is accompanied by increased NK cell-mediated killing. In summary, our results highlight the complex role of HLA-C in lentiviral infections and demonstrate that HIV-1 and HIV-2 have evolved at least two independent mechanisms to decrease HLA-C levels on infected cells.IMPORTANCE Genome-wide association studies suggest that HLA-C expression is a major determinant of viral load set points and CD4+ T cell counts in HIV-infected individuals. On the one hand, efficient HLA-C expression enables the killing of infected cells by cytotoxic T lymphocytes (CTLs). On the other hand, HLA-C sends inhibitory signals to natural killer (NK) cells and enhances the infectivity of newly produced HIV particles. HIV-1 group M viruses modulate HLA-C expression using the accessory protein Vpu, possibly to balance CTL- and NK cell-mediated immune responses. Here, we show that the second human immunodeficiency virus, HIV-2, can use its accessory protein Vif to evade HLA-C-mediated restriction. Furthermore, our mutational analyses provide insights into the underlying molecular mechanisms. In summary, our results reveal how the two human AIDS viruses modulate HLA-C, a key component of the antiviral immune response.

Differential Vpu-Mediated CD4 and Tetherin Downregulation Functions among Major HIV-1 Group M Subtypes.

Downregulation of BST-2/tetherin and CD4 by HIV-1 viral protein U (Vpu) promotes viral egress and allows infected cells to evade host immunity. Little is known however about the natural variability in these Vpu functions among the genetically diverse viral subtypes that contribute to the HIV-1 pandemic. We collected Vpu isolates from 332 treatment-naive individuals living with chronic HIV-1 infection in Uganda, Rwanda, South Africa, and Canada. Together, these Vpu isolates represent four major HIV-1 group M subtypes (A [n = 63], B [n = 84], C [n = 94], and D [n = 59]) plus intersubtype recombinants and uncommon strains (n = 32). The ability of each Vpu clone to downregulate endogenous CD4 and tetherin was quantified using flow cytometry following transfection into an immortalized T-cell line and compared to that of a reference Vpu clone derived from HIV-1 subtype B NL4.3. Overall, the median CD4 downregulation function of natural Vpu isolates was similar to that of NL4.3 (1.01 [interquartile range {IQR}, 0.86 to 1.18]), while the median tetherin downregulation function was moderately lower than that of NL4.3 (0.90 [0.79 to 0.97]). Both Vpu functions varied significantly among HIV-1 subtypes (Kruskal-Wallis P < 0.0001). Specifically, subtype C clones exhibited the lowest CD4 and tetherin downregulation activities, while subtype D and B clones were most functional for both activities. We also identified Vpu polymorphisms associated with CD4 or tetherin downregulation function and validated six of these using site-directed mutagenesis. Our results highlight the marked extent to which Vpu function varies among global HIV-1 strains, raising the possibility that natural variation in this accessory protein may contribute to viral pathogenesis and/or spread.IMPORTANCE The HIV-1 accessory protein Vpu enhances viral spread by downregulating CD4 and BST-2/tetherin on the surface of infected cells. Natural variability in these Vpu functions may contribute to HIV-1 pathogenesis, but this has not been investigated among the diverse viral subtypes that contribute to the HIV-1 pandemic. In this study, we found that Vpu function differs significantly among HIV-1 subtypes A, B, C, and D. On average, subtype C clones displayed the lowest ability to downregulate both CD4 and tetherin, while subtype B and D clones were more functional. We also identified Vpu polymorphisms that associate with functional differences among HIV-1 isolates and subtypes. Our study suggests that genetic diversity in Vpu may play an important role in the differential pathogenesis and/or spread of HIV-1.

Lymphocyte‐specific protein 1 (LSP1) regulates bone marrow stromal cell antigen 2 (BST‐2)‐mediated intracellular trafficking of HIV‐1 in dendritic cells

Human immunodeficiency virus type 1 (HIV-1) subverts intracellular trafficking pathways to avoid its degradation and elimination, thereby enhancing its survival and spread. The molecular mechanisms involved in intracellular transport of HIV-1 are not yet fully defined. We demonstrate that the actin-binding protein lymphocyte-specific protein 1 (LSP1) interacts with the interferon-inducible protein bone marrow stromal antigen 2 (BST-2) in dendritic cells (DCs) to facilitate both endocytosis of surface-bound HIV-1 and the formation of early endosomes. Analysis of the molecular interaction between LSP1 and BST-2 reveals that the N terminus of LSP1 interacts with BST-2. Overall, we identify a novel mechanism of intracellular trafficking of HIV-1 in DCs centering on the LSP1/BST-2 complex. We also show that the HIV-1 accessory protein Vpu subverts this pathway by inducing proteasomal degradation of LSP1, augmenting cell-cell transmission of HIV-1.

The HIV-1 Accessory Protein Vpu Downregulates Peroxisome Biogenesis.

Human immunodeficiency virus type 1 (HIV-1) establishes lifelong infections in humans, a process that relies on its ability to thwart innate and adaptive immune defenses of the host. Recently, we reported that HIV-1 infection results in a dramatic reduction of the cellular peroxisome pool. Peroxisomes are metabolic organelles that also function as signaling platforms in the innate immune response. Here, we show that the HIV-1 accessory protein Vpu is necessary and sufficient for the depletion of cellular peroxisomes during infection. Vpu induces the expression of four microRNAs that target mRNAs encoding proteins required for peroxisome formation and metabolic function. The ability of Vpu to downregulate peroxisomes was found to be dependent upon the Wnt/β-catenin signaling pathway. Given the importance of peroxisomes in innate immune signaling and central nervous system function, the roles of Vpu in dampening antiviral signaling appear to be more diverse than previously realized. Finally, our findings highlight a potential role for Wnt/β-catenin signaling in peroxisome homeostasis through modulating the production of biogenesis factors.IMPORTANCE People living with HIV can experience accelerated aging and the development of neurological disorders. Recently, we reported that HIV-1 infection results in a dramatic loss of peroxisomes in macrophages and brain tissue. This is significant because (i) peroxisomes are important for the innate immune response and (ii) loss of peroxisome function is associated with cellular aging and neurodegeneration. Accordingly, understanding how HIV-1 infection causes peroxisome depletion may provide clues regarding how the virus establishes persistent infections and, potentially, the development of neurological disorders. Here, we show that the accessory protein Vpu is necessary and sufficient for the induction of microRNAs that target peroxisome biogenesis factors. The ability of Vpu to downregulate peroxisome formation depends on the Wnt/β-catenin pathway. Thus, in addition to revealing a novel mechanism by which HIV-1 uses intracellular signaling pathways to target antiviral signaling platforms (peroxisomes), we have uncovered a previously unknown link between the Wnt/β-catenin pathway and peroxisome homeostasis.

Plasma Membrane-Associated Restriction Factors and Their Counteraction by HIV-1 Accessory Proteins.

The plasma membrane is a site of conflict between host defenses and many viruses. One aspect of this conflict is the host’s attempt to eliminate infected cells using innate and adaptive cell-mediated immune mechanisms that recognize features of the plasma membrane characteristic of viral infection. Another is the expression of plasma membrane-associated proteins, so-called restriction factors, which inhibit enveloped virions directly. HIV-1 encodes two countermeasures to these host defenses: The membrane-associated accessory proteins Vpu and Nef. In addition to inhibiting cell-mediated immune-surveillance, Vpu and Nef counteract membrane-associated restriction factors. These include BST-2, which traps newly formed virions at the plasma membrane unless counteracted by Vpu, and SERINC5, which decreases the infectivity of virions unless counteracted by Nef. Here we review key features of these two antiviral proteins, and we review Vpu and Nef, which deplete them from the plasma membrane by co-opting specific cellular proteins and pathways of membrane trafficking and protein-degradation. We also discuss other plasma membrane proteins modulated by HIV-1, particularly CD4, which, if not opposed in infected cells by Vpu and Nef, inhibits viral infectivity and increases the sensitivity of the viral envelope glycoprotein to host immunity.

Hydrophobic matching of HIV-1 Vpu transmembrane helix-helix interactions is optimized for subcellular location

The HIV-1 accessory protein Vpu mediates the downregulation of several host cell proteins, an activity that is critical for viral replication in vivo. As the first step in directing cell-surface proteins to internal cellular compartments, and in many cases degradation, Vpu binds a subset of its target proteins through their transmembrane domains. Each of the known targets of Vpu are synthesized in the ER, and must traverse the different membrane environments found along the secretory pathway, thus it is important to consider how membrane composition might influence the interactions between Vpu and its targets. We have used Förster resonance energy transfer (FRET) to measure the oligomerization of Vpu with the transmembrane domains of target proteins in model membranes of varying lipid composition. Our data show that both lipid bilayer thickness and acyl chain order can significantly influence monomer-oligomer equilibria within the Vpu-target system. Changes in oligomerization levels were found to be non-specific with no single Vpu-target interaction being favored under any condition. Our analysis of the influence of the membrane environment on the strength of helix-helix interactions between Vpu and its targets in vitro suggests that the strength of Vpu-target interactions in vivo will be partially dependent on the membrane environment found in specific membrane compartments.

Contribution of the Cytoplasmic Determinants of Vpu to the Expansion of Virus-Containing Compartments in HIV-1-Infected Macrophages.

HIV-1 infection of macrophages leads to the sequestration of newly formed viruses in intracellular plasma membrane-connected structures termed virus-containing compartments (VCCs), where virions remain infectious and hidden from immune surveillance. The cellular restriction factor bone marrow stromal cell antigen 2 (BST2), which prevents HIV-1 dissemination by tethering budding viral particles at the plasma membrane, can be found in VCCs. The HIV-1 accessory protein Vpu counteracts the restriction factor BST2 by downregulating its expression and removing it from viral budding sites. Numerous studies described these Vpu countermeasures in CD4+ T cells or model cell lines, but the interplay between Vpu and BST2 in VCC formation and HIV-1 production in macrophages is less explored. Here, we show that Vpu expression in HIV-1-infected macrophages enhances viral release. This effect is related to Vpu's ability to circumvent BST2 antiviral activity. We show that in absence of Vpu, BST2 is enriched in VCCs and colocalizes with capsid p24, whereas Vpu expression significantly reduces the presence of BST2 in these compartments. Furthermore, our data reveal that BST2 is dispensable for the formation of VCCs and that Vpu expression impacts the volume of these compartments. This Vpu activity partly depends on BST2 expression and requires the integrity of the Vpu transmembrane domain, the dileucine-like motif E59XXXLV64 and phosphoserines 52 and 56 of Vpu. Altogether, these results highlight that Vpu controls the volume of VCCs and promotes HIV-1 release from infected macrophages.IMPORTANCE HIV-1 infection of macrophages leads to the sequestration of newly formed viruses in virus-containing compartments (VCCs), where virions remain infectious and hidden from immune surveillance. The restriction factor BST2, which prevents HIV-1 dissemination by tethering budding viral particles, can be found in VCCs. The HIV-1 Vpu protein counteracts BST2. This study explores the interplay between Vpu and BST2 in the viral protein functions on HIV-1 release and viral particle sequestration in VCCs in macrophages. The results show that Vpu controls the volume of VCCs and favors viral particle release. These Vpu functions partly depend on Vpu's ability to antagonize BST2. This study highlights that the transmembrane domain of Vpu and two motifs of the Vpu cytoplasmic domain are required for these functions. These motifs were notably involved in the control of the volume of VCCs by Vpu but were dispensable for the prevention of the specific accumulation of BST2 in these structures.