A novel Vpr peptide interactor fused to integrase (IN) restores integration activity to IN-defective HIV-1 virions

A novel approach to complement human immunodeficiency virus type I (HIV-1) integrase (IN)-defective virions has been identified. The approach involves fusion of a 23-amino-acid stretch to the N-terminus of wild-type IN and coexpression of this chimera with the IN-defective proviral template in virus producing cells. The 23-amino-acid peptide represents a Vpr "interactor," referred to as the the WxxF or WF domain, which apparently leads to docking of the domain along with the fusion partner onto HIV-1 Vpr, thus permitting virion incorporation of the chimeric protein when expressed, in trans, with other viral products. Transfection of the WF-IN expression plasmid along with HIV-1 viral clones that produce Vpr, but bear an IN mutation, results in the release of a proportion of viral particles that are competent for integration. The extent of complementation was assessed using the MAGI cell assay, where integration of viral DNA results in the eventual appearance of easily visible multinucleated blue syncytia. The efficiency of dWF-IN (double copy of WF domain) complementation is not improved markedly by incorporation of a HIV-1 protease cleavage site (PR) between the dWF domain and IN (dWF-PR-IN), unlike that observed with Vpr fusions to IN. Furthermore, the ability of Vpr-PR-IN and dWF-PR-IN to complement IN-defective proviral clones, both of which bear an intervening protease cleavage site, appear comparable. Western blotting analyses using virions isolated through sucrose cushions demonstrate clearly the incorporation of the dWF-IN fusion protein into Vpr containing HIV-1 particles but not in Vpr-deficient virions. Additional Western blotting analyses indicate that all Vpr-IN and dWF-IN chimeras, with or without a PR site, are packaged into virions. The efficiency of virion incorporation of Vpr-IN and dWF-IN chimeras appears approximately comparable by Western blotting analysis. The ability of dWF-IN to complement IN-defective proviruses with efficiency similar to that of Vpr-PR-IN and dWF-PR-IN indicates that dWF-IN retains the full complement of functions necessary for integration of proviral DNA and is likely due to the benign nature of this small domain at the amino-terminus of IN.     

HIV-1 Vpr does not inhibit CTL-mediated apoptosis of HIV-1 infected cells

HIV-1 infected persons develop a robust CTL response to HIV antigens, yet HIV-1 is able to evade this host response and successfully replicate. The mechanism(s) of evasion is not completely defined but has been suggested to include resistance of infected cells to CTL-mediated apoptosis. The HIV-1 Vpr protein induces G2 arrest by indirectly inhibiting activation of cyclin B/p34cdc2 kinase. Granzyme B, the principle mediator of CTL-induced apoptosis, prematurely activates this same kinase complex. Therefore, we assessed the susceptibility of HIV-1 infected cells to CTL-mediated apoptosis to determine whether the expression of Vpr protected the infected cells from CTL-induced apoptosis. Antigen-specific CD8(+) CTL were able to induce apoptosis in HIV-1 infected cells and cells labeled with peptide corresponding to the CTL epitope with equivalent efficiency. This demonstrates that neither HIV-1 Vpr nor any other HIV protein directly inhibits CTL effector functions. Furthermore, we confirm that HIV-1 Nef is able to provide partial protection from CTL recognition of infected cells. Thus, the inability of CTL to control HIV-1 infection is likely not due to direct inhibition of CTL-mediated apoptosis.     

HIV-1 Vpr: mechanisms of G2 arrest and apoptosis

Since the first isolation of HIV-1 from a patient with generalized lymphadenopathy in 1983, great progress has been made in understanding the viral life cycle and the functional nuances of each of the nine genes encoded by HIV-1. Considerable attention has been paid to four small HIV-1 open reading frames, vif, vpr, vpu and nef. These genes were originally termed “accessory” because their deletion failed to completely disable viral replication in vitro. More than twenty years after the cloning and sequencing of HIV-1, a great deal of information is available regarding the multiple functions of the accessory proteins and it is well accepted that, collectively, these gene products modulate the host cell biology to favor viral replication, and that they are largely responsible for the pathogenesis of HIV-1. Expression of Vpr, in particular, leads to cell cycle arrest in G₂, followed by apoptosis. Here we summarize our current understanding of Vpr biology with a focus on Vpr-induced G₂ arrest and apoptosis.     

HIV-1 Vpr蛋白对C8166细胞毒性研究

目的 利用腺病毒系统研究HIV-1 Vpr蛋白在T细胞来源的C8166细胞内的高表达以及细胞内该蛋白对T细胞毒性.方法 将表达目的 蛋白Vpr的重组腺病毒rAd-vpr和空白载体病毒rAd-vector分别感染对数生长期的C8166细胞,流式细胞术(FCM)检测细胞周期分布、凋亡和坏死.用Hoechst-PI荧光染色观察细胞的凋亡和坏死,JC-1荧光染色测定线粒体膜电势.结果 Annexin V-PI染色及Hoechst-PI染色一致显示HIV-1 Vpr能显著诱导C8166细胞凋亡和坏死;PI细胞周期结果表明HIV-1 Vpr能阻滞C8166细胞于G2期;JC-1荧光染色法测定HIV-1 Vpr致C8166线粒体膜电势下降.结论 细胞内HIV-1 Vpr介导的T细胞毒性包括线粒体功能障碍、细胞周期的G2期阻滞和细胞的死亡.     

HIV-1 Vpr interacts with the nuclear transport pathway to promote macrophage infection

HIV-1 Vpr promotes nuclear entry of viral nucleic acids in nondividing macrophages and also causes a G₂ cell-cycle arrest. Consistent with its role in nuclear transport, we show Vpr localizes to the nuclear envelope in both human and yeast cells. Like the importin-β subunit of the nuclear import receptor, Vpr also interacts with the yeast importin-α subunit and nucleoporins. Moreover, overexpression of either Vpr or importin-β in yeast blocks nuclear transport of mRNAs. A mutant form of Vpr (Vpr F34I) that does not localize at the nuclear envelope, or bind to importin-α and nucleoporins, renders HIV-1 incapable of infecting macrophages efficiently. Vpr F34I, however, still causes a G₂ arrest, demonstrating that the dual functions of Vpr are genetically separable. Our data suggest Vpr functionally resembles importin-β in nuclear import of the HIV-1 pre-integration complex and this function is essential for the role of Vpr in macrophage infection, but not G₂ arrest.     

重组慢病毒表达载体介导HIV-1 Vpr蛋白调控KSHV潜伏感染的初探

目的 利用慢病毒载体系统包装含人类免疫缺陷病毒1型(HIV-1)病毒蛋白R(Vpr)的重组慢病毒,使之感染原发性渗出性淋巴瘤(PEL)细胞BCBL-1,并检测Vpr蛋白对细胞中卡波济肉瘤相关疱疹病毒(KSHV)潜伏感染与裂解性复制的影响.方法 从实验室先前构建的重组真核表达质粒pCI-neo-Vpr中扩增出Vpr基因,插入到pHAGE-CMV-MCS-IzsGreen载体中构建成重组慢病毒质粒pHAGE-Vpr,利用脂质体将其与包装质粒psPAX2及包膜质粒pMD2.G共转染293T细胞,荧光显微镜观察293T细胞中绿色荧光蛋白(GFP)的表达,293T细胞培养上清经0.45 μm滤器过滤后即获得重组病毒悬液.慢病毒系列稀释后感染293T细胞,荧光计数法测定病毒滴度,逆转录PCR(RT-PCR)检测Vpr基因在293T细胞中的转录.以感染复数(MOI)为1的病毒量感染靶细胞BCBL-1,荧光显微镜观察靶细胞中GFP的表达,并利用RT-PCR和和免疫印迹(Western blot)技术分别检测Vpr基因在靶细胞中的转录和表达情况,同时检测KSHV裂解期基因复制与转录激活子(Rta)mRNA转录及蛋白表达.结果 经酶切鉴定、核酸序列测定和293T细胞中GFP表达证实成功包装了携带HIV-1Vpr基因的重组慢病毒,滴度为4×107TU/ml.重组慢病毒感染BCBL-1细胞后,细胞中有GFP表达,RT-PCR和Western blot均能够在相应位置检测到目的 基因Vpr的条带,并且Vpr降低了KSHV RtamRNA转录及蛋白表达水平.结论 重组慢病毒介导的HIV-1 Vpr蛋白过表达能够抑制KSHV裂解性复制、增强病毒的潜伏感染.     

HIV-1 Vpr transactivates LTR-directed expression through sequences present within -278 to -176 and increases virus replication in vitro.

Human immunodeficiency virus type 1 (HIV-1) Vpr, a 14-kDa virion-associated protein, plays an important role in the viral life cycle. Using a panel of truncated HIV-1 LTR-CAT constructs and Vpr expression plasmid, we have identified sequences from nucleotide -278 to -176 in LTR as Vpr-mediated transactivation domain. This region includes the glucocorticoid response element (GRE) in HIV-1 LTR. Transactivation by Vpr was noted with the HIV-1 LTR reporter constructs containing CAT or luciferase. A similar effect was also observed with a construct in which the GRE motif was linked to CAT. Studies involving Vpr mutants identified that helical domains I and III, and amino acid residues at G75 and C76, are responsible for GRE-mediated LTR transactivation. The transactivation function of Vpr is independent of its cell cycle arrest activity. Further, viral replication studies indicated that Vpr-mediated increase in viral replication is directly correlated with the ability of Vpr to transactivate HIV-1 LTR. The results presented here demonstrate that Vpr activates HIV-1 LTR through the host GR pathway and suggest that an intact GRE in the LTR is critical for Vpr activity.     

HIV-1 Vpr促进Vif和APOBEC3G蛋白表达的研究

目的 观察人获得性免疫缺陷病毒蛋白Vpr对Vif蛋白和APOBEC3G蛋白表达水平的影响.方法 使用电转化法将携带Vif基因的酵母表达载体转化到裂殖酵母中,使用脂质体转染法将表达Vif蛋白、APOBEC3G蛋白的哺乳动物表达载体转染到可诱导稳定表达Vpr蛋白的哺乳动物HEK293细胞中;使用Western Blot方法检测目标蛋白表达水平的变化.结果 在裂殖酵母和哺乳动物细胞中,Vpr蛋白的表达能够提高细胞内的Vif蛋白的表达水平,在哺乳动物细胞中,Vpr蛋白对APOBEC3G蛋白的表达亦有促进作用,Vpr蛋白引起的Vif蛋白量的提高并没有导致APOBEC3G蛋白的减少.结论 Vpr蛋白具有调节细胞内蛋白表达的功能.     

Cell-free transmission of Vif mutants of HIV-1.

To determine the phenotype of human immunodeficiency virus type 1 (HIV-1) defective in the production of Vif, mutations were introduced into the vif gene of infectious molecular clones of the LAI, MAL, and ELI strains. Previous results had demonstrated that viruses derived from these wild type clones display different replicative capacities on peripheral blood mononuclear cells (PBMC) and different tropisms for CD4-positive cell lines. Using cell-free infection, Vif mutants of LAI, MAL, and ELI were found to have delayed kinetics and to produce less virus than their corresponding wild type viruses when propagated on most permissive cell lines. An additional mutation in the vpr gene in the LAI strain had no effect on this phenotype. However, on one T cell line, the H9 cell line, two Vif mutants of LAI and a Vif.Vpr double mutant were unable to replicate. Furthermore, Vif mutants from all three strains were only able to establish a productive infection on PBMC by coculture and not by cell-free infection. No defects in the processing of the virion proteins or the release of particles in the Vif mutants were detected.     

Inhibition of HIV-1 expression by HIV-2

HIV-1 and HIV-2 are co-endemic in certain geographic areas. HIV-2 is more weakly pathogenic than HIV-1, and progression to AIDS occurs less frequently and over a longer period of time. Recent epidemiologic studies suggest that individuals infected with HIV-2 have a lower risk of HIV-1 infection. Both immune mechanisms and various modes of viral interference have been proposed to account for these results. Our findings, described in this paper, suggest that HIV-2 inhibits HIV-1 replication. To study the molecular interactions between HIV-1 and HIV-2, proviral clones were transfected alone or in combination into the human T cell line CEM. LTR-CAT indicator constructs were included for the purpose of monitoring viral promoter activity. Viral replication in transfected cells was monitored by p24 antigen capture assay of cell culture supernatants and Western blot analysis of cell extracts. HIV-2 inhibited HIV-1 replication as determined by intracellular and extracellular p24 antigen levels. Similar results were obtained with simultaneous virus infection using HIV-1 and HIV-2, rather than transfections of proviral DNA. Using cotransfection of HIV-1 and HIV-2 LTR indicator gene constructs, the mechanism of inhibition was found to be suppression of the HIV-1 LTR by HIV-2. The inhibitory effect of HIV-2 is not due to Tat-2, but appears to discriminate between the HIV-1 and HIV-2 LTRs based on differences in the Tat activation response element, TAR. These results suggest both a molecular mechanism for HIV-2 interference with HIV-1 replication and a potential molecular approach to therapy.     

Functional conservation of HIV-1 Vpr and variability in a mother-child pair of long-term non-progressors

Increasing evidence suggests that HIV-1 Vpr is required in vivo for viral pathogenesis. Since Vpr displays multiple activities, little is known about which Vpr-specific activities are conserved in naturally occurring viruses or how natural mutations in Vpr might modulate viral pathogenesis in HIV-infected individuals. The goals of this study were to evaluate the functional variability of Vpr in naturally occurring viruses. The Vpr-specific activities of nuclear localization, induction of cell cycle G2 arrest and cell death were compared between viruses isolated from the fast progressing AIDS patients and a mother-child pair of long-term non-progressors (LTNPs). Wild-type Vpr activities were found in all of the viruses that were isolated from the fast progressing AIDS patients except for the truncated Vpr(IIIB) which lacked these activities. In contrast, defective Vpr were readily detected in viral populations isolated, over an 11-year period, from the mother-child pair. Sequence analyses indicated that these Vpr carried unique amino acid substitutions that frequently interrupted a highly conserved domain containing an N-terminal alpha-helix-turn-alpha-helix. Thus, Vpr activities are generally conserved in naturally occurring viruses. The functionally defective Vpr identified in the mother-child pair of LTNPs are likely to be unique and may possibly contribute to the slow disease progression.     

A human immunodeficiency virus Env inducible transcription system to examine consequences of gp120 expression

According to several studies, the HIV-1 envelope gp120 protein and the co-receptor CXCR4 play an essential role in HIV-1 induced cell toxicity. Characterisation of the CD4-independent m7NDK isolate provided the opportunity of studying the effects of direct interactions between m7NDK gp120 and CXCR4. Therefore, an inducible expression system was designed enabling synthesis of HIV-1 Env proteins upon doxycycline induction. Analysis of the expression of the env gene of the m7NDK HIV-1 isolate revealed, unexpectedly, that even long-term expression of m7NDK gp120 did not result in cytotoxycity in CXCR4-positive or -negative cell lines. This is the first report of a CD4-independent HIV-1-protein inducible expression regulated through the Tet-On system and by an alternative splicing. Env inducible expression cell lines could constitute a useful cellular tool to undertake analysis of HIV Env protein expression.     

Mu-opioid modulation of HIV-1 coreceptor expression and HIV-1 replication

A substantial proportion of HIV-1-infected individuals are intravenous drug users (i.v.DUs) who abuse opiates. Opioids induce a number of immunomodulatory effects that may directly influence HIV-1 disease progression. In the present report, we have investigated the effect of opioids on the expression of the major HIV-1 coreceptors CXCR4 and CCR5. For these studies we have focused on opiates which are ligands for the mu-opioid receptor. Our results show that DAMGO, a selective mu-opioid agonist, increases CXCR4 and CCR5 expression in both CD3(+) lymphoblasts and CD14(+) monocytes three- to fivefold. Furthermore, DAMGO-induced elevation of HIV-1 coreceptor expression translates into enhanced replication of both X4 and R5 viral strains of HIV-1. We have confirmed the role of the mu-opioid receptor based on the ability of a mu-opioid receptor-selective antagonist to block the effects of DAMGO. We have also found that morphine enhances CXCR4 and CCR5 expression and subsequently increases both X4 and R5 HIV-1 infection. We suggest that the capacity of mu-opioids to increase HIV-1 coreceptor expression and replication may promote viral binding, trafficking of HIV-1-infected cells, and enhanced disease progression.