Vpu Exploits the Cross-Talk between BST2 and the ILT7 Receptor to Suppress Anti-HIV-1 Responses by Plasmacytoid Dendritic Cells.

Plasmacytoid dendritic cells (pDCs) constitute a major source of type-I interferon (IFN-I) production during acute HIV infection. Their activation results primarily from TLR7-mediated sensing of HIV-infected cells. However, the interactions between HIV-infected T cells and pDCs that modulate this sensing process remain poorly understood. BST2/Tetherin is a restriction factor that inhibits HIV release by cross-linking virions onto infected cell surface. BST2 was also shown to engage the ILT7 pDC-specific inhibitory receptor and repress TLR7/9-mediated IFN-I production by activated pDCs. Here, we show that Vpu, the HIV-1 antagonist of BST2, suppresses TLR7-mediated IFN-I production by pDC through a mechanism that relies on the interaction of BST2 on HIV-producing cells with ILT7. Even though Vpu downregulates surface BST2 as a mean to counteract the restriction on HIV-1 release, we also find that the viral protein re-locates remaining BST2 molecules outside viral assembly sites where they are free to bind and activate ILT7 upon cell-to-cell contact. This study shows that through a targeted regulation of surface BST2, Vpu promotes HIV-1 release and limits pDC antiviral responses upon sensing of infected cells. This mechanism of innate immune evasion is likely to be important for an efficient early viral dissemination during acute infection.

HIV-1 Vpu antagonizes BST-2 by interfering mainly with the trafficking of newly synthesized BST-2 to the cell surface.

Bone marrow stromal cell antigen-2 (BST-2) inhibits human immunodeficiency virus type 1 (HIV-1) release by cross-linking nascent virions on infected cell surface. HIV-1 Vpu is thought to antagonize BST-2 by downregulating its surface levels via a mechanism that involves intracellular sequestration and lysosomal degradation. Here, we investigated the functional importance of cell-surface BST-2 downregulation and the BST-2 pools targeted by Vpu using an inducible proviral expression system. Vpu established a surface BST-2 equilibrium at ∼60% of its initial levels within 6 h, a condition that coincided with detection of viral release. Analysis of BST-2 post-endocytic trafficking revealed that the protein is engaged in a late endosomal pathway independent of Vpu. While Vpu moderately enhanced cell-surface BST-2 clearance, it strongly affected the protein resupply to the plasma membrane via newly synthesized proteins. Noticeably, Vpu affected clearance of surface BST-2 more substantially in Jurkat T cells than in HeLa cells, suggesting a cell-dependent impact of Vpu on the pool of surface BST-2. Collectively, our data reveal that Vpu imposes a new BST-2 equilibrium, incompatible with efficient restriction of HIV-1 release, by combining an acceleration of surface BST-2 natural clearance, whose degree might be cell-type dependent, to a severe impairment of the protein resupply to the plasma membrane.

Virus-activated interferon regulatory factor 7 upregulates expression of the interferon-regulated BST2 gene independently of interferon signaling.

BST-2/tetherin is an interferon (IFN)-inducible host restriction factor that inhibits the release of many enveloped viruses and functions as a negative-feedback regulator of IFN production by plasmacytoid dendritic cells. Currently, mechanisms underlying BST2 transcriptional regulation by type I IFN remain largely unknown. Here, we demonstrate that the BST2 promoter is a secondary target of the IFN cascade and show that a single IRF binding site is sufficient to render this promoter responsive to IFN-α. Interestingly, expression of IRF-1 or virus-activated forms of IRF-3 and IRF-7 stimulated the BST2 promoter even under conditions where type I IFN signaling was inhibited. Indeed, vesicular stomatitis virus could directly upregulate BST-2 during infection of mouse embryonic fibroblasts through a process that required IRF-7 but was independent from the type I IFN cascade; however, in order to achieve optimal BST-2 induction, the type I IFN cascade needed to be engaged through activation of IRF-3. Furthermore, using human peripheral blood mononuclear cells, we show that BST-2 upregulation is part of an early intrinsic immune response since TLR8 and TLR3 agonists, known to trigger pathways that mediate activation of IRF proteins, could upregulate BST-2 prior to engagement of the type I IFN pathway. Collectively, our findings reveal that BST2 is activated by the same signals that trigger type I IFN production, outlining a regulatory mechanism ensuring that production of type I IFN and expression of a host restriction factor involved in the IFN negative-feedback loop are closely coordinated.

Antagonism of tetherin restriction of HIV-1 release by Vpu involves binding and sequestration of the restriction factor in a perinuclear compartment.

The Vpu accessory protein promotes HIV-1 release by counteracting Tetherin/BST-2, an interferon-regulated restriction factor, which retains virions at the cell-surface. Recent reports proposed beta-TrCP-dependent proteasomal and/or endo-lysosomal degradation of Tetherin as potential mechanisms by which Vpu could down-regulate Tetherin cell-surface expression and antagonize this restriction. In all of these studies, Tetherin degradation did not, however, entirely account for Vpu anti-Tetherin activity. Here, we show that Vpu can promote HIV-1 release without detectably affecting Tetherin steady-state levels or turnover, suggesting that Tetherin degradation may not be necessary and/or sufficient for Vpu anti-Tetherin activity. Even though Vpu did not enhance Tetherin internalization from the plasma membrane (PM), it did significantly slow-down the overall transport of the protein towards the cell-surface. Accordingly, Vpu expression caused a specific removal of cell-surface Tetherin and a re-localization of the residual pool of Tetherin in a perinuclear compartment that co-stained with the TGN marker TGN46 and Vpu itself. This re-localization of Tetherin was also observed with a Vpu mutant unable to recruit beta-TrCP, suggesting that this activity is taking place independently from beta-TrCP-mediated trafficking and/or degradation processes. We also show that Vpu co-immunoprecipitates with Tetherin and that this interaction involves the transmembrane domains of the two proteins. Importantly, this association was found to be critical for reducing cell-surface Tetherin expression, re-localizing the restriction factor in the TGN and promoting HIV-1 release. Overall, our results suggest that association of Vpu to Tetherin affects the outward trafficking and/or recycling of the restriction factor from the TGN and as a result promotes its sequestration away from the PM where productive HIV-1 assembly takes place. This mechanism of antagonism that results in TGN trapping is likely to be augmented by beta-TrCP-dependent degradation, underlining the need for complementary and perhaps synergistic strategies to effectively counteract the powerful restrictive effects of human Tetherin.

Effect of calcium-modulating cyclophilin ligand on human immunodeficiency virus type 1 particle release and cell surface expression of tetherin.

The human immunodeficiency virus type 1 (HIV-1) accessory protein Vpu enhances virus particle release by counteracting a host factor that retains virions at the surfaces of infected cells. It was recently demonstrated that cellular protein BST-2/CD317/Tetherin restricts HIV-1 release in a Vpu-dependent manner. Calcium-modulating cyclophilin ligand (CAML) was also proposed to be involved in this process. We investigated whether CAML is involved in cell surface expression of Tetherin. Here, we show that CAML overexpression in permissive Cos-7 cells or CAML depletion in restrictive HeLa cells has no effect on HIV-1 release or on Tetherin surface expression, indicating that CAML is not required for Tetherin-mediated restriction of HIV-1 release.

Suppression of Tetherin-restricting activity upon human immunodeficiency virus type 1 particle release correlates with localization of Vpu in the trans-Golgi network.

Vpu promotes the efficient release of human immunodeficiency virus type 1 (HIV-1) by overcoming the activity of tetherin, a host cell restriction factor that retains assembled virions at the cell surface. In this study, we analyzed the intracellular localization and trafficking of subtype B Vpu in HIV-1-producing human cells. We found that mutations of conserved positively charged residues (R30 and K31) within the putative overlapping tyrosine- and dileucine-based sorting motifs of the Vpu hinge region affected both the accumulation of the protein in the trans-Golgi network (TGN) and its efficient delivery to late endosomal degradative compartments. A functional characterization of this mutant revealed that the mislocalization of Vpu from the TGN correlated with an attenuation of HIV-1 release. Interestingly, clathrin light chain small interfering RNA-directed disruption of Vpu trafficking from the TGN to the endosomal system slightly stimulated Vpu-mediated HIV-1 release and completely restored the activity of the Vpu R30A,K31A mutant. An analysis of the C-terminal deletion mutants of Vpu identified an additional determinant in the second helical structure of the protein, which regulated TGN retention/localization, and further revealed the functional importance of Vpu localization in the TGN. Finally, we show that a large fraction of Vpu colocalizes with tetherin in the TGN and provide evidence that the degree of Vpu colocalization with tetherin in the TGN is important for efficient HIV-1 release. Taken together, our results reveal that Vpu traffics between the TGN and the endosomal system and suggest that the proper distribution of Vpu in the TGN is critical to overcome the restricting activity of tetherin on HIV-1 release.

HIV-1 Vpr-mediated G2 arrest involves the DDB1-CUL4AVPRBP E3 ubiquitin ligase.

Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) has been shown to cause G2 cell cycle arrest in human cells by inducing ATR-mediated inactivation of p34cdc2, but factors directly engaged in this process remain unknown. We used tandem affinity purification to isolate native Vpr complexes. We found that damaged DNA binding protein 1 (DDB1), viral protein R binding protein (VPRBP), and cullin 4A (CUL4A)--components of a CUL4A E3 ubiquitin ligase complex, DDB1-CUL4A(VPRBP)--were able to associate with Vpr. Depletion of VPRBP by small interfering RNA impaired Vpr-mediated induction of G2 arrest. Importantly, VPRBP knockdown alone did not affect normal cell cycle progression or activation of ATR checkpoints, suggesting that the involvement of VPRBP in G2 arrest was specific to Vpr. Moreover, leucine/isoleucine-rich domain Vpr mutants impaired in their ability to interact with VPRBP and DDB1 also produced strongly attenuated G2 arrest. In contrast, G2 arrest-defective C-terminal Vpr mutants were found to maintain their ability to associate with these proteins, suggesting that the interaction of Vpr with the DDB1-VPRBP complex is necessary but not sufficient to block cell cycle progression. Overall, these results point toward a model in which Vpr could act as a connector between the DDB1-CUL4A(VPRBP) E3 ubiquitin ligase complex and an unknown cellular factor whose proteolysis or modulation of activity through ubiquitination would activate ATR-mediated checkpoint signaling and induce G2 arrest.

Requirements for the selective degradation of CD4 receptor molecules by the human immunodeficiency virus type 1 Vpu protein in the endoplasmic reticulum.

BACKGROUND: HIV-1 Vpu targets newly synthesized CD4 receptor for rapid degradation by a process reminiscent of endoplasmic reticulum (ER)-associated protein degradation (ERAD). Vpu is thought to act as an adaptor protein, connecting CD4 to the ubiquitin (Ub)-proteasome degradative system through an interaction with beta-TrCP, a component of the SCFbeta-TrCP E3 Ub ligase complex. RESULTS: Here, we provide direct evidence indicating that Vpu promotes trans-ubiquitination of CD4 through recruitment of SCFbeta-TrCP in human cells. To examine whether Ub conjugation occurs on the cytosolic tail of CD4, we substituted all four Ub acceptor lysine residues for arginines. Replacement of cytosolic lysine residues reduced but did not prevent Vpu-mediated CD4 degradation and ubiquitination, suggesting that Vpu-mediated CD4 degradation is not entirely dependent on the ubiquitination of cytosolic lysines and as such might also involve ubiquitination of other sites. Cell fractionation studies revealed that Vpu enhanced the levels of ubiquitinated forms of CD4 detected in association with not only the ER membrane but also the cytosol. Interestingly, significant amounts of membrane-associated ubiquitinated CD4 appeared to be fully dislocated since they could be recovered following sodium carbonate salt treatment. Finally, expression of a transdominant negative mutant of the AAA ATPase Cdc48/p97 involved in the extraction of ERAD substrates from the ER membrane inhibited Vpu-mediated CD4 degradation. CONCLUSION: Taken together, these results are consistent with a model whereby HIV-1 Vpu targets CD4 for degradation by an ERAD-like process involving most likely poly-ubiquitination of the CD4 cytosolic tail by SCFbeta-TrCP prior to dislocation of receptor molecules across the ER membrane by a process that depends on the AAA ATPase Cdc48/p97.

Association between disruption of CD4 receptor dimerization and increased human immunodeficiency virus type 1 entry.

BACKGROUND: Human immunodeficiency virus (HIV) enters target cells by a membrane fusion process that involves a series of sequential interactions between its envelope glycoproteins, the CD4 receptor and CXCR4/CCR5 coreceptors. CD4 molecules are expressed at the cell surface of lymphocytes and monocytes mainly as monomers, but basal levels of CD4 dimers are also present at the cell surface of these cells. Previous evidence indicates that the membrane distal and proximal extracellular domains of CD4, respectively D1 and D4, are involved in receptor dimerization. RESULTS: Here, we have used A201 cell lines expressing two CD4 mutants, CD4-E91K, E92K (D1 mutant) and CD4-Q344E (D4 mutant), harboring dimerization defects to analyze the role of CD4 dimerization in HIV-1 entry. Using entry assays based on beta-lactamase-Vpr or luciferase reporter activities, as well as virus encoding envelope glycoproteins derived from primary or laboratory-adapted strains, we obtained evidence suggesting an association between disruption of CD4 dimerization and increased viral entry efficiency. CONCLUSION: Taken together, our results suggest that monomeric forms of CD4 are preferentially used by HIV-1 to gain entry into target cells, thus implying that the dimer/monomer ratio at the cell surface of HIV-1 target cells may modulate the efficiency of HIV-1 entry.

Productive human immunodeficiency virus type 1 assembly takes place at the plasma membrane.

Gag proteins are necessary and sufficient to direct human immunodeficiency virus type 1 (HIV-1) particle assembly and budding. Recent evidence suggests that Gag targeting to late endosomal/multivesicular body (LE/MVB) compartments occurs prior to viral particle budding at the plasma membrane (PM). However, the route that Gag follows before reaching its steady-state destinations still remains a subject of debate. Using a subcellular fractionation method that separates PM from LE/MVB combined with pulse-chase labeling, we analyzed Gag trafficking in HIV-1-producing HEK 293T cells. Our results reveal that the majority of newly synthesized Gag is primarily targeted to the PM. While PM-targeted Gag was efficiently released, a significant fraction of the remaining cell surface-associated Gag was found to be subsequently internalized to LE/MVB, where it accumulated, thus accounting for the majority of LE/MVB-associated Gag. Importantly, this accumulation of Gag in LE/MVB was found to be cholesterol dependent since it was sensitive to the sterol-binding drugs filipin and methyl-beta-cyclodextrin. These results point towards the PM as being the primary site of productive HIV-1 assembly in cells that also support Gag accumulation in intracellular compartments.