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Objectives: The objective of this article is to investigate the contribution of colon and blood CD4+ T-cell subsets expressing the chemokine receptor CCR6 to HIV persistence during antiretroviral therapy. Design: Matched sigmoid biopsies and blood samples (n = 13) as well as leukapheresis (n = 20) were collected from chronically HIV-infected individuals receiving antiretroviral therapy. Subsets of CD4+ T cells with distinct differentiation/polarization profiles were identified using surface markers as follows: memory (TM, CD45RA−), central memory (TCM; CD45RA−CCR7+), effector (TEM/TM; CD45RA−CCR7−), Th17 (CCR6+CCR4+), Th1Th17 (CCR6+CXCR3+), Th1 (CCR6−CXCR3+), and Th2 (CCR6−CCR4+). Methods: We used polychromatic flow cytometry for cell sorting, nested real-time PCR for HIV DNA quantification, ELISA and flow cytometry for HIV p24 quantification. HIV reactivation was induced by TCR triggering in the presence/absence of all-trans retinoic acid. Results: Compared with blood, the frequency of CCR6+ TM was higher in the colon. In both colon and blood compartments, CCR6+ TM were significantly enriched in HIV DNA when compared with their CCR6− counterparts (n = 13). In blood, integrated HIV DNA levels were significantly enriched in CCR6+ versus CCR6− TCM of four of five individuals and CCR6+ versus CCR6− TEM of three of five individuals. Among blood TCM, Th17 and Th1Th17 contributed the most to the pool of cells harboring integrated HIV DNA despite their reduced frequency compared with Th2, which were infected the least. HIV reactivation was induced by TCR triggering and/or retinoic acid exposure at higher levels in CCR6+ versus CCR6− TM, TCM, and TEM. Conclusion: CCR6 is a marker for colon and blood CD4+ T cells enriched for replication-competent HIV DNA. Novel eradication strategies should target HIV persistence in CCR6+CD4+ T cells from various anatomic sites.

The development of an effective vaccine against HIV infection remains a global priority. Dendritic cell (DC)-based HIV immunotherapeutic vaccine is a promising approach which aims at optimizing the HIV-specific immune response using primed DCs to promote and enhance both the cellular and humoral arms of immunity. Since the Ebola virus envelope glycoprotein (EboGP) has strong DC-targeting ability, we investigated whether EboGP is able to direct HIV particles towards DCs efficiently and promote potent HIV-specific immune responses. Our results indicate that the incorporation of EboGP into non-replicating virus-like particles (VLPs) enhances their ability to target human monocyte-derived dendritic cells (MDDCs) and monocyte-derived macrophages (MDMs). Also, a mucin-like domain deleted EboGP (EboGPΔM) can further enhanced the MDDCs and MDMs-targeting ability. Furthermore, we investigated the effect of EboGP on HIV immunogenicity in mice, and the results revealed a significantly stronger HIV-specific humoral immune response when immunized with EboGP-pseudotyped HIV VLPs compared with those immunized with HIV VLPs. Splenocytes harvested from mice immunized with EboGP-pseudotyped HIV VLPs secreted increased levels of macrophage inflammatory proteins-1α (MIP-1α) and IL-4 upon stimulation with HIV Env and/or Gag peptides compared with those harvested from mice immunized with HIV VLPs. Collectively, this study provides evidence for the first time that the incorporation of EboGP in HIV VLPs can facilitate DC and macrophage targeting and induce more potent immune responses against HIV.

Productive assembly of human immunodeficiency virus type 1 (HIV-1) takes place, primarily, at the plasma membrane. However, depending on the cell types, a significant proportion of nascent virus particles are internalized and routed to late endosomes. We previously reported that expression of human leucocyte antigen (HLA)-DR promoted a redistribution of Gag in late endosomes and an increased detection of mature virions in these compartments in HeLa and human embryonic kidney 293T model cell lines. Although this redistribution of Gag resulted in a marked decrease of HIV-1 release, the underlying mechanism remained undefined. Here, we provide evidence that expression of HLA-DR at the cell surface induces a redistribution of mature Gag products into late endosomes by enhancing nascent HIV-1 particle internalization from the plasma membrane through a process that relies on the presence of intact HLA-DR α and β-chain cytosolic tails. These findings raise the possibility that major histocompatibility complex class-II molecules might influence endocytic events at the plasma membrane and as a result promote endocytosis of progeny HIV-1 particles.

Myeloid cells display a differential permissivity to primate lentivirus infection that is related to their ability to encode the Vpx and to a lesser extent the Vpr accessory proteins. Vpr is encoded by all primate lentiviruses, including HIV-1 and HIV-2, while its paralog, Vpx, is unique to HIV-2 and a subset of simian lentiviruses. Both proteins usurp the CRL4A (DCAF1) E3 ligase to fulfil their functions. Vpx induces the degradation of SAMHD1, a nucleotide triphosphohydrolase that blocks lentiviral reverse transcription in myeloid cells via depletion of the intracellular pool of dNTPs. Vpr engages CRL4A (DCAF1) to degrade a yet unknown factor(s), whose proteolysis induces a G2 cell-cycle arrest in dividing cells. Although the identification of the host protein(s) targeted for degradation by Vpr will be necessary to understand its actual function, the discovery of SAMHD1 has already shed light into a new mechanism of restriction that limits infection of myeloid cells by HIV-1.

Binding of anti-HIV antibodies (Abs) to envelope (Env) glycoproteins on infected cells can mark them for elimination via antibody-dependent cell-mediated cytotoxicity (ADCC). BST2, a type I interferon (IFN)-stimulated restriction factor that anchors nascent Env-containing virions at the surface of infected cells has been shown to enhance ADCC functions. In a comprehensive analysis of ADCC potency by neutralizing anti-HIV Abs (NAbs), we show in this study that NAbs are capable of mediating ADCC against HIV-infected T cells with 3BNC117, PGT126 and PG9 being most efficient. We demonstrate that HIV-induced BST2 antagonism effectively attenuates Ab binding and ADCC responses mediated by all classes of NAbs that were tested. Interestingly, IFNα treatment can reverse this effect in a BST2-dependent manner. Importantly, while reactivated latent T cell lines display some susceptibility to ADCC mediated by broadly NAbs, inactivating BST2 viral countermeasures and/or exogenous IFNα augment their elimination. Overall, our findings support the notion that NAbs can induce ADCC. They highlight that while BST2 antagonism by HIV promotes ADCC evasion, strategies aimed at restoring BST2 restriction could improve anti-HIV responses and potentially provide a means to eliminate reactivated cells in latent reservoirs.

Human Immunodeficiency virus (HIV) enters the brain soon after seroconversion and induces chronic neuroinflammation by infecting and activating brain macrophages. Inflammasomes are cytosolic protein complexes that mediate caspase-1 activation and ensuing cleavage and release of IL-1β and -18 by macrophages. Our group recently showed that HIV-1 infection of human microglia induced inflammasome activation in NLRP3-dependent manner. The HIV-1 viral protein R (Vpr) is an accessory protein that is released from HIV-infected cells, although its effects on neuroinflammation are undefined. Infection of human microglia with Vpr-deficient HIV-1 resulted in reduced caspase-1 activation and IL-1β production, compared to cells infected with a Vpr-encoding HIV-1 virus. Vpr was detected at low nanomolar concentrations in cerebrospinal fluid from HIV-infected patients and in supernatants from HIV-infected primary human microglia. Exposure of human macrophages to Vpr caused caspase-1 cleavage and IL-1β release with reduced cell viability, which was dependent on NLRP3 expression. Increased NLRP3, caspase-1, and IL-1β expression was evident in HIV-1 Vpr transgenic mice compared to wild-type littermates, following systemic immune stimulation. Treatment with the caspase-1 inhibitor, VX-765, suppressed NLRP3 expression with reduced IL-1β expression and associated neuroinflammation. Neurobehavioral deficits showed improvement in Vpr transgenic animals treated with VX-765. Thus, Vpr-induced NLRP3 inflammasome activation, which contributed to neuroinflammation and was abrogated by caspase-1 inhibition. This study provides a new therapeutic perspective for HIV-associated neuropsychiatric disease.

The plasma membrane protects the cell from its surroundings and regulates cellular communication, homing, and metabolism. Not surprisingly, the composition of this membrane is highly controlled through the vesicular trafficking of proteins to and from the cell surface. As intracellular pathogens, most viruses exploit the host plasma membrane to promote viral replication while avoiding immune detection. This is particularly true for the enveloped human immunodeficiency virus (HIV), which assembles and obtains its lipid shell directly at the plasma membrane. HIV-1 encodes two proteins, negative factor (Nef) and viral protein U (Vpu), which function primarily by altering the quantity and localization of cell surface molecules to increase virus fitness despite host antiviral immune responses. These proteins are expressed at different stages in the HIV-1 life cycle and employ a variety of mechanisms to target both unique and redundant surface proteins, including the viral receptor CD4, host restriction factors, immunoreceptors, homing molecules, tetraspanins and membrane transporters. In this review, we discuss recent progress in the study of the Nef and Vpu targeting of host membrane proteins with an emphasis on how remodeling of the cell membrane allows HIV-1 to avoid host antiviral immune responses leading to the establishment of systemic and persistent infection.

Patients infected with human immunodeficiency virus type 1 (HIV-1) often display neurological complications in late stage disease and increased viral loads directly correlated with higher concentrations of extracellular HIV-1 viral protein r (Vpr) in the blood serum and cerebrospinal fluid. Additionally, HIV-1-infected patients with a low CD4+ T-lymphocyte count displayed lower concentrations of reduced glutathione (GSH), the main intracellular antioxidant molecule, and lower level of survival. To establish a correlation between increased concentrations of extracellular Vpr and an oxidative stress-induced phenotype, the U-87 MG astroglioma cell line has been used to determine the downstream effects induced by Vpr. Conditioned media obtained from the human endothelial kidney (HEK) 293 T cell line transfected either in the absence or presence of HIV-1 Vpr contained free Vpr. Exposure of U-87 MG to this conditioned media decreased intracellular levels of both adenosine triphosphate (ATP) and GSH. These observations were recapitulated using purified recombinant HIV-1 Vpr both in U-87 MG and primary human fetal astrocytes in a dose- and time-dependent manner. Vpr-induced oxidative stress could be partly restored by co-treatment with the antioxidant molecule N-acetyl-cysteine (NAC). In addition, free Vpr augmented production of reactive oxygen species due to an increase in the level of oxidized glutathione (GSSG). This event was almost entirely suppressed by treatment with an anti-Vpr antibody or co-treatment with NAC. These studies confirm a role of extracellular Vpr in impairing astrocytic levels of intracellular ATP and GSH. Studies are underway to better understand the intricate correlation between reductions in ATP and GSH metabolites and how they affect neuronal survival in end-stage disease.

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.

Immunosuppressive lentivirus infections, including human, simian, and feline immunodeficiency viruses (HIV, SIV, and FIV, respectively), cause the acquired immunodeficiency syndrome (AIDS), frequently associated with AIDS enteropathy. Herein, we investigated the extent to which lentivirus infections affected mucosal integrity and intestinal permeability in conjunction with immune responses and activation of endoplasmic reticulum (ER) stress pathways. Duodenal biopsies from individuals with HIV/AIDS exhibited induction of IL-1β, CD3ε, HLA-DRA, spliced XBP-1(Xbp-1s), and CHOP expression compared to uninfected persons (P

HIV-1 employs the cellular nuclear import machinery to actively transport its preintegration complex (PIC) into the nucleus for integration of the viral DNA. Several viral karyophilic proteins and cellular import factors have been suggested to contribute to HIV-1 PIC nuclear import and replication. However, how HIV interacts with different cellular machineries to ensure efficient nuclear import of its preintegration complex in dividing and nondividing cells is still not fully understood. In this study, we have investigated different importin α (Impα) family members for their impacts on HIV-1 replication, and we demonstrate that short hairpin RNA (shRNA)-mediated Impα3 knockdown (KD) significantly impaired HIV infection in HeLa cells, CD4 + C8166 T cells, and primary macrophages. Moreover, quantitative real-time PCR analysis revealed that Impα3-KD resulted in significantly reduced levels of viral 2-long-terminal repeat (2-LTR) circles but had no effect on HIV reverse transcription. All of these data indicate an important role for Impα3 in HIV nuclear import. In an attempt to understand how Impα3 participates in HIV nuclear import and replication, we first demonstrated that the HIV-1 karyophilic protein integrase (IN) was able to interact with Impα3 both in a 293T cell expression system and in HIV-infected CD4 + C8166 T cells. Deletion analysis suggested that a region (amino acids [aa] 250 to 270) in the C-terminal domain of IN is involved in this viral-cellular protein interaction. Overall, this study demonstrates for the first time that Impα3 is an HIV integrase-interacting cofactor that is required for efficient HIV-1 nuclear import and replication in both dividing and nondividing cells.

HIV up-regulates cell-surface expression of specific ligands for the activating NKG2D receptor, including ULBP-1, -2, and -3, but not MICA or MICB, in infected cells both in vitro and in vivo. However, the viral factor(s) involved in NKG2D ligand expression still remains undefined. HIV-1 Vpr activates the DNA damage/stress-sensing ATR kinase and promotes G2 cell-cycle arrest, conditions known to up-regulate NKG2D ligands. We report here that HIV-1 selectively induces cell-surface expression of ULBP-2 in primary CD4+ T lymphocytes by a process that is Vpr dependent. Importantly, Vpr enhanced the susceptibility of HIV-1–infected cells to NK cell–mediated killing. Strikingly, Vpr alone was sufficient to up-regulate expression of all NKG2D ligands and thus promoted efficient NKG2D-dependent NK cell–mediated killing. Delivery of virion-associated Vpr via defective HIV-1 particles induced analogous biologic effects in noninfected target cells, suggesting that Vpr may act similarly beyond infected cells. All these activities relied on Vpr ability to activate the ATR-mediated DNA damage/stress checkpoint. Overall, these results indicate that Vpr is a key determinant responsible for HIV-1–induced up-regulation of NKG2D ligands and further suggest an immunomodulatory role for Vpr that may not only contribute to HIV-1–induced CD4+ T-lymphocyte depletion but may also take part in HIV-1–induced NK-cell dysfunction.

MicroRNAs (miRNAs) are small noncoding RNA molecules, which are known to regulate gene expression in physiological and pathological conditions. miRNA profiling was performed using brain tissue from patients with HIV encephalitis (HIVE), a neuroinflammatory/degenerative disorder caused by HIV infection of the brain. Microarray analysis showed differential expression of multiple miRNAs in HIVE compared to control brains. Target prediction and gene ontology enrichment analysis disclosed targeting of several gene families/biological processes by differentially expressed miRNAs (DEMs), with cell death-related genes, including caspase-6, showing a bias toward down-regulated DEMs. Consistent with the miRNA data, HIVE brains exhibited higher levels of caspase-6 transcripts compared with control patients. Immunohistochemical analysis showed localization of the cleaved form of caspase-6 in astrocytes in HIVE brain sections. Exposure of cultured human primary astrocytes to HIV viral protein R (Vpr) induced p53 up-regulation, loss of mitochondrial membrane potential, and caspase-6 activation followed by cell injury. Transgenic mice, expressing Vpr in microglial cells, demonstrated astrocyte apoptosis in brain, which was associated with caspase-6 activation and neurobehavioral abnormalities. Overall, these data point to previously unrecognized alterations in miRNA profile in the brain during HIV infection, which contribute to cell death through dysregulation of cell death machinery. © FASEB.

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.

Increasing evidence suggests that extracellular Vpr could contribute to HIV pathogenesis through its effect on bystander cells. Soluble forms of Vpr have been detected in the sera and cerebrospinal fluids of HIV-1-infected patients, and in vitro studies have implicated extracellular Vpr as an effector of cellular responses, including G2 arrest, apoptosis and induction of cytokines and chemokines production, presumably through its ability to transduce into multiple cell types. However, the mechanism underlying Vpr release from HIV-1-producing cells remains undefined and the biological modifications that the extracellular protein may undergo are largely unknown. We provide evidence indicating that soluble forms of Vpr are present in the extracellular medium of HIV-1-producing cells. Release of Vpr in the extracellular medium did not originate from decaying or disrupted HIV-1 virions that package Vpr but rather appeared associated with HIV-1-mediated cytopathicity. Interestingly, Vpr was found to undergo proteolytic processing at a very well conserved proprotein convertase (PC) cleavage site, R(85)QRR(88) downward arrow, located within the functionally important C-terminal arginine-rich domain of the protein. Vpr processing occurred extracellularly upon close contact to cells and most likely involved a cell surface-associated PC. Consistently, PC inhibitors suppressed Vpr processing, while expression of extracellular matrix-associated PC5 and PACE4 enhanced Vpr cleavage. PC-mediated processing of extracellular Vpr led to the production of a truncated Vpr product that was defective for the induction of cell cycle arrest and apoptosis when expressed in human cells. Collectively, these results suggest that cell surface processing of extracellular Vpr by PCs might regulate the levels of active soluble Vpr.

Despite the introduction of highly active antiretroviral therapy, dementia caused by human immunodeficiency virus-1 (HIV-1) infection remains a devastating and common neurological disorder. Although the mechanisms governing neurodegeneration during HIV-1 infection remain uncertain, the HIV-1 accessory protein, viral protein R (Vpr), has been proposed as a neurotoxic protein. Herein, we report that Vpr protein and transcript were present in the brains of HIV-infected persons. Moreover, soluble Vpr caused neuronal apoptosis, involving cytochromecextravasation, p53 induction, and activation of caspase-9 while exerting a depressive effect on whole-cell currents in neurons (p< 0.05), which was inhibited by iberiotoxin. Vpr-activated glial cells secreted neurotoxins in a concentration-dependent manner (p< 0.001). Transgenic (Tg) mice expressing Vpr in brain monocytoid cells displayed the transgene principally in the basal ganglia (p< 0.05) and cerebral cortex (p< 0.01) compared with hindbrain expression. Vpr was released from cultured transgenic macrophages, which was cytotoxic to neurons and was blocked by anti-Vpr antibody (p< 0.05). Neuronal injury was observed in Tg animals compared with wild-type littermates, chiefly affecting GAD65 (p< 0.01) and vesicular acetylcholine transferase (p< 0.001) immunopositive neuronal populations in the basal ganglia. There was also a loss of subcortical synaptophysin (p< 0.001) immunoreactivity as well as an increase in activated caspase-3, which was accompanied by a hyperexcitable neurobehavioral phenotype (p< 0.05). Thus, HIV-1 Vpr caused neuronal death through convergent pathogenic mechanisms with ensuingin vivoneurodegeneration, yielding new insights into the mechanisms by which HIV-1 injures the nervous system.

Human immunodeficiency virus type 1 (HIV-1) entry into target cells is directed by the envelope (Env) glycoproteins, which are present on the surface of HIV-1 virion or infected cells in the form of trimers consisting of gp120/gp41 complexes. The surface subunit, gp120, initiates the entry process by interacting sequentially with the CD4 receptor and a co-receptor, thereby inducing a conformational change that allows the transmembrane (TM) gp41 subunit to mediate fusion between viral and target cell membranes. Cleavage of Env into its gp120 and gp41 components is necessary for activation of its fusogenic activity. Here, the gp41 TM glycoprotein was altered by either deleting an isoleucine residue (DeltaI642) in a critical region of its ectodomain or by substituting its membrane spanning domain (MSD) by that of the influenza hemagglutinin (HA) glycoprotein (TM-HA) to examine the contribution of these regions to Env functions. Characterization of these mutant forms of gp41 revealed that they both affected the infectivity of pseudotyped virions, however, through distinct defects in Env functions. While deletion of Ile 642 drastically altered processing of Env, replacement of gp41 MSD by that of HA led to a marked fusion defect even though the TM-HA Env was efficiently processed and incorporated into viral particles. Interestingly, both DeltaI642 and TM-HA Env were found to act as trans dominant-negative mutant of viral infectivity, presumably via their ability to form hetero-oligomers with wild type Env. Together, these results support a previously proposed model whereby all three gp120-gp41 monomers must be cleaved for the Env homo-trimer to function and suggest that the gp41 MSD plays a critical role in the formation of fusion-competent Env trimers.

Summary Critical cell surface immunoreceptors downregulated during HIV infection have previously been identified using non-systematic, candidate approaches. To gain a comprehensive, unbiased overview of how HIV infection remodels the T cell surface, we took a distinct, systems-level, quantitative proteomic approach. >100 plasma membrane proteins, many without characterized immune functions, were downregulated during HIV infection. Host factors targeted by the viral accessory proteins Vpu or Nef included the amino acid transporter SNAT1 and the serine carriers SERINC3/5. We focused on SNAT1, a β-TrCP-dependent Vpu substrate. SNAT1 antagonism was acquired by Vpu variants from the lineage of SIVcpz/HIV-1 viruses responsible for pandemic AIDS. We found marked SNAT1 induction in activated primary human CD4+ T cells, and used Consumption and Release (CoRe) metabolomics to identify alanine as an endogenous SNAT1 substrate required for T cell mitogenesis. Downregulation of SNAT1 therefore defines a unique paradigm of HIV interference with immunometabolism., Graphical Abstract, Highlights • Unbiased global analysis of T cell surface proteome remodeling during HIV infection • >100 proteins downregulated, including Nef targets SERINC3/5 and Vpu target SNAT1 • β-TrCP-dependent SNAT1 downregulation acquired by pandemic SIVcpz/HIV-1 viruses • Uptake of exogenous alanine by SNAT1 critical for primary CD4+ T cell mitogenesis, Viruses manipulate host factors to enhance their replication. Matheson et al. use functional proteomics to analyze plasma membrane proteins downregulated during HIV-1 infection. Serine carriers SERINC3/5 and alanine transporter SNAT1 were identified as Nef and Vpu targets, respectively. Antagonism of SNAT1-mediated alanine transport enables viral interference with T cell immunometabolism

© 2015, American Society for Microbiology. The proprotein convertases (PCs) furin, PC5, PACE4, and PC7 cleave secretory proteins after basic residues, including the HIV envelope glycoprotein (gp160) and Vpr. We evaluated the abundance of PC mRNAs in postmortem brains of individuals exhibiting HIV-associated neurocognitive disorder (HAND), likely driven by neuroinflammation and neurotoxic HIV proteins (e.g., envelope and Vpr). Concomitant with increased inflammation-related gene expression (interleukin-1ß [IL-1ß]), the mRNA levels of the above PCs are significantly increased, together with those of the proteinase-activated receptor 1 (PAR1), an inflammation-associated receptor that is cleaved by thrombin at ProArg41↓(where the down arrow indicates the cleavage location), and potentially by PCs at Arg41XXXXArg46↓. The latter motif in PAR1, but not its R46A mutant, drives its interactions with PCs. Indeed, PAR1 upregulation leads to the inhibition of membrane-bound furin, PC5B, and PC7 and inhibits gp160 processing and HIV infectivity. Additionally, a proximity ligation assay revealed that furin and PC7 interact with PAR1. Reciprocally, increased furin expression reduces the plasma membrane abundance of PAR1 by trapping it in the trans-Golgi network. Furthermore, soluble PC5A/PACE4 can target/disarm cell surface PAR1 through cleavage at Arg46↓. PACE4/PC5A decreased calcium mobilization induced by thrombin stimulation. Our data reveal a new PC-PAR1-interaction pathway, which offsets the effects of HIV-induced neuroinflammation, viral infection, and potentially the development of HAND.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-α/β cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

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., Author Summary Plasmacytoid dendritic cells (pDCs) produce large quantities of type I interferon (IFN-I) upon stimulation by many viruses, including HIV. Their activation is very effective following cell contacts with HIV-1-infected CD4+ T cells. We investigated whether HIV-1 could regulate the antiviral responses of pDCs triggered upon sensing of infected cells. We show that HIV-1 suppresses the levels of IFN-I produced by pDCs through a process that requires expression of the Vpu accessory protein in virus-producing cells. A well-described role of Vpu is to promote efficient HIV-1 production by counteracting BST2, a host factor that entraps nascent viral particle at the cell surface. Apart from its antiviral activity, BST2 was reported to inhibit IFN-I production by pDCs through binding and activation of the ILT7 pDC-specific inhibitory receptor. Our results reveal that through a highly sophisticated targeted regulation of BST2 levels at the surface of infected cells, Vpu promotes HIV-1 release and limits IFN-I production by pDCs via the negative signaling exerted by the BST2-ILT7 pair. Overall, this study sheds light on a novel Vpu-BST2 interaction that allows HIV-1 to escape pDC antiviral responses. This modulation of pDC antiviral response by HIV Vpu may facilitate the initial viral expansion during acute infection.

The Madin-Darby canine kidney (MDCK) cell line has become the prototypic cell type for studying the mechanisms involved in viral glycoproteins transport and viral assembly in polarized cells. This cell line has been used in our laboratories for studying human immunodeficiency virus (HIV-1), despite the fact that MDCK cells cannot be infected by HIV. In transfected MDCK cells, HIV-1 glycoproteins are specifically transported to the basolateral cell surface where viral budding also mostly occurs. However, this model is of limited use when viral propagation, infection of most cells, or larger production of virions, is needed. The initial objective of this work was thus to establish an MDCK-derived cell line that could be productively infected by HIV-1, in order to pursue our studies on the polarization of viral budding. Expression of both receptor and co-receptor for T-tropic strains of the virus showed that canine cells are rendered permissive once virus binding and entry is allowed. In addition, a reduced infectivity of the viral particles released from the basolateral surface was observed. This observation most likely reflects the interference mediated by CD4 molecules that accumulate at the basolateral domain. Accordingly, this effect was largely prevented when using viruses that down-regulate cell surface CD4 by expression of both viral accessory proteins Vpu and Nef. This is a further evidence that the function of different viral proteins depends of the site of viral budding, which is itself determined by the presence of targeting signal(s) harbored by viral envelope glycoproteins.

Two cis-acting RNA trafficking sequences (heterogenous ribonucleoprotein A2 (hnRNP A2)-response elements 1 and 2 or A2RE-1 and A2RE-2) have been identified in HIV-1 vpr and gag mRNAs and were found to confer cytoplasmic RNA trafficking in a murine oligodendrocyte assay. Their activities were assessed during HIV-1 proviral gene expression in COS7 cells. Single point mutations that were shown to severely block RNA trafficking were introduced into each of the A2REs. In both cases, this resulted in a marked decrease in hnRNP A2 binding to HIV-1 genomic RNA in whole cell extracts and hnRNP A2-containing polysomes. This also resulted in an accumulation of HIV-1 genomic RNA in the nucleus and a significant reduction in genomic RNA encapsidation levels. Immunofluorescence analyses revealed altered expression patterns for pr55Gag and particularly that for Vpr. Vpr localization became almost completely nuclear and this was reflected in a significant reduction in virion-associated Vpr levels. These effects coincided with late steps of the viral replication cycle and were not seen at early time points post-transfection. Transcription, splicing, steady state RNA levels, and pr55Gag processing were not affected. On the other hand, viral replication was markedly compromised in A2RE-2 mutant viruses and this correlated with lowered genomic RNA encapsidation levels. These data reveal new insights into the virus-host interactions between hnRNP A2 and the HIV-1 A2REs and their influence on the patterns of HIV-1 gene expression and viral assembly.

Retroviruses, such as human and simian immunodeficiency viruses (HIV and SIV), and murine leukemia viruses (MuLV), harbor a tyrosine-based motif in the intracytoplasmic domain of their envelope glycoprotein. This motif can act as an endocytosis signal or as a targeting signal, restricting viral budding at specific cell surface membrane domains. In the present study, proviral DNA of the ecotropic Cas-Br-E strain of MuLV was modified by substitution or deletion of the critical tyrosine residue. Mutant viruses lost basolateral targeting in polarized MDCK epithelial cells while expression level of the glycoprotein at the cell surface was not affected. This suggests that the tyrosine-based motif in MuLV does not act as an endocytosis signal. Only a small delay in the appearance of disease was observed in inoculated mice. In contrast, a striking change in the pathology was observed with enlarged thymus and lymph nodes in animals inoculated with mutant viruses.

In this study, reverse transcriptase (RT)- and integrase (IN)-defective human immunodeficiency virus type 1 (HIV-1) was transcomplemented with Vpr-RT-IN fusion proteins to delineate pol sequences important for HIV-1 replication. Our results reveal that a 194-bp sequence encompassing the 3′end of the IN gene and containing the central DNA flap is necessary and sufficient for efficient HIV-1 single-cycle replication in dividing and nondividing cells. Furthermore, we show that the central DNA flap enhances HIV-1 single-round replication by five- to sevenfold, primarily by facilitating nuclear import of proviral DNA. In agreement with previous reports, our data support a functional role of the central DNA flap during the early stages of HIV-1 infection.

Human immunodeficiency virus has evolved several redundant mechanisms to remove its receptor, the CD4 molecule, from the cell surface. Indeed, HIV-1 encodes three proteins, Nef, Vpu and Env, that have a profound effect on CD4 trafficking and catabolism. Given this functional convergence, it is believed that cell surface CD4 regulation constitutes an important determinant of viral replication and pathogenesis in vivo. This review highlights recent progress made in our understanding of the molecular mechanisms underlying the down-regulation of the CD4 receptor by HIV-1 and describes our current comprehension of the role of CD4 down-regulation during HIV-1 infection.

In late April, the laboratory of immunologist Cheolho Cheong, Assistant Professor at the IRCM in Montreal, was celebrating that their work was scheduled for publication in the May 2016 issue of Cell Metabolism. The article, “Indoleamine 2,3-Dioxygenase-Expressing Aortic Plasmacytoid Dendritic Cells Protect against Atherosclerosis by Induction of Regulatory T Cells,” was the first senior author publication from the nascent independent laboratory of Dr. Cheong. Cheolho was so excited about the work, exclaiming, “Now I can show my friends I am still alive, still in science!” With profound sadness and shock, we report that, on April 24, days after celebrating his 41st birthday and returning the page proofs, Cheolho collapsed at home, passing from this life suddenly and unexpectedly.

The Nef protein encoded by human immunodeficiency virus type 1 (HIV-1) has been shown to be an important factor of progression of viral growth and pathogenesis in both in vitro and in vivo. The lack of a simple procedure to purify Nef in its native conformation has limited molecular studies on Nef function. A two-step procedure that includes heparin and immobilized metal ion affinity chromatographies (IMACs) was developed to purify His-tagged Nef (His(6)-Nef) expressed in bacteria in native condition. During the elaboration of this purification procedure, we identified two closely SDS-PAGE-migrating contaminating bacterial proteins, SlyD and GCHI, that co-eluted with His(6)-Nef in IMAC in denaturing condition and developed purification steps to eliminate these contaminants in native condition. Overall, this study describes a protocol that allows rapid purification of His(6)-Nef protein expressed in bacteria in native condition and that removes metal affinity resin-binding bacterial proteins that can contaminate recombinant His-tagged protein preparation.

The absence of immune defects that occurs in the syndrome of long-term nonprogressive (LTNP) HIV infection offers insights into the pathophysiology of HIV-induced immune disease. The (H[F/S]RIG)(2) domain of viral protein R (Vpr) induces apoptosis and may contribute to HIV-induced T cell depletion. We demonstrate a higher frequency of R77Q Vpr mutations in patients with LTNP than in patients with progressive disease. In addition, T cell infections using vesicular stomatitis virus G (VSV-G) pseudotyped HIV-1 Vpr R77Q result in less (P = 0.01) T cell death than infections using wild-type Vpr, despite similar levels of viral replication. Wild-type Vpr-associated events, including procaspase-8 and -3 cleavage, loss of mitochondrial transmembrane potential (deltapsi(m)), and DNA fragmentation factor activation are attenuated by R77Q Vpr. These data highlight the pathophysiologic role of Vpr in HIV-induced immune disease and suggest a novel mechanism of LTNP.

The CD4 molecule plays a key role in the development and activation of helper T cells. Dimerization and oligomerization is often a necessary step in the function of several cell surface receptors. Herein, we provide direct biochemical evidence confirming the presence of CD4 as dimers in transfected cells from hemopoetic and fibroblastic origin as well as in primary T cells. Such dimers are also observed with murine CD4 confirming selective pressure during evolution to maintain such a structure. Using a series of point mutations, we have precisely mapped the dimerization site at residues K318 and Q344 within the fourth extracellular domain of CD4. These residues are highly conserved and their mutation results in interference with dimer formation. More importantly, we demonstrate that dimer formation is essential for the coligand and coreceptor functions of CD4 in T cell activation. These data strongly suggest that CD4 dimerization is necessary for helper T cell function.

Primary monocytes are refractory to HIV-1 infection and become permissive upon differentiation into monocyte-derived dendritic cells (MDDCs) or macrophages. Multiple mechanisms have been proposed to interpret HIV-1 restriction in monocytes. Human cellular miRNAs can modulate HIV-1 infection by targeting either conserved regions of the HIV-1 genome or host gene transcripts. We have recently reported that the translation of host protein pur-alpha is repressed by abundant cellular miRNAs to inhibit HIV-1 infection in monocytes. Here, we report that the transcript of another cellular factor, VprBP [Vpr (HIV-1)-binding protein], was repressed by cellular miRNA-1236, which contributes to HIV-1 restriction in monocytes. Transfection of miR-1236 inhibitors enhanced translation of VprBP in monocytes and significantly promoted viral infection; exogenous input of synthesized miR-1236 mimics into MDDCs suppressed translation of VprBP, and, accordingly, significantly impaired viral infection. Our data emphasize the role of miRNA in modulating differentiation-dependent susceptibility of the host cell to HIV-1 infection. Understanding the modulation of HIV-1 infection by cellular miRNAs may provide key small RNAs or the identification of new important protein targets regulated by miRNAs for the development of antiviral strategies.

The HIV auxiliary protein Vpr potently blocks the cell cycle at the G2/M transition. Here, we show that G2/M arrest results from untimely activation of the structure-specific endonuclease (SSE) regulator SLX4 complex (SLX4com) by Vpr, a process that requires VPRBP-DDB1-CUL4 E3-ligase complex. Direct interaction of Vpr with SLX4 induced the recruitment of VPRBP and kinase-active PLK1, enhancing the cleavage of DNA by SLX4-associated MUS81-EME1 endonucleases. G2/M arrest-deficient Vpr alleles failed to interact with SLX4 or to induce recruitment of MUS81 and PLK1. Furthermore, knockdown of SLX4, MUS81, or EME1 inhibited Vpr-induced G2/M arrest. In addition, we show that the SLX4com is involved in suppressing spontaneous and HIV-1-mediated induction of type 1 interferon and establishment of antiviral responses. Thus, our work not only reveals the identity of the cellular factors required for Vpr-mediated G2/M arrest but also identifies the SLX4com as a regulator of innate immunity.

HIV viral protein R (Vpr) induces a cell cycle arrest at the G2/M phase by activating the ATR DNA damage/replication stress signalling pathway through engagement of the DDB1-CUL4A-DCAF1 E3 ubiquitin ligase via a direct binding to the substrate specificity receptor DCAF1. Since no high resolution structures of the DDB1-DCAF1-Vpr substrate recognition module currently exist, we used a mutagenesis approach to better define motifs in DCAF1 that are crucial for Vpr and DDB1 binding. Herein, we show that the minimal domain of DCAF1 that retained the ability to bind Vpr and DDB1 was mapped to residues 1041 to 1393 (DCAF1 WD). Mutagenic analyses identified an α-helical H-box motif and F/YxxF/Y motifs located in the N-terminal domain of DCAF1 WD that are involved in exclusive binding to DDB1. While we could not identify elements specifically involved in Vpr binding, overall, the mutagenesis data suggest that the predicted β-propeller conformation of DCAF1 is likely to be critical for Vpr association. Importantly, we provide evidence that binding of Vpr to DCAF1 appears to modulate the formation of a DDB1/DCAF1 complex. Lastly, we show that expression of DCAF1 WD in the absence of endogenous DCAF1 was not sufficient to enable Vpr-mediated G2 arrest activity. Overall, our results reveal that Vpr and DDB1 binding on DCAF1 can be genetically separated and further suggest that DCAF1 contains determinants in addition to the Vpr and DDB1 minimal binding domain, which are required for Vpr to enable the induction of a G2 arrest.

Intracellular trafficking of retroviral RNAs is a potential mechanism to target viral gene expression to specific regions of infected cells. Here we show that the human immunodeficiency virus type 1 (HIV-1) genome contains two sequences similar to the hnRNP A2 response element (A2RE), a cis-acting RNA trafficking sequence that binds to the trans-acting trafficking factor, hnRNP A2, and mediates a specific RNA trafficking pathway characterized extensively in oligodendrocytes. The two HIV-1 sequences, designated A2RE-1, within the major homology region of the gag gene, and A2RE-2, in a region of overlap between the vpr and tat genes, both bind to hnRNP A2 in vitro and are necessary and sufficient for RNA transport in oligodendrocytes in vivo. A single base change (A8G) in either sequence reduces hnRNP A2 binding and, in the case of A2RE-2, inhibits RNA transport. A2RE-mediated RNA transport is microtubule and hnRNP A2 dependent. Differentially labelled gag and vpr RNAs, containing A2RE-1 and A2RE-2, respectively, coassemble into the same RNA trafficking granules and are cotransported to the periphery of the cell. tat RNA, although it contains A2RE-2, is not transported as efficiently as vpr RNA. An A2RE/hnRNP A2-mediated trafficking pathway for HIV RNA is proposed, and the role of RNA trafficking in targeting HIV gene expression is discussed.

Natural killer (NK) cell degranulation in response to virus-infected cells is triggered by interactions between invariant NK cell surface receptors and their ligands on target cells. Although HIV-1 Vpr induces expression of ligands for NK cell activation receptor, NKG2D, on infected cells, this is not sufficient to promote lytic granule release. We show that triggering the NK cell coactivation receptor NK-T- and -B cell antigen (NTB-A) alongside NKG2D promotes NK cell degranulation. Normally, NK cell surface NTB-A binds to NTB-A on CD4+ T cells. However, HIV-1 Vpu downmodulates NTB-A on infected T cells. Vpu associates with NTB-A through its transmembrane region without promoting NTB-A degradation. Cells infected with HIV-1 Vpu mutant elicited at least 50% more NK cells to degranulate than wild-type virus. Moreover, NK cells have a higher capacity to lyse HIV-infected cells with a mutant Vpu. Thus, Vpu downmodulation of NTB-A protects the infected cell from lysis by NK cells.

The Vpr protein of primate lentiviruses arrests cell cycling at the G(2)/M phase through an inactivation of cyclin B-p34(cdc2) and its upstream regulator cdc25. We provide here biochemical and functional evidence demonstrating that human immunodeficiency virus type 1 (HIV-1) Vpr mediates G(2) arrest by forming a complex with protein phosphatase 2A (PP2A), an upstream regulator of cdc25. Vpr associates with PP2A through a specific interaction with the B55 regulatory subunit. This interaction is necessary but not sufficient for G(2) arrest. Interestingly, we found that Vpr association with B55-containing PP2A targets the enzymatic complex to the nucleus and, importantly, enhances the recruitment and dephosphorylation of the cdc25 substrate. Our data suggest that Vpr mediates G(2) arrest by enhancing the nuclear import of PP2A and by positively modulating its catalytic activity towards active phosphorylated nuclear cdc25.

Human immunodeficiency virus (HIV)-positive individuals express elevated levels of interleukin-8 (IL-8), which is believed to be responsible for some of the clinical manifestations occurring during AIDS. We report here that virion-derived HIV type 1 (HIV-1) protein R (Vpr) increased IL-8 expression in primary T cells and macrophages, as well as in the T-cell line Jurkat, the monocytic cell line U937, and the epithelial cell line A549. Vpr appeared to increase IL-8 expression and IL-8 promoter activity by activating transcription factors NF-κB and NF-IL-6. Elevated Vpr was also shown to increase transcription of the NF-κB and NF-IL-6 enhancer-containing viral promoters for HIV, cytomegalovirus, and simian virus 40, as well as increase the expression of IL-6 and IL-10 in primary macrophages and in A549 cells, tumor necrosis factor alpha expression in primary T cells, and IL-6 and gamma interferon expression in U937 cells. These results suggest a new role for Vpr in the pathogenesis of HIV infection, namely, the activation of transcription factors NF-IL-6 and NF-κB.

The human immunodeficiency virus type 1 Vpr is a virion-associated protein that is incorporated in trans into viral particles, presumably via an interaction with the p6 domain of the Gag polyprotein precursor. Recently, several studies demonstrated that Vpr fusion proteins could be used as intravirion inactivating agents. In this study, we compared different Vpr-chloramphenicol acetyltransferase (CAT) fusion proteins for their virion incorporation ability and their effect on the infectivity of HIV viruses. Our deletion analysis indicates that both the N-terminal alpha-helical domain and the leucine/isoleucine-rich (LR) domain located in the middle region of Vpr are required for optimal virion incorporation of Vpr-CAT fusion proteins. The C-terminal basic region, associated with Vpr's ability to mediate cell cycle arrest in G2, was not required for virion incorporation, thus allowing the development of Vpr-based chimeric proteins devoid of any effect on cell growth. The fusion of Vpr at the N- or C-terminus of CAT targeted with equal efficiency the chimeric protein into virions. While the virion incorporation of most Vpr-CAT fusion proteins tested in this study was dependent on the presence of an intact p6 domain, fusion proteins containing only the N-terminal alpha-helical domain of Vpr (amino acid 1 to 42) were incorporated into virions in a p6-independent manner. Virion incorporation of Vpr-CAT fusion proteins was shown to decrease viral infectivity. Moreover, the insertion of HIV protease-cleavage sites between Vpr and CAT not only efficiently delivered and released the cleaved CAT product into HIV viral particles, but also greatly potentiated the inhibition of progeny virion infectivity. Overall, our study: (1) defines the Vpr sequence requirement and configuration necessary for the specific and optimal incorporation of Vpr fusion protein into HIV particles; (2) shows that Vpr fusion proteins have the ability to suppress HIV infectivity by targeting multiple steps of viral morphogenesis.

Human immunodeficiency virus type 1 (HIV-1) Vpr is a virion-associated protein which facilitates HIV-1 infection of nondividing cells by contributing to the nuclear transport of the preintegration complex (PIC). Vpr was also shown to induce a cell cycle G2 arrest in infected proliferating cells that optimizes HIV-1 long terminal repeat (LTR)-directed gene expression and viral production. However, it is unclear whether this activity is mediated primarily early by virion-associated Vpr or alternatively late during infection when Vpr is de novo expressed. We report here that in the absence of de novo expression, virion-associated Vpr induces a transient G2 arrest that can subsequently lead to cell killing by apoptosis. Interestingly, the induction of both cell cycle G2 arrest and apoptosis by virion-associated Vpr requires viral entry but not viral replication, since reverse transcriptase and protease inhibitor treatments do not prevent these Vpr effects. These results raise the possibility that in vivo both infectious and noninfectious viruses contribute to the dysfunction and killing of CD4 1 cells. In addition, our results reveal that virion-associated Vpr stimulates viral replication in proliferating cells after establishing a cell cycle G2 arrest by increasing LTR-directed gene expression. Importantly, this Vpr-mediated LTR activation appears to be a requirement for subsequent optimal Tat transactivation. Taken together, these results strongly suggest that in addition to participating in the HIV PIC nuclear transport in nondividing cells, virion-associated Vpr activates HIV-1 LTR-directed gene expression by manipulating the host cell cycle. From this, we conclude that Vpr functions as an immediate-early protein during HIV-1 infection. Human immunodeficiency virus type 1 (HIV-1), the etiological agent of AIDS, infects and ultimately incapacitates critical cellular components of the immune system. Part of the explanation for the complex HIV-host interaction lies in the complex genetic organization of the viral genome. In addition to gag, pol, and env structural gene products, HIV encodes six regulatory or accessory proteins that are not found in the other classes of retroviruses. Some of these products (Tat and Rev) are essential for HIV replication, while others (Vif, Vpr, Vpu, and Nef) appear to optimally modulate the infection and replication processes (7, 9). Vpr is a 14-kDa, 96-amino-acid protein that is highly conserved among HIV-1, HIV-2, and simian immunodeficiency virus. The importance of Vpr during HIV infection and pathogenesis has been suggested by a number of in vitro and in vivo studies (reviewed in references 7 and 9). Vpr was shown to be packaged in significant quantities into viral particles (4, 47). These observations suggested that Vpr may play a role in early events during infection. Indeed, recent studies have demonstrated that Vpr participates in the active nuclear translocation of the HIV-1 preintegration complex (PIC) in nondividing cells by interacting with the nuclear transport pathway (12, 17, 29, 34, 41). This function of Vpr appears essential for HIV infection of macrophages under conditions that closely mimic the in vivo situation (39). A recently discovered second function of Vpr is to promote cell differentiation and growth arrest at the G2/M phase of the cell cycle (16, 21

The accessory protein, Vpr, is a virion-associated protein that is required for HIV-1 replication in macrophages and regulates viral gene expression in T cells. Vpr causes arrest of cell cycle progression at G 2 /M, presumably through its effect on cyclin B1⋅Cdc2 activity. Here, we show that the ability of Vpr to activate HIV transcription correlates with its ability to induce G 2 /M growth arrest, and this effect is mediated by the p300 transcriptional co-activator, which promotes cooperative interactions between the Rel A subunit of NF-κB and cyclin B1⋅Cdc2. Vpr cooperates with p300, which regulates NF-κB and the basal transcriptional machinery, to increase HIV gene expression. Similar effects are seen in the absence of Vpr with a kinase-deficient Cdc2, and overexpression of p300 increases levels of HIV Vpr + replication. Taken together, these data suggest that p300, through its interactions with NF-κB, basal transcriptional components, and Cdks, is modulated by Vpr and regulates HIV replication. The regulation of p300 by Vpr provides a mechanism to enhance viral replication in proliferating cells after growth arrest by increasing viral transcription.

The HIV-1-specific vpu gene encodes an integral membrane phosphoprotein which affects three aspects of the HIV-1 infectious cycle: it enhances virion release from infected cells; it causes degradation of the CD4 protein in the endoplasmic reticulum; and it delays syncytia formation in HIV-1-infected CD4 + T-cells. Although little is known about how Vpu mediates these effects, it has been proposed to function as a nonspecific cation channel. In this report, voltage clamp measurements of Xenopus oocytes show that Vpu expression is not associated with increased transmembrane currents. Instead, Vpu expression diminishes membrane conductance. Injection of 4.6 ng of Vpu mRNA into these cells reduces endogenous potassium conductance by 50%. Only Vpu mutants which retain the ability to degrade CD4 can diminish K + conductance. Inhibition by Vpu is not unique to K + channels as it is also observed on several coexpressed membrane proteins but not on a coexpressed cytoplasmic protein. These results indicate that the CD4 degradative capability of Vpu and the Vpu-mediated modulation of membrane protein expression are mechanistically coupled and that Vpu may contribute to HIV pathogenesis by altering plasma membrane protein expression at the cell surface.

We previously demonstrated that infection of primary human thymic dendritic cells (DCs) with laboratory strains of HIV leads to the release of soluble factor(s) which induced thymocyte killing. In the present paper, we extend the characterization of this process. Our results reveal that primary HIV-1 isolates are similarly able to induce the production of cytotoxic factor(s) from thymic DCs and that the release of such factor(s) is dependent on viral infection. Interestingly, we observed that CD4 + and CD8 + purified thymocyte subsets, and activated PBMCs are susceptible to the cytotoxic activity, whereas freshly isolated resting PBMCs are resistant to this effect. Cycloheximide treatment prevents the killing of thymocytes exposed to HIV-infected DC supernatant, revealing that this form of cell death is an active biological process requiring protein synthesis. Finally, our data suggest that FasL and TNFα could both participate in the killing process. These in vitro observations provide a plausible model, whereby HIV-infected DCs can play a role in vivo in the induction of uninfected thymocyte killing.

It is currently well established that HIV-1 Vpr augments viral replication in primary human macrophages. In its virion-associated form, Vpr has been suggested to aid efficient translocation of the proviral DNA into the cell nucleus. Although Vpr growth-arrests dividing T cells, the relevance of this biological activity in nondividing macrophages is unclear. Here we use Vpr-mutants to demonstrate that the molecular determinants involved in G2-arresting T cells are also involved in increasing viral transcription in macrophages, even though these cells are refractive to the diploid DNA status typical of G2 phase. Our results suggest that the two phenotypes, namely the nuclear localization and the G2-arrest activity of the protein, segregate functionally among the late and early functions of Vpr. The nuclear localization property of Vpr correlates with its ability to effectively target the proviral DNA to the cell nucleus early in the infection, whereas the G2-arrest phenotype correlates with its ability to activate viral transcription after establishment of an infection. These two functions may render Vpr's role essential and not accessory under infection conditions that closely mimic the in vivo situation, that is, primary cells being infected at low viral inputs.