HIV-1 Tat对人脑内皮细胞MMP-9蛋白的影响及作用机制

目的 探讨HIV-1 Tat对人脑内皮细胞基质金属蛋白-9(MMP-9)的影响及作用机制。方法 按照Weksler's方法分离和处理人脑血管内皮细胞。小分子干扰RNA(siRNA)沉寂MMP-9,根据报告的序列设计MMP-9的特异性siRNA和Scramble II对照,MMP-9的活性采用明胶酶谱法检测,进行内皮细胞渗透性检测。结果 实验结果显示,Tat增强MMP-9的表达和活性,siRNA靶向作用于MMP-9以减少可溶性闭锁蛋白的浓度,Tat可以使HBMECs渗透性显著增强。MMP-9干扰可以恢复Tat处理后细胞的完整性。结论 Tat可以抑制闭锁蛋白的表达,并通过增强MMP的表达和激活而间接破坏闭锁蛋白,使血脑屏障(BBB)渗透性增强。     

HIV-1 Tat Activates Neuronal Ryanodine Receptors with Rapid Induction of the Unfolded Protein Response and Mitochondrial Hyperpolarization

Neurologic disease caused by human immunodeficiency virus type 1 (HIV-1) is ultimately refractory to highly active antiretroviral therapy (HAART) because of failure of complete virus eradication in the central nervous system (CNS), and disruption of normal neural signaling events by virally induced chronic neuroinflammation. We have previously reported that HIV-1 Tat can induce mitochondrial hyperpolarization in cortical neurons, thus compromising the ability of the neuron to buffer calcium and sustain energy production for normal synaptic communication. In this report, we demonstrate that Tat induces rapid loss of ER calcium mediated by the ryanodine receptor (RyR), followed by the unfolded protein response (UPR) and pathologic dilatation of the ER in cortical neurons in vitro. RyR antagonism attenuated both Tat-mediated mitochondrial hyperpolarization and UPR induction. Delivery of Tat to murine CNS in vivo also leads to long-lasting pathologic ER dilatation and mitochondrial morphologic abnormalities. Finally, we performed ultrastructural studies that demonstrated mitochondria with abnormal morphology and dilated endoplasmic reticulum (ER) in brain tissue of patients with HIV-1 inflammation and neurodegeneration. Collectively, these data suggest that abnormal RyR signaling mediates the neuronal UPR with failure of mitochondrial energy metabolism, and is a critical locus for the neuropathogenesis of HIV-1 in the CNS.     

RNA interference as an antiviral approach: targeting HIV-1

RNA interference (RNAi) is an evolutionary conserved gene-silencing mechanism in which 21- to 23-mer double-stranded short interfering RNA (siRNA) mediates the sequence-specific degradation of mRNA. The recent discovery that exogenously delivered siRNA can trigger RNAi in mammalian cells raises the possibility of using this technology as a therapeutic tool against pathogenic viruses. This review describes the antiviral RNAi field, which is barely two years-old, with an emphasis on recent studies aimed at suppression of HIV-1.     

Gene therapy as a vaccine for HIV-1

Introduction: An effective vaccine that can protect people against infection of the human immunodeficiency virus type 1 (HIV-1) remains elusive. HIV-1 vaccine research has encountered several false starts and a few causes for hope over the last 28 years, but no real success stories. Thus, it is time to think out of the box and design and test unorthodox vaccination strategies. Areas covered: Recent studies in mice and monkeys have revealed the potential of a gene therapy that provides vaccine-like protection against HIV-1 infection by producing a potent vector-encoded antibody that neutralizes the invading viruses. This novel strategy is called Vectored Immuno Prophylaxis or VIP, and it circumvents the sometimes difficult phases of regular vaccination protocols, that is, antigen design and induction of protective immune responses. Expert opinion: VIP is a prolonged form of passive immunization by means of a gene therapy. We will discuss the ins and outs of VIP and the therapeutic possibilities and challenges.     

Galectin-1-specific inhibitors as a new class of compounds to treat HIV-1 infection

Despite significant improvements, antiretroviral therapies against HIV-1 are plagued by a high frequency of therapeutic failures that have been associated with acquisition of drug resistance. We recently reported that HIV-1 exploits a host glycan binding protein, galectin-1, to increase its attachment to host cells, thereby increasing its overall infectivity in susceptible cells. This finding suggests that host molecules such as galectin-1 could reduce the expected efficiency of HIV-1 drugs targeting early steps of the replicative cycle, such as attachment and entry processes. Thus, new classes of drugs that would interfere with galectin-1/HIV-1 interactions could benefit the current antiretroviral therapy. To further explore this possibility, experiments were conducted to discover leading compounds showing specific inhibition of galectin-1 activity in a cellular model of HIV-1 infection. Three lactoside compounds were found to modestly inhibit the interaction of galectin-1 with primary human CD4(+) T cells. Interestingly, these same inhibitors reduced the galectin-1-mediated increase in HIV-1 attachment to target cells in a much more efficient manner. More important, the tested lactoside derivatives also significantly decreased the galectin-1-dependent enhancement of HIV-1 infection. These observations deserve further attention when considering that the development of new drugs to prevent and treat HIV-1 infection remains a priority.     

Structure and function of HIV-1 and SIV Tat proteins based on carboxy-terminal truncations, chimeric Tat constructs, and NMR modeling

To further define the structure and function of the domains in HIV-1 and SIV Tat proteins, chimeric Tat cDNA expression constructs were generated with crossover points at the carboxy-terminal end of the cysteine rich domain. The chimera containing the amino-terminal region of SIV and carboxy-terminal region of HIV exhibited activity similar to HIV-1 Tat and SIV Tat on both the HIV-1 and SIV LTRs. In contrast, the reciprocal chimera functioned poorly. As determined by the activity of carboxy-terminal truncation mutants, the region immediately downstream of the basic domain is critical for efficient transactivation by HIV-1 Tat, but not SIV Tat protein. In this report, we present a model for Tat domains based on NMR data and the known functional properties of Tat protein. According to our modeling two sites for protein :protein interactions are present in HIV-1 and SIV Tat proteins. Site I, which is presumably involved in cyclin T binding, is similar in both HIV-1 and SIV Tat proteins as well as in Tat chimeras. Site II, however appears structurally different in HIV-1 and SIV Tat models, although in both cases is comprised of amino and carboxy-terminal residues. Differences in Site II may thus account for the differential activities of HIV-1 and SIV Tat carboxy-terminal truncations. Site II in the poorly active chimera differs significantly from that found in HIV-1 and SIV Tat proteins. The two site structural model presented here may have important implications for the role of Tat in HIV pathogenesis and may provide insights for the design of Tat vaccines and targeted therapeutics.     

High-throughput human immunodeficiency virus type 1 (HIV-1) full replication assay that includes HIV-1 Vif as an antiviral target

Antiviral screens have proved useful for the identification of novel human immunodeficiency virus type 1 (HIV-1) inhibitors. In this study, we describe an HIV-1 full replication (HIV-1 Rep) assay that incorporates all of the targets required for replication in T-cell lines, including the HIV-1 Vif gene. The HIV-1 Rep assay was designed to exhibit optimal sensitivity to late-stage as well as early-stage inhibitors to maximize the likelihood of identification of novel target antiviral compounds in a screen. In addition, the flexibility of the HIV-1 Rep assay allows the rapid evaluation of antiviral compounds against different virus strains in different T-cell lines without significant modification of the assay format. We demonstrate that the HIV-1 Rep assay exhibits characteristics (e.g., a favorable Z' value) compatible with high-throughput screening in a 384-well format. The utility of the HIV-1 Rep assay was demonstrated in a high-throughput screen of >10(6) compounds. To our knowledge, this study represents the first example of an HIV-1 antiviral screen that includes Vif as a functional target and was executed on an industrial scale.     

In Vivo Selection of CD4(+) T Cells Transduced with a Gamma-Retroviral Vector Expressing a Single-Chain Intrabody Targeting HIV-1 Tat

Abstract We evaluated the potential of an anti-human immunodeficiency virus (HIV) Tat intrabody (intracellular antibody) to promote the survival of CD4(+) cells after chimeric simian immunodeficiency virus (SIV)/HIV (SHIV) infection in rhesus macaques. Following optimization of stimulation and transduction conditions, purified CD4(+) T cells were transduced with GaLV-pseudotyped retroviral vectors expressing either an anti-HIV-1 Tat or a control single-chain intrabody. Ex vivo intrabody-gene marking was highly efficient, averaging four copies per CD4(+) cell. Upon reinfusion of engineered autologous CD4(+) cells into two macaques, high levels of gene marking (peak of 0.6% and 6.8% of peripheral blood mononuclear cells (PBMCs) and 0.3% or 2.2% of the lymph node cells) were detected in vivo. One week post cell infusion, animals were challenged with SHIV 89.6p and the ability of the anti-HIV Tat intrabody to promote cell survival was evaluated. The frequency of genetically modified CD4(+) T cells progressively decreased, concurrent with loss of CD4(+) cells and elevated viral loads in both animals. However, CD4(+) T cells expressing the therapeutic anti-Tat intrabody exhibited a relative survival advantage over an 8- and 21-week period compared with CD4(+) cells expressing a control intrabody. In one animal, this survival benefit of anti-Tat transduced cells was associated with a reduction in viral load. Overall, these results indicate that a retrovirus-mediated anti-Tat intrabody provided significant levels of gene marking in PBMCs and peripheral tissues and increased relative survival of transduced cells in vivo.     

HIV-1 proprotein processing as a target for gene therapy

The central role of endoconvertases and HIV-1 protease (HIV-1 PR) in the processing of HIV proproteins makes the design of specific inhibitors important in anti-HIV gene therapy. Accordingly, we tested native alpha(1) antitrypsin (alpha(1)AT) delivered by a recombinant simian virus-40-based vector, SV(AT), as an inhibitor of HIV-1 proprotein maturation. Cell lines and primary human lymphocytes were transduced with SV(AT) without selection and detectable toxicity. Expression of alpha(1)AT was confirmed by Northern blotting, immunoprecipitation and immunostaining. SV(AT)-transduced cells showed no evidence of HIV-1-related cytopathic effects when challenged with high doses of HIV-1(NL4-3). As measured by HIV-1 p24 assay, SV(AT)-transduced cells were protected from HIV-1(NL4-3) at challenge dose of 40 000 TCID(50) (MOI = 0.04). In addition, peripheral blood lymphocytes treated with SV(AT) were protected from HIV doses challenge up to 40 000 TCID(50) (MOI = 0.04). By Western blot analyses, the delivered alpha(1)AT inhibited cellular processing of gp160 to gp120 and decreased HIV-1 virion gp120. SV(AT) inhibited processing of p55(Gag) as well. Furthermore, high levels of uncleaved p55(Gag) protein were detected in HIV virus particles recovered from SV(AT)-transduced cells lines and primary lymphocytes. Thus, delivering alpha(1)AT using SV(AT) to human lymphocytes strongly inhibits replication of HIV-1, most likely by inhibiting the activities both of the cellular serine proteases involved in processing gp160 and of the aspartyl protease, HIV-1 PR, which cleaves p55(Gag). alpha(1)AT delivered by SV(AT) may represent a novel and effective strategy for gene therapy to interfere with HIV replication, by blocking a stage in the virus replicative cycle that has until now been inaccessible to gene therapeutic intervention.     

建立用于检测H IV-1的基于核酸序列扩增的酶联免疫吸附测定法

目的：建立用于检测 HIV-1的基于核酸序列扩增的酶联免疫吸附测定（NASBA-ELISA）法。方法根据 HIV-1基因组长末端重复序列设计引物对 HIV-1 RNA进行基于核酸序列扩增（NASBA）,结合微孔板中液相杂交和酶标显色反应检测核酸扩增物,并对该检测方法的灵敏度、特异度及其对临床样本的检测结果进行评价。结果该方法可检测到0．1 fg/μL的RNA,且对 HIV-1具有特异性。在120例临床样本的检测中,其检测准确度高于常规的实时荧光定量反转录聚合酶链反应（fqRT-PCR）法。结论 NASBA-ELISA方法能灵敏并特异地检测 HIV-1。     

Development of immunotherapeutic strategies for HIV-1

In the majority of untreated patients, HIV-1 infection presents as a progressive disease of the immune system. Recent studies indicate that immune responses can be induced in HIV-1 infected individuals, leading to some immune control of virus replication. Such immune responses are also observed in small numbers of untreated HIV-1 infected long-term non-progressor (LTNP) patients, as well as in other viral infections (including those with human herpesviruses). Emerging novel technologies, animal studies and detailed immunological studies have proven invaluable in defining the immune responses that are associated with a favourable clinical outcome. Central effector and regulatory cells are HIV-1-specific CD8+ cytotoxic T-lymphocytes (CTL) and CD4+ helper T-lymphocytes respectively. Fully functional antigen-presenting cells (APC) are also essential in all stages of HIV-1 infection and possibly some (but not all) antibody responses contribute to beneficial immunity. The availability of combination anti-retroviral drug therapy, which successfully controls viraemia, has enabled a beneficial outcome in many HIV-1 infected individuals. Since no chronically HIV-1 infected patient has been shown to eradicate virus, novel approaches utilising therapeutic immunisation and various cytokines to manipulate immune responses and to induce and steer immunity towards a desired phenotype are required. There is a clear rationale for immunotherapeutic intervention in chronic progressive HIV-1 infection, which forms the foundation for novel approaches aimed at inducing and maintaining immune control. Here we review the immunopathogenesis of HIV-1 infection and discuss the promises of therapeutic immunisation and immunotherapy in general and their potential in the treatment of chronic HIV-1 disease.     

Development of a confirmatory enzyme-linked immunosorbent assay for HIV-1 antibodies

We subcloned six discrete protein-coding regions representing the gag (Kp24 and Kp55), env (Kp41, Kp120N, and Kp120CC), and pol (Kp66/31) gene products of the human immunodeficiency virus type 1 (HIV-1) and expressed them in Escherichia coli as fusion proteins with the first 56 residues of galactokinase. An enzyme-linked immunosorbent assay for confirming the presence of HIV-1 antibodies was developed by coating the six purified antigens on individual wells of a microtiter plate (the HIVAGEN assay). This assay yielded no false-negative results and fewer indeterminate results than the Western blot assay for 143 specimens from patients with acquired immunodeficiency syndrome (AIDS) or AIDS-related complex (ARC). Analysis of 1016 specimens from seronegative donors by the HIVAGEN assay yielded no false-positive results, and the rate of indeterminate results was substantially lower than for the Western blot assay. The HIVAGEN assay is well suited for routine confirmation of the presence of HIV-1 antibodies because it is objective, quantitative, rapid, precise, and readily automatable.     

Reliability of a new recombinant immunoblot assay (RIBA HIV-1/HIV-2 SIA) as a supplemental (confirmatory) test for HIV-1 and HIV-2 infections

The diagnosis of HIV infection is usually determined using serological methods. Positive results in a screening test should be confirmed with more specific supplementary assays, and the Western blot (WB) is the most frequently used. However, problems of WB, particularly in regard to the significance of indeterminate results, still remain. We evaluate a new recombinant HIV-1/2 antigen-based immunoblot (RIBA) as confirmatory assay in a panel of 190 clinical samples. The RIBA was much more sensitive and specific than WB. In particular, the test allows for a significant reduction in the number of indeterminate results and for more accurate distinction between HIV-1 and/or HIV-2 infections. Thus, these results make tests such as the RIBA instead of WB particularly attractive for validating anti-HIV EIA reactivity.     

Actinohivin,a Broadly Neutralizing Prokaryotic Lectin,Inhibits HIV-1 Infection by Specifically Targeting High-Mannose-Type Glycans on the gp120 Envelope

The lectin actinohivin (AH) is a monomeric carbohydrate-binding agent (CBA) with three carbohydrate-binding sites. AH strongly interacts with gp120 derived from different X4 and R5 human immunodeficiency virus (HIV) strains, simian immunodeficiency virus (SIV) gp130, and HIV type 1 (HIV-1) gp41 with affinity constants (K_D) in the lower nM range. The gp120 and gp41 binding of AH is selectively reversed by (α1,2-mannose)₃ oligosaccharide but not by α1,3/α1,6-mannose- or GlcNAc-based oligosaccharides. AH binding to gp120 prevents binding of α1,2-mannose-specific monoclonal antibody 2G12, and AH covers a broader epitope on gp120 than 2G12. Prolonged exposure of HIV-1-infected CEM T-cell cultures with escalating AH concentrations selects for mutant virus strains containing N-glycosylation site deletions (predominantly affecting high-mannose-type glycans) in gp120. In contrast to 2G12, AH has a high genetic barrier, since several concomitant N-glycosylation site deletions in gp120 are required to afford significant phenotypic drug resistance. AH is endowed with broadly neutralizing activity against laboratory-adapted HIV strains and a variety of X4 and/or R5 HIV-1 clinical clade isolates and blocks viral entry within a narrow concentration window of variation (∼5-fold). In contrast, the neutralizing activity of 2G12 varied up to 1,000-fold, depending on the virus strain. Since AH efficiently prevents syncytium formation in cocultures of persistently HIV-1-infected HuT-78 cells and uninfected CD4⁺ T lymphocytes, inhibits dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin-mediated capture of HIV-1 and subsequent virus transmission to CD4⁺ T lymphocytes, does not upregulate cellular activation markers, lacks mitogenic activity, and does not induce cytokines/chemokines in peripheral blood mononuclear cell cultures, it should be considered a potential candidate drug for microbicidal use.The envelope glycoproteins of human immunodeficiency virus (HIV) mediate attachment and viral entry into susceptible target cells. The initial steps in HIV infection include binding of the trimeric gp120 with three CD4 receptor molecules, a number of conformational changes in gp120, and interactions with chemokine receptor CCR5 or CXCR4, followed by the unfolding of gp41, which anchors the virion to the target membrane and which brings the viral and cellular membranes in close contact for further virus-cell fusion (17).The HIV type 1 (HIV-1) gp120 envelope glycoprotein is highly glycosylated. Approximately half of its molecular weight is contributed by its carbohydrate content (22, 36). The recombinant HIV-1(III_B) gp120 expressed in Chinese hamster ovary (CHO) cells is occupied by 11 high-mannose- or hybrid-type glycans and 13 complex-type glycans (29).A variety of carbohydrate-binding agents (CBAs), such as the prokaryotic agent cyanovirin-N (CV-N) (7-10), the plant lectins Hippeastrum hybrid agglutinin (HHA) (1) and Galanthus nivalis agglutinin (GNA) (1), and the antibiotics pradimicin A and S (PRM-A and PRM-S, respectively) (4, 7), have been described to inhibit viral entry, presumably by their interaction with the glycans on HIV gp120. It has indeed been demonstrated that CBAs block virus entry by inhibiting the fusion of cell-free HIV virions with their target cells. They prevent the capture of virus particles by the dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) and the macrophage mannose receptor, which are present on cells of the innate immune system. The subsequent transmission of the virus to CD4⁺ T cells is also efficiently hampered by CBAs, pointing to their potential as HIV microbicide drug candidates (6). By the lack of an efficient HIV vaccine, microbicides can evolve to be important tools for the prevention of HIV transmission and infection (23, 37, 38).One CBA, namely, the prokaryotic agent CV-N, has been tested in animal vaginal and rectal virus transmission models (43, 44) and shown to successfully inhibit viral infections in the animals, suggesting that CV-N has the potential to act as an anti-HIV microbicide. However, recently, it was described that CV-N induced the production of a variety of cytokines and cellular activation markers in peripheral blood mononuclear cells (PBMCs), and in addition, the pronounced mitogenic activity of CV-N was also observed (2, 26). These potential side effects can compromise the application of CV-N as an efficient microbicide.Actinohivin (AH) is an anti-HIV protein that has been isolated from a prokaryotic microorganism (the actinomycete Longispora albida K97-0003^T) (14, 27). AH consists of 114 amino acids (12,524.3 Da) and exhibits a unique highly conserved internal sequence triplication (60% identity and ∼70% homology on each segment). It was shown that the three repeats of AH are necessary to block syncytium formation in recombinant cell cultures (HeLa /T env tat and HeLa CD4 lacZ cells) (40, 41). Recently, the crystal structure of AH was solved, and it was revealed that AH has three sugar-binding pockets (41). It was suggested before that AH-gp120 interactions are accomplished by N-linked high-mannose-type carbohydrates that are abundantly present on gp120 (15, 41).We have now performed a detailed investigation of AH regarding its anti-HIV activity spectrum, potential side effects, kinetic interaction with the HIV-1 envelope proteins gp120 and gp41, and resistance spectrum and also added data on other CBAs that are related to AH in terms of their carbohydrate specificities (i.e., HHA and monoclonal antibody [MAb] 2G12) for comparative reasons. Based on its safety profile and unique kinetic/antiviral properties our data revealed that AH qualifies as a potential drug lead for further preclinical investigations.