Sulforaphane Reduces SAMHD1 Phosphorylation To Protect Macrophages from HIV-1 Infection.

The cellular protein SAMHD1 is important for DNA repair, suppressing LINE elements, controlling deoxynucleoside triphosphate (dNTP) concentrations, maintaining HIV-1 latency, and preventing excessive type I interferon responses. SAMHD1 is also a potent inhibitor of HIV-1 and other significant viral pathogens. Infection restriction is due in part to the deoxynucleoside triphosphatase (dNTPase) activity of SAMHD1 but is also mediated through a dNTPase-independent mechanism that has been described but not explored. The phosphorylation of SAMHD1 at threonine 592 (T592) controls many of its functions. Retroviral restriction, irrespective of dNTPase activity, is linked to unphosphorylated T592. Sulforaphane (SFN), an isothiocyanate, protects macrophages from HIV infection by mobilizing the transcription factor and antioxidant response regulator Nrf2. Here, we show that SFN and other clinically relevant Nrf2 mobilizers reduce SAMHD1 T592 phosphorylation to protect macrophages from HIV-1. We further show that SFN, through Nrf2, triggers the upregulation of the cell cycle control protein p21 in human monocyte-derived macrophages to contribute to SAMHD1 activation. We additionally present data that support another, potentially redox-dependent mechanism employed by SFN to contribute to SAMHD1 activation through reduced phosphorylation. This work establishes the use of exogenous Nrf2 mobilizers as a novel way to study virus restriction by SAMHD1 and highlights the Nrf2 pathway as a potential target for the therapeutic control of SAMHD1 cellular and antiviral functions. IMPORTANCE Here, we show, for the first time, that the treatment of macrophages with Nrf2 mobilizers, known activators of antioxidant responses, increases the fraction of SAMHD1 without a regulatory phosphate at position 592. We demonstrate that this decreases infection of macrophages by HIV-1. Phosphorylated SAMHD1 is important for DNA repair, the suppression of LINE elements, the maintenance of HIV-1 in a latent state, and the prevention of excessive type I interferon responses, while unphosphorylated SAMHD1 blocks HIV infection. SAMHD1 impacts many viruses and is involved in various cancers, so knowledge of how it works and how it is regulated has broad implications for the development of therapeutics. Redox-modulating therapeutics are already in clinical use or under investigation for the treatment of many conditions. Thus, understanding the impact of redox modifiers on controlling SAMHD1 phosphorylation is important for many areas of research in microbiology and beyond.

Sulforaphane Inhibits HIV Infection of Macrophages through Nrf2.

Marburg virus, the Kaposi's sarcoma-associated herpesvirus (KSHV) and Dengue virus all activate, and benefit from, expression of the transcription regulator nuclear erythroid 2-related factor 2 (Nrf2). The impact of Nrf2 activation on human immunodeficiency virus (HIV) infection has not been tested. Sulforaphane (SFN), produced in cruciferous vegetables after mechanical damage, mobilizes Nrf2 to potently reprogram cellular gene expression. Here we show for the first time that SFN blocks HIV infection in primary macrophages but not in primary T cells. Similarly SFN blocks infection in PMA-differentiated promonocytic cell lines, but not in other cell lines tested. siRNA-mediated depletion of Nrf2 boosted HIV infectivity in primary macrophages and reduced the anti-viral effects of SFN treatment. This supports a model in which anti-viral activity is mediated through Nrf2 after it is mobilized by SFN. We further found that, like the type I interferon-induced cellular anti-viral proteins SAMHD1 and MX2, SFN treatment blocks infection after entry, but before formation of 2-LTR circles. Interestingly however, neither SAMHD1 nor MX2 were upregulated. This shows for the first time that Nrf2 action can potently block HIV infection and highlights a novel way to trigger this inhibition.

Inhibition of HIV early replication by the p53 and its downstream gene p21.

BACKGROUND: The tumor suppressor gene p53 has been found to suppress HIV infection by various mechanisms, but the inhibition of HIV at an early stage of replication by host cell p53 and its downstream gene p21 has not been well studied. METHOD: VSV-G pseudotyped HIV-1 or HIV-2 viruses with GFP or luciferase reporter gene were used to infect HCT116 p53+/+ cells, HCT116 p53-/- cells and hMDMs. The infections were detected by flow cytometry or measured by luciferase assay. Reverse transcription products were quantified by a TaqMan real time PCR. siRNA knockdown experiments were applied to study potential roles of p53 and p21 genes in their restriction to HIV infection. Western blot experiments were used to analyze changes in gene expression. RESULTS: The infection of HIV-1 was inhibited in HCT116 p53+/+ cells in comparison to HCT116 p53-/- cells. The fold of inhibition was largely increased when cell cycle switched from cycling to non-cycling status. Further analysis showed that both p53 and p21 expressions were upregulated in non-cycling HCT116 p53+/+ cells and HIV-1 reverse transcription was subsequently inhibited. siRNA knockdown of either p53 or p21 rescued HIV-1 reverse transcription from the inhibition in non-cycling HCT116 p53+/+ cells. It was identified that the observed restrictions by p53 and p21 were associated with the suppression of RNR2 expression and phosphorylation of SAMHD1. These observations were confirmed by using siRNA knockdown experiments. In addition, p53 also inhibited HIV-2 infection in HCT116 p53+/+ cells and siRNA knockdown of p21 increased HIV-2 infection in hMDMs. Finally the expressions of p53 and p21 were found to be induced in hMDMs shortly after HIV-1 infection. CONCLUSIONS: The p53 and its downstream gene p21 interfere with HIV early stage of replication in non-cycling cells and hMDMs.

Cullin4A and cullin4B are interchangeable for HIV Vpr and Vpx action through the CRL4 ubiquitin ligase complex.

UNLABELLED: Human immunodeficiency virus (HIV) seizes control of cellular cullin-RING E3 ubiquitin ligases (CRLs) to promote viral replication. HIV-1 Vpr and HIV-2/simian immunodeficiency virus (SIV) Vpr and Vpx engage the cullin4 (CUL4)-containing ubiquitin ligase complex (CRL4) to cause polyubiquitination and proteasomal degradation of host proteins, including ones that block infection. HIV-1 Vpr engages CRL4 to trigger the degradation of uracil-N-glycosylase 2 (UNG2). Both HIV-1 Vpr and HIV-2/SIV Vpr tap CRL4 to initiate G2 cell cycle arrest. HIV-2/SIV Vpx secures CRL4 to degrade the antiviral protein SAMHD1. CRL4 includes either cullin4A (CUL4A) or cullin4B (CUL4B) among its components. Whether Vpr or Vpx relies on CUL4A, CUL4B, or both to act through CRL4 is not known. Reported structural, phenotypic, and intracellular distribution differences between the two CUL4 types led us to hypothesize that Vpr and Vpx employ these in a function-specific manner. Here we determined CUL4 requirements for HIV-1 and HIV-2/SIV Vpr-mediated G2 cell cycle arrest, HIV-1 Vpr-mediated UNG2 degradation, and HIV-2 Vpx-mediated SAMHD1 degradation. Surprisingly, CUL4A and CUL4B are exchangeable for CRL4-dependent Vpr and Vpx action, except in primary macrophages, where Vpx relies on both CUL4A and CUL4B for maximal SAMHD1 depletion. This work highlights the need to consider both CUL4 types for Vpr and Vpx functions and also shows that the intracellular distribution of CUL4A and CUL4B can vary by cell type. IMPORTANCE: The work presented here shows for the first time that HIV Vpr and Vpx do not rely exclusively on CUL4A to cause ubiquitination through the CRL4 ubiquitin ligase complex. Furthermore, our finding that intracellular CUL4 and SAMHD1 distributions can vary with cell type provides the basis for reconciling previous disparate findings regarding the site of SAMHD1 depletion. Finally, our observations with primary immune cells provide insight into the cell biology of CUL4A and CUL4B that will help differentiate the functions of these similar proteins.

HIV relies on neddylation for ubiquitin ligase-mediated functions.

BACKGROUND: HIV and SIV defeat antiviral proteins by usurping Cullin-RING E3 ubiquitin ligases (CRLs) and likely influence other cellular processes through these as well. HIV-2 viral protein X (Vpx) engages the cullin4-containing CRL4 complex to deplete the antiviral protein SAMHD1. Vif expressed by HIV-1 and HIV-2 taps a cullin5 ubiquitin ligase complex to mark the antiviral protein APOBEC3G for destruction. Viral Protein R of HIV-1 (Vpr) assembles with the CRL4 ubiquitin ligase complex to deplete uracil-N-glycosylase2 (UNG2). Covalent attachment of the ubiquitin-like protein side-chain NEDD8 functionally activates cullins which are common to all of these processes. RESULTS: The requirement for neddylation in HIV-1 and HIV-2 infectivity was tested in the presence of APOBEC3G and SAMHD1 respectively. Further the need for neddylation in HIV-1 Vpr-mediated depletion of UNG2 was probed. Treatment with MLN4924, an adenosine sulfamate analog which hinders the NEDD8 activating enzyme NAE1, blocked neddylation of cullin4A (CUL4A). The inhibitor hindered HIV-1 infection in the presence of APOBEC3G, even when Vif was expressed, and it stopped HIV-2 infection in the presence of SAMHD1 and Vpx. Consistent with these findings, MLN4924 prevented Vpx-mediated depletion of SAMHD1 in macrophages infected with Vpx-expressing HIV-2, as well as HIV-1 Vif-mediated destruction of APOBEC3G. It also stemmed Vpr-mediated UNG2 elimination from cells infected with HIV-1. CONCLUSIONS: Neddylation plays an important role in HIV-1 and HIV-2 infection. This observation is consistent with the essential parts that cullin-based ubiquitin ligases play in overcoming cellular anti-viral defenses.

CIITA enhances HIV-1 attachment to CD4+ T cells leading to enhanced infection and cell depletion.

Activated CD4(+) T cells are more susceptible to HIV infection than resting T cells; the reason for this remains unresolved. Induction of CIITA and subsequent expression of the MHC class II isotype HLA-DR are hallmarks of CD4(+) T cell activation; therefore, we investigated the role of CIITA expression in T cells during HIV infection. CIITA-expressing SupT1 cells display enhanced virion attachment in a gp160/CD4-dependent manner, which results in increased HIV infection, virus release, and T cell depletion. Although increased attachment and infection of T cells correlated with HLA-DR surface expression, Ab blocking, transient expression of HLA-DR without CIITA, and short hairpin RNA knockdown demonstrate that HLA-DR does not directly enhance susceptibility of CIITA-expressing cells to HIV infection. Further analysis of the remaining MHC class II isotypes, HLA-DP and HLA-DQ, MHC class I isotypes, HLA-A, HLA-B, and HLA-C, and the class II Ag presentation genes, invariant chain and HLA-DM, demonstrate that these proteins likely do not contribute to CIITA enhancement of HIV infection. Finally, we demonstrate that in activated primary CD4(+) T cells as HLA-DR/CIITA expression increases there is a corresponding increase in virion attachment. Overall, this work suggests that induction of CIITA expression upon CD4(+) T cell activation contributes to enhanced attachment, infection, virus release, and cell death through an undefined CIITA transcription product that may serve as a new antiviral target.

xCT/SLC7A11 antiporter function inhibits HIV-1 infection.

Human macrophages are protected by intrinsic antiviral defenses that provide moderate protection against HIV-1 infection. Macrophages that do become infected can serve as long-lived reservoirs, to disseminate HIV-1 to CD4+ T cells. Infection of macrophages with HIV-1 and HIV-2 is inhibited by constitutive mobilization of antioxidant response master transcription regulator Nrf2. The downstream mediator of this restriction was not identified. Among the tens of genes controlled directly by Nrf2 in macrophages, we found that xCT/SLC7A11, a 12-transmembrane, cystine-glutamate antiporter promotes antiretroviral activity. We show here that depletion of xCT mRNA increases HIV-1 infection. Reconstitution of xCT knock out cells with wild-type xCT but not a transport-deficient mutant restores anti-HIV-1 activity. Pharmacological inhibitors of xCT amino acid transport also increase infection. The block is independent of known restriction factors and acts against HIV-1 and HIV-2. Like the block triggered through Nrf2, xCT function impedes infection immediately before 2-LTR circle formation.

The functions of the HIV1 protein Vpr and its action through the DCAF1.DDB1.Cullin4 ubiquitin ligase.

Among the proteins encoded by human and simian immunodeficiency viruses (HIV and SIV) at least three, Vif, Vpu and Vpr, subvert cellular ubiquitin ligases to block the action of anti-viral defenses. This review focuses on Vpr and its HIV2/SIV counterparts, Vpx and Vpr, which all engage the DDB1.Cullin4 ubiquitin ligase complex through the DCAF1 adaptor protein. Here, we discuss the multiple functions that have been linked to Vpr expression and summarize the current knowledge on the role of the ubiquitin ligase complex in carrying out a subset of these activities.

Analysis of Early Phase HIV-1 Replication and Integration Events by Using Real-time PCR.

Upon entry into a host cell, the HIV-1 virus undergoes a series of critical early replication events including reverse transcription, nuclear import, and integration of its cDNA into the host genome. Molecular assays used to detect and analyze changes in HIV-1 early phase replication events are valuable tools in developing potential antiretroviral drugs, as well as studying the pathogenesis of HIV. Described here are the molecular assays utilized to detect and quantify HIV-1 early, intermediate, and late reverse transcription (RT) products. In addition to this, protocols for quantifying HIV-1 2-LTR circle DNA and proviral DNA after integration are also included. In these protocols, the optimized TaqMan Real-time PCR reagent is used to increase assay sensitivity and reproducibility. Furthermore, a nested PCR is applied to HIV-1 integration quantification with increased accuracy.

A sensitive and specific enzyme-based assay detecting HIV-1 virion fusion in primary T lymphocytes.

As an early event in the viral life cycle, the entry of enveloped viruses into target cells has received considerable attention. Viral fusion to cellular targets has been studied principally with fusion assays in which cells engineered to express the viral envelope are cultured with the target cells. These assays yield valuable information but do not fully recapitulate all of the variables governing the fusion of actual virions to their cellular targets. The virion membrane and the plasma membrane, for example, differ strikingly in their lipid and protein compositions. Two virion-based fusion assays have been described. One is based on the redistribution of a self-quenching fluorophore, whereas the second depends on photosensitized activation of a hydrophobic probe by a fluorescent lipid loaded into the target membrane. These assays are complex and have not been adapted to study fusion in complex cell populations. We have developed a simple, rapid assay allowing the detection of HIV-1 virion fusion to biologically relevant target cells, including primary CD4(+) T lymphocytes. It is based on the incorporation of beta-lactamase-Vpr chimeric proteins (BlaM-Vpr) into HIV-1 virions and their subsequent delivery into the cytoplasm of target cells as a result of virion fusion. This transfer is then detected by enzymatic cleavage of the CCF2 dye, a fluorescent substrate of beta-lactamase (BlaM), loaded in the target cells. BlaM cleaves the beta-lactam ring in CCF2, changing its fluorescence emission spectrum from green (520 nm) to blue (447 nm) and thereby allowing fusion to be detected by fluorescence microscopy, flow cytometry, or UV photometry.

Insights into the biology of HIV-1 viral protein R.

HIV-1 viral protein R (Vpr) is a small, highly conserved accessory protein encoded by the HIV genome that serves many functions in the viral life cycle. Vpr induces G2 cell cycle arrest, which is thought to indirectly enhance viral replication by increasing transcription from the LTR. Vpr has also been implicated in facilitating infection of nondividing cells, most notably macrophages. Because Vpr is a nucleo-cytoplasmic shuttling protein, its role in enhancing viral replication in macrophages may be mediated through enhanced entry of the HIV preintegration complex through the limiting nuclear pore. Free Vpr is detectable in the serum of patients, and in vitro studies implicate extracellular forms of Vpr as an effector of cellular responses mediated through its ability to transduce through intact cytoplasmic membranes. We review the biologic properties of Vpr, focusing on its mechanism of action, role in HIV replication, and significance for host pathogenesis.

The HIV-1 protein Vpr targets the endoribonuclease Dicer for proteasomal degradation to boost macrophage infection.

The HIV-1 protein Vpr enhances macrophage infection, triggers G2 cell cycle arrest, and targets cells for NK-cell killing. Vpr acts through the CRL4(DCAF1) ubiquitin ligase complex to cause G2 arrest and trigger expression of NK ligands. Corresponding ubiquitination targets have not been identified. UNG2 and SMUG1 are the only known substrates for Vpr-directed depletion through CRL4(DCAF1). Here we identify the endoribonuclease Dicer as a target of HIV-1 Vpr-directed proteasomal degradation through CRL4(DCAF1). We show that HIV-1 Vpr inhibits short hairpin RNA function as expected upon reduction of Dicer levels. Dicer inhibits HIV-1 replication in T cells. We demonstrate that Dicer also restricts HIV-1 replication in human monocyte-derived macrophages (MDM) and that reducing Dicer expression in MDMs enhances HIV-1 infection in a Vpr-dependent manner. Our results support a model in which Vpr complexes with human Dicer to boost its interaction with the CRL4(DCAF1) ubiquitin ligase complex and its subsequent degradation.

The role of HIV-1 Vpr in promoting the infection of nondividing cells and in cell cycle arrest.

PURPOSE OF REVIEW: The search for the role(s) that HIV-1 Vpr and its HIV2/SIV paralogs Vpr and Vpx play in viral infection and pathogenesis showed that all three engage CRL4 ubiquitin ligase complexes. This association triggers ubiquitination and degradation of cellular substrates. The identity of the ubiquitin ligase substrates is only now beginning to be revealed. This review focuses on recent work that has identified one such substrate and exposed new cellular restrictions to infection. RECENT FINDINGS: Three groups have now described cellular factors that restrict HIV-1 infection in cells of the myeloid lineage. One of these factors, sterile alpha motif- and metal-dependent phosphohydrolase domain-containing protein 1 (SAMHD1), was shown to be depleted through the CRL4 ubiquitin ligase complex in the presence of HIV-2/SIV Vpx. The other restriction can be defeated by Vpx in the absence of at least one part of the ubiquitin ligase complex that triggers SAMHD1 depletion.Another group has shown that the previously described upregulation of natural killer-cell ligands on the surface of HIV-1-infected cells requires the actions of both the cytidine deaminase APOBEC3G and uracil-N-glycosylase 2 in association with HIV-1 Vpr. SUMMARY: As more cellular interaction partners are identified for HIV-1 Vpr and its paralogs from other viruses, details are emerging about Vpr function. The recent findings have highlighted the existence of two new human proteins that can act to combat HIV infection and have revealed how HIV-1 proteins act in concert to modulate the interaction between natural killer cells and HIV-1 infected cells.

The HIV1 protein Vpr acts to enhance constitutive DCAF1-dependent UNG2 turnover.

BACKGROUND: The HIV1 protein Vpr assembles with and acts through an ubiquitin ligase complex that includes DDB1 and cullin 4 (CRL4) to cause G2 cell cycle arrest and to promote degradation of both uracil DNA glycosylase 2 (UNG2) and single-strand selective mono-functional uracil DNA glycosylase 1 (SMUG1). DCAF1, an adaptor protein, is required for Vpr-mediated G2 arrest through the ubiquitin ligase complex. In work described here, we used UNG2 as a model substrate to study how Vpr acts through the ubiquitin ligase complex. We examined whether DCAF1 is essential for Vpr-mediated degradation of UNG2 and SMUG1. We further investigated whether Vpr is required for recruiting substrates to the ubiquitin ligase or acts to enhance its function and whether this parallels Vpr-mediated G2 arrest. METHODOLOGY/PRINCIPAL FINDINGS: We found that DCAF1 plays an important role in Vpr-independent UNG2 and SMUG1 depletion. UNG2 assembled with the ubiquitin ligase complex in the absence of Vpr, but Vpr enhanced this interaction. Further, Vpr-mediated enhancement of UNG2 degradation correlated with low Vpr expression levels. Vpr concentrations exceeding a threshold blocked UNG2 depletion and enhanced its accumulation in the cell nucleus. A similar dose-dependent trend was seen for Vpr-mediated cell cycle arrest. CONCLUSIONS/SIGNIFICANCE: This work identifies UNG2 and SMUG1 as novel targets for CRL4(DCAF1)-mediated degradation. It further shows that Vpr enhances rather than enables the interaction between UNG2 and the ubiquitin ligase. Vpr augments CRL4(DCAF1)-mediated UNG2 degradation at low concentrations but antagonizes it at high concentrations, allowing nuclear accumulation of UNG2. Further, the protein that is targeted to cause G2 arrest behaves much like UNG2. Our findings provide the basis for determining whether the CRL4(DCAF1) complex is alone responsible for cell cycle-dependent UNG2 turnover and will also aid in establishing conditions necessary for the identification of additional targets of Vpr-enhanced degradation.

The HIV1 protein Vpr acts to promote G2 cell cycle arrest by engaging a DDB1 and Cullin4A-containing ubiquitin ligase complex using VprBP/DCAF1 as an adaptor.

The roles of the HIV1 protein Vpr in virus replication and pathogenesis remain unclear. Expression of Vpr in dividing cells causes cell cycle arrest in G(2). Vpr also facilitates low titer infection of terminally differentiated macrophages, enhances transcription, promotes apoptosis, and targets cellular uracil N-glycosylase for degradation. Using co-immunoprecipitation and tandem mass spectroscopy, we found that HIV1 Vpr engages a DDB1- and cullin4A-containing ubiquitin-ligase complex through VprBP/DCAF1. HIV2 Vpr has two Vpr-like proteins, Vpr and Vpx, which cause G(2) arrest and facilitate macrophage infection, respectively. HIV2 Vpr, but not Vpx, engages the same set of proteins. We further demonstrate that the interaction between Vpr and the ubiquitin-ligase components as well as further assembly of the ubiquitin-ligase are necessary for Vpr-mediated G(2) arrest. Our data support a model in which Vpr engages the ubiquitin ligase to deplete a cellular factor that is required for cell cycle progression into mitosis. Vpr, thus, functions like the HIV1 proteins Vif and Vpu to usurp cellular ubiquitin ligases for viral functions.

Human immunodeficiency virus type 1 Vpr induces DNA replication stress in vitro and in vivo.

The human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) causes cell cycle arrest in G2. Vpr-expressing cells display the hallmarks of certain forms of DNA damage, specifically activation of the ataxia telangiectasia mutated and Rad3-related kinase, ATR. However, evidence that Vpr function is relevant in vivo or in the context of viral infection is still lacking. In the present study, we demonstrate that HIV-1 infection of primary, human CD4+ lymphocytes causes G2 arrest in a Vpr-dependent manner and that this response requires ATR, as shown by RNA interference. The event leading to ATR activation in CD4+ lymphocytes is the accumulation of replication protein A in nuclear foci, an indication that Vpr likely induces stalling of replication forks. Primary macrophages are refractory to ATR activation by Vpr, a finding that is consistent with the lack of detectable ATR, Rad17, and Chk1 protein expression in these nondividing cells. These observations begin to explain the remarkable resilience of macrophages to HIV-1-induced cytopathicity. To study the in vivo consequences of Vpr function, we isolated CD4+ lymphocytes from HIV-1-infected individuals and interrogated the cell cycle status of anti-p24Gag-immunoreactive cells. We report that infected cells in vivo display an aberrant cell cycle profile whereby a majority of cells have a 4N DNA content, consistent with the onset of G2 arrest.

Nef is physically recruited into the immunological synapse and potentiates T cell activation early after TCR engagement.

The HIV-1 protein Nef enhances viral pathogenicity and accelerates disease progression in vivo. Nef potentiates T cell activation by an unknown mechanism, probably by optimizing the intracellular environment for HIV replication. Using a new T cell reporter system, we have found that Nef more than doubles the number of cells expressing the transcription factors NF-kappaB and NFAT after TCR stimulation. This Nef-induced priming of TCR signaling pathways occurred independently of calcium signaling and involved a very proximal step before protein kinase C activation. Engagement of the TCR by MHC-bound Ag triggers the formation of the immunological synapse by recruiting detergent-resistant membrane microdomains, termed lipid rafts. Approximately 5-10% of the total cellular pool of Nef is localized within lipid rafts. Using confocal and real-time microscopy, we found that Nef in lipid rafts was recruited into the immunological synapse within minutes after Ab engagement of the TCR/CD3 and CD28 receptors. This recruitment was dependent on the N-terminal domain of Nef encompassing its myristoylation. Nef did not increase the number of cell surface lipid rafts or immunological synapses. Recently, studies have shown a specific interaction of Nef with an active subpopulation of p21-activated kinase-2 found only in the lipid rafts. Thus, the corecruitment of Nef and key cellular partners (e.g., activated p21-activated kinase-2) into the immunological synapse may underlie the increased frequency of cells expressing transcriptionally active forms of NF-kappaB and NFAT and the resultant changes in T cell activation.

HIV-1 Vif blocks the antiviral activity of APOBEC3G by impairing both its translation and intracellular stability.

The human immunodeficiency virus type 1 (HIV-1) relies on Vif (viral infectivity factor) to overcome the potent antiviral function of APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G, also known as CEM15). Using an APOBEC3G-specific antiserum, we now show that Vif prevents virion incorporation of endogenous APOBEC3G by effectively depleting the intracellular levels of this enzyme in HIV-1-infected T cells. Vif achieves this depletion by both impairing the translation of APOBEC3G mRNA and accelerating the posttranslational degradation of the APOBEC3G protein by the 26S proteasome. Vif physically interacts with APOBEC3G, and expression of Vif alone in the absence of other HIV-1 proteins is sufficient to cause depletion of APOBEC3G. These findings highlight how the bimodal translational and posttranslational inhibitory effects of Vif on APOBEC3G combine to markedly suppress the expression of this potent antiviral enzyme in virally infected cells, thereby effectively curtailing the incorporation of APOBEC3G into newly formed HIV-1 virions.

HIV-1 Vpr displays natural protein-transducing properties: implications for viral pathogenesis.

The 14-kDa Vpr protein of human immunodeficiency virus type 1 (HIV-1) serves multiple functions in the retroviral life cycle, including the enhancement of viral replication in nondividing macrophages, the induction of G2 cell-cycle arrest in proliferating T lymphocytes, and the modulation of HIV-1-induced apoptosis. Extracellular Vpr has been detected in the sera and cerebral spinal fluid of HIV-infected patients. However, it is not known whether such forms of Vpr are biologically active. Vpr contains a carboxy-terminal basic amino acid rich segment stretch that is homologous to domains that mediate the energy- and receptor-independent cellular uptake of polypeptides by a process termed protein transduction. Similar functional protein-transducing domains are present in HIV-1 Tat, herpes simplex virus-1 DNA-binding protein VP22, and the Drosophila antennapedia homeotic transcription factor. We now demonstrate effective transduction of biologically active, synthetic Vpr (sVpr) as well as the Vpr-beta-galactosidase fusion protein. However, in contrast to other transducing proteins, Vpr transduction is not enhanced by protein denaturation, and Vpr's carboxy-terminal basic domain alone is not sufficient for its transduction across biological membranes. In contrast, the full-length Vpr protein effectively transduces a broad array of cells, leading to dose-dependent G2 cell-cycle arrest and apoptosis. Addition of Vpr into the extracellular medium also rescues the replication of Vpr-deficient strains of HIV-1 in human macrophage cultures. Native Vpr may thus be optimized for protein transduction, a feature that might enhance and extend the pathological effects of HIV infection.