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Background: BST2 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., Results: We 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., Conclusions: Altogether, 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.

Background: Tetherin is an intrinsic anti-viral factor impairing the release of nascent HIV-1 particles from infected cells. Vpu, an HIV-1 accessory protein, antagonizes the anti-viral action of tetherin. Although previous studies using in vitro cell culture systems have revealed the molecular mechanisms of the anti-viral action of tetherin and the antagonizing action of Vpu against tetherin, it still remains unclear how Vpu affects the kinetics of HIV-1 replication in vivo., Results: To quantitatively assess the role of Vpu in viral replication in vivo, we analyzed time courses of experimental data with viral load and target cell levels in the peripheral blood of humanized mice infected with wild-type and vpu-deficient HIV-1. Our recently developed mathematical model describes the acute phase of this infection reasonably, and allowed us to estimate several parameters characterizing HIV-1 infection in mice. Using a technique of Bayesian parameter estimation, we estimate distributions of the basic reproduction number of wild-type and vpu-deficient HIV-1. This reveals that Vpu markedly increases the rate of viral replication in vivo., Conclusions: Combining experiments with mathematical modeling, we provide an estimate for the contribution of Vpu to viral replication in humanized mice.

Backgroud: BST-2 is an interferon-induced host restriction factor that inhibits the release of diverse mammalian enveloped viruses from infected cells by physically trapping the newly formed virions onto the host cell surface. Human Immunodeficiency Virus-1 (HIV-1) encodes an accessory protein Vpu that antagonizes BST-2 by down-regulating BST-2 from the cell surface., Results: Using a cell-based ELISA screening system, we have discovered a lead compound, 2-thio-6-azauridine, that restores cell surface BST-2 level in the presence of Vpu. This compound has no effect on the expression of BST-2 and Vpu, but inhibits Vpu-mediated BST-2 down-regulation and exerts no effect on Vpu-induced down-regulation of CD4 or KSHV K5 protein induced BST-2 down-regulation. 2-thio-6-azauridine suppresses HIV-1 production in a BST-2-dependent manner. Further results indicate that 2-thio-6-azauridine does not interrupt the interaction of BST-2 with Vpu and β-TrCP2, but decreases BST-2 ubiquitination., Conclusion: Our study demonstrates the feasibility of using small molecules to target Vpu function and sensitize wild type HIV-1 to BST-2-mediated host restriction.

Background: Hijacking 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 SCF(β-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., Results: We 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., Conclusions: We 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.

Background: Vpu is a multifunctional accessory protein that enhances the release of HIV-1 by counteracting the entrapment of nascent virions on infected cell surface mediated by BST2/Tetherin. Vpu-mediated BST2 antagonism involves physical association with BST2 and subsequent mislocalization of the restriction factor to intracellular compartments followed by SCF(β-TrCP) E3 ligase-dependent lysosomal degradation. Apart from BST2 antagonism, Vpu also induces down regulation of several immune molecules, including CD4 and SLAMF6/NTB-A, to evade host immune responses and promote viral dissemination. However, it should be noted that the multiple functions of Vpu have been studied in cell-based assays, and thus it remains unclear how Vpu influences the dynamic of HIV-1 infection in in vivo conditions., Results: Using a humanized mouse model of acute infection as well as CCR5-tropic HIV-1 that lack Vpu or encode WT Vpu or Vpu with mutations in the β-TrCP binding domain, we provide evidence that Vpu-mediated BST2 antagonism plays a crucial role in establishing early plasma viremia and viral dissemination. Interestingly, we also find that efficient HIV-1 release and dissemination are directly related to functional strength of Vpu in antagonizing BST2. Thus, reduced antagonism of BST2 due to β-TrCP binding domain mutations results in decreased plasma viremia and frequency of infected T cells, highlighting the importance of Vpu-mediated β-TrCP-dependent BST-2 degradation for optimal initial viral propagation., Conclusions: Overall, our findings suggest that BST2 antagonism by Vpu is critical for efficient early viral expansion and dissemination during acute infection and as such is likely to confer HIV-1 increased transmission fitness.

Background: BST-2 (bone marrow stromal cell antigen 2) is an interferon-inducible protein that inhibits virus release by tethering viral particles to the cell surface. This antiviral activity of BST-2 is antagonized by HIV-1 accessory protein Vpu. Vpu physically interacts with BST-2 through their mutual transmembrane (TM) domains. In this study, we utilized the BRET assay and molecular dynamics (MD) simulation method to further characterize the interaction of BST-2 and Vpu., Results: Amino acids I34, L37, P40 and L41 in the TM domain of BST-2, and L11, A18 and W22 in the TM domain of Vpu were identified to be critical for the interaction between BST-2 and Vpu. The residues P40 in the TM domain of BST-2 and L11 in the TM domain of Vpu were shown, for the first time, to be important for their interaction. Furthermore, triple-amino-acid substitutions, 14-16 (AII to VAA) and 26-28 (IIE to AAA) in Vpu TM, not the single-residue mutation, profoundly disrupted BST-2/Vpu interaction. The results of MD simulation revealed significant conformational changes of the BST-2/Vpu complex as a result of mutating P40 of BST-2 and L11, 14-16 (AII to VAA) and 26-28 (IIE to AAA) of Vpu. In addition, disrupting the interaction between BST-2 and Vpu rendered BST-2 resistant to Vpu antagonization., Conclusions: Through use of the BRET assay, we identified novel key residues P40 in the TM domain of BST-2 and L11 in the TM domain of Vpu that are important for their interaction. These results add new insights into the molecular mechanism behind BST-2 antagonization by HIV-1 Vpu.

Background: The 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., Results: To 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., Conclusions: Our 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.

Background: A new subgroup of HIV-1, designated Group P, was recently detected in two unrelated patients of Cameroonian origin. HIV-1 Group P phylogenetically clusters with SIVgor suggesting that it is the result of a cross-species transmission from gorillas. Until today, HIV-1 Group P has only been detected in two patients, and its degree of adaptation to the human host is largely unknown. Previous data have shown that pandemic HIV-1 Group M, but not non-pandemic Group O or rare Group N viruses, efficiently antagonize the human orthologue of the restriction factor tetherin (BST-2, HM1.24, CD317) suggesting that primate lentiviruses may have to gain anti-tetherin activity for efficient spread in the human population. Thus far, three SIV/HIV gene products (vpu, nef and env) are known to have the potential to counteract primate tetherin proteins, often in a species-specific manner. Here, we examined how long Group P may have been circulating in humans and determined its capability to antagonize human tetherin as an indicator of adaptation to humans., Results: Our data suggest that HIV-1 Group P entered the human population between 1845 and 1989. Vpu, Env and Nef proteins from both Group P viruses failed to counteract human or gorilla tetherin to promote efficient release of HIV-1 virions, although both Group P Nef proteins moderately downmodulated gorilla tetherin from the cell surface. Notably, Vpu, Env and Nef alleles from the two HIV-1 P strains were all able to reduce CD4 cell surface expression., Conclusions: Our analyses of the two reported HIV-1 Group P viruses suggest that zoonosis occurred in the last 170 years and further support that pandemic HIV-1 Group M strains are better adapted to humans than non-pandemic or rare Group O, N and P viruses. The inability to antagonize human tetherin may potentially explain the limited spread of HIV-1 Group P in the human population.

Background: HIV-1 viruses are categorized into four distinct groups: M, N, O and P. Despite the same genomic organization, only the group M viruses are responsible for the world-wide pandemic of AIDS, suggesting better adaptation to human hosts. Previously, it has been reported that the group M Vpu protein is capable of both down-modulating CD4 and counteracting BST-2/tetherin restriction, while the group O Vpu cannot antagonize tetherin. This led us to investigate if group O, and the related group P viruses, possess functional anti-tetherin activities in Vpu or another viral protein, and to further map the residues required for group M Vpu to counteract human tetherin., Results: We found a lack of activity against human tetherin for both the Vpu and Nef proteins from group O and P viruses. Furthermore, we found no evidence of anti-human tetherin activity in a fully infectious group O proviral clone, ruling out the possibility of an alternative anti-tetherin factor in this virus. Interestingly, an activity against primate tetherins was retained in the Nef proteins from both a group O and a group P virus. By making chimeras between a functional group M and non-functional group O Vpu protein, we were able to map the first 18 amino acids of group M Vpu as playing an essential role in the ability of the protein to antagonize human tetherin. We further demonstrated the importance of residue alanine-18 for the group M Vpu activity. This residue lies on a diagonal face of conserved alanines in the TM domain of the protein, and is necessary for specific Vpu-tetherin interactions., Conclusions: The absence of human specific anti-tetherin activities in HIV-1 group O and P suggests a failure of these viruses to adapt to human hosts, which may have limited their spread.

Background: The 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., Results: To 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., Conclusions: These 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.

Background: Tetherin (BST-2/CD317/HM1.24) is an interferon (IFN)-inducible factor of the innate immune system, recently shown to exert antiviral activity against HIV-1 and other enveloped viruses by tethering nascent viral particles to the cell surface, thereby inhibiting viral release. In HIV-1 infection, the viral protein U (Vpu) counteracts this antiviral action by down-modulating tetherin from the cell surface. Viral dissemination between T-cells can occur via cell-free transmission or the more efficient direct cell-to-cell route through lipid raft-rich virological synapses, to which tetherin localizes., Results: We established a flow cytometry-based co-culture assay to distinguish viral transfer from viral transmission and investigated the influence of tetherin on cell-to-cell spread of HIV-1. Sup-T1 cells inducible for tetherin expression were used to examine the impact of effector and target cell tetherin expression on virus transfer and transmission. Using this assay, we showed that tetherin inhibits direct cell-to-cell virus transfer and transmission. Viral Vpu promoted viral transmission from tetherin-expressing cells by down-modulating tetherin from the effector cell surface. Further, we showed that tetherin on the target cell promotes viral transfer and transmission. Viral infectivity in itself was not affected by tetherin., Conclusion: In addition to inhibiting viral release, tetherin also inhibits direct cell-to-cell spread. Viral protein Vpu counteracts this restriction, outweighing its possible cost of fitness in cell-to-cell transmission. The differential role of tetherin in effector and target cells suggest a role for tetherin in cell-cell contacts and virological synapses.

Viral protein U (Vpu) is a type 1 membrane-associated accessory protein that is unique to human immunodeficiency virus type 1 (HIV-1) and a subset of related simian immunodeficiency virus (SIV). The Vpu protein encoded by HIV-1 is associated with two primary functions during the viral life cycle. First, it contributes to HIV-1-induced CD4 receptor downregulation by mediating the proteasomal degradation of newly synthesized CD4 molecules in the endoplasmic reticulum (ER). Second, it enhances the release of progeny virions from infected cells by antagonizing Tetherin, an interferon (IFN)-regulated host restriction factor that directly cross-links virions on host cell-surface. This review will mostly focus on recent advances on the role of Vpu in CD4 downregulation and Tetherin antagonism and will discuss how these two functions may have impacted primate immunodeficiency virus cross-species transmission and the emergence of pandemic strain of HIV-1.

Background: In the absence of the Vpu protein, newly formed HIV-1 particles can remain attached to the surface of human cells due to the action of an interferon-inducible cellular restriction factor, BST-2/tetherin. Tetherin also restricts the release of other enveloped viral particles and is counteracted by a several viral anti-tetherin factors including the HIV-2 Env, SIV Nef and KSHV K5 proteins., Results: We observed that a fraction of tetherin is located at the surface of restricting cells, and that co-expression of both HIV-1 Vpu and HIV-2 Env reduced this population. In addition, Vpu, but not the HIV-2 Env, reduced total cellular levels of tetherin. An additional effect observed for both Vpu and the HIV-2 Env was to redirect tetherin to an intracellular perinuclear compartment that overlapped with markers for the TGN (trans-Golgi network). Sequestration of tetherin in this compartment was independent of tetherin's normal endocytosis trafficking pathway., Conclusions: Both HIV-1 Vpu and HIV-2 Env redirect tetherin away from the cell surface and sequester the protein in a perinuclear compartment, which likely blocks the action of this cellular restriction factor. Vpu also promotes the degradation of tetherin, suggesting that it uses more than one mechanism to counteract tetherin restriction.

Background: The gibbon ape leukemia virus (GaLV) Env protein mediates entry into a wide range of human cells and is frequently used to pseudotype retroviral vectors. However, an incompatibility exists between GaLV Env and lentiviral vectors that results in decreased steady-state levels of the mature GaLV Env in cells and prevents its incorporation into lentiviral vector particles., Results: We identified the HIV-1 Vpu protein as the major cause of the depletion in GaLV Env levels that occurs when lentiviral vector components are present. This activity of Vpu targeted the mature (cleaved) form of the GaLV Env that exists within or beyond the trans-Golgi. The activity required two conserved phospho-serines in the cytoplasmic tail of Vpu that are known to recruit beta TrCP, a substrate adaptor for an SCF E3 ubiquitin ligase complex, and could be blocked by mutation of lysine 618 in the GaLV Env tail. Moreover, the Vpu-mediated decrease of GaLV Env levels was inhibited by the lysosomal inhibitor, bafilomycin A1. Interestingly, this activity of Vpu was only observed in the presence of other lentiviral vector components., Conclusions: Similar to the mechanism whereby Vpu targets BST-2/tetherin for degradation, these findings implicate beta-TrCP-mediated ubiquitination and the endo-lysosomal pathway in the degradation of the GaLV Env by lentiviral vector components. Possibly, the cytoplasmic tail of the GaLV Env contains features that mimic bona fide targets of Vpu, important to HIV-1 replication. Furthermore, the lack of effect of Vpu on GaLV Env in the absence of other HIV-1 proteins, suggests that a more complex interaction may exist between Vpu and its target proteins, with the additional involvement of one or more component(s) of the HIV-1 replication machinery.

Background: Isolation of novel retroviral restriction factors will open new avenues for anti-HIV/AIDS treatment. Although HIV-1 replication is restricted by APOBEC3G/APOBEC3F, TRIM5alpha, and CD317, none defend HIV-1 infection under natural conditions. Previously, we demonstrated a host factor from the human T cell line CEM.NKR that potently restricted wild-type HIV-1 replication. Interestingly, this restriction resembled the APOBEC3G/APOBEC3F pattern in that viral replication was inhibited from the second round of replication cycle at a post-entry step., Results: Here, we further characterized this factor and found it distinguishable from the known anti-HIV APOBEC3 proteins. Although CEM.NKR cells expressed both APOBEC3G and APOBEC3F, their levels were at least 10 or 4-fold lower than those in H9 cells, and importantly, Vif effectively neutralized their activity. Among eight subclones isolated from CEM.NKR cells, one was relatively permissive, four were semi-permissive, and three were completely non-permissive for HIV-1 replication. When the levels of APOBEC3 expression were determined, all these clones retained similar low levels of APOBEC3DE, APOBEC3F, APOBEC3G and APOBEC3H expression, and no APOBEC3B expression was detected. Since the vif from SIVmac can effectively neutralize APOBEC3B and APOBEC3H, recombinant HIV-1 expressing this SIV gene were created. However, these viruses still failed to replicate in CEM.NKR cells. We also confirmed that HIV-1 restriction in CEM.NKR was not due to a loss of calnexin expression., Conclusion: Taken together, these results not only demonstrate that all these aforementioned anti-HIV APOBEC3 proteins do not contribute to this HIV-1 restriction, but also shed light on a novel and potent HIV-1 inhibitor in CEM.NKR cells.

Accumulation of high levels of unintegrated viral DNA is a common feature of retroviral infection. It was recently discovered that coinfection of cells with integrated and unintegrated HIV-1 can result in complementation, allowing viral replication in the absence of integration. This new mode of HIV-1 replication has numerous implications for the function of unintegrated viral DNA and its application as a therapeutic vector.

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.