HIV-1整合酶链转移反应抑制剂的荧光筛选方法

整合酶被认为是抗HIV-1药物研究的理想靶点之一。为了建立便捷高效的整合酶链转移反应抑制剂筛选方法,首先将HIV-1整合酶原核表达载体pNL-IN转化入大肠杆菌感受态细胞BL21(DE3)进行原核表达,并用镍琼脂糖凝胶进行亲和纯化,获得了纯度和活性均较高的整合酶重组蛋白;然后设计了生物素标记的供体DNA和FITC标记的靶DNA,用链霉亲和素磁珠捕获反应体系中的DNA产物;最后用荧光分析仪检测DNA产物的荧光信号,并计算待测样品的抑制率。用已知整合酶抑制剂S-1360和MK-0518对筛选方法进行了验证,测定结果与已有实验数据相当,表明本筛选方法能够有效应用于HIV-1整合酶链转移反应抑制剂的筛选。与现有的整合酶链转移反应抑制剂筛选方法相比,本筛选方法步骤更为简化、耗时更短、成本更低。     

HIV-1复制抑制剂的新靶标

现在市场上的多种治疗ＡＩＤＳ的药物都是病毒逆转录酶和蛋白水解酶的特异性抑制剂 ,但是发现有 2 0 %以上的病人不能在短期内承受这些药物的治疗 ,而且长期使用蛋白水解酶抑制剂对人体正常新陈代谢的影响也越来越突出 ,现已发现ＨＩＶ 1的耐药变种已经出现并正以很快的速度传播。另外 ,这些药物不能直接清除感染ＨＩＶ 1病人体内的病毒。所以现在需要发现新型的治疗ＡＩＤＳ病的药物 ,这些药物可以长期使用 ,能补充或者部分代替已有的药物 ,而这就需要先找到ＨＩＶ 1复制抑制剂的新靶标。ＨＩＶ 1病毒的生活周期是这样的 :病毒被膜和细胞表…     

HIV-1整合酶核心区可溶性表达及抑制剂筛选

为了实现HIV-1整合酶蛋白核心区 (central core domain of integrase, IN-CCD) 的可溶性表达,并建立以IN-CCD为靶点的抑制剂体外筛选方法,从包含F185K突变HIV-1 IN基因的质粒中经PCR扩增得到含有F185K突变的IN-CCD基因,克隆到pET28b载体上构建重组质粒pIN-CCD,转化pIN-CCD至E. coli BL21 (DE3)中经IPTG诱导、表达,Ni-亲和层析纯化,获得IN-CCD蛋白。修饰DNA底物,以链亲和素包被的磁珠为载体捕获DNA产物,结合酶联免疫吸附测定法(ELISA)检测IN-CCD的去整合活性,并筛选以IN-CCD为靶点的抑制剂。结果表明重组蛋白IN-CCD实现了高效可溶性表达,纯化后蛋白纯度达95%。建立的ELISA可以检测IN-CCD的去整合活性,且方法特异性和灵敏度好,可以实现高通量抑制剂筛选。从100个样品中筛选得到5个具有初步抑制IN-CCD去整合活性的样品。     

HIV-1 Tat蛋白在细胞核中的功能

HIV-1 Tat蛋白是HIV-1病毒基因表达的重要调控蛋白,其通过与不同的细胞分子及信号通路相互作用来调控细胞过程。Tat蛋白由感染细胞产生,也可由感染细胞产生后分泌而作用于其他细胞;因此,它既能影响感染细胞,也能影响未感染细胞。Tat蛋白积聚在细胞核中,但根据其表达水平的不同,其可能会定位于核质或核仁,并发挥不同的核效应。该文针对Tat蛋白的结构、核输入机制及细胞核效应的研究进展进行概述。     

HIV-1蛋白酶的表达、纯化及其抑制剂体外筛选方法的建立

从HIV-1IIIB病毒RNA经RT-PCR得到HIV-1蛋白酶编码序列,克隆到pet28a质粒中构建HIV-1蛋白酶表达载体。阳性克隆转染E.coliBL21DE3,经IPTG诱导,蛋白酶以包涵体的形式表达,表达量占菌体总蛋白量的40%。包涵体经TritonX-100洗涤后溶解于8M尿素,溶解后的蛋白溶液经sephacyls-200H.R分子筛柱纯化后纯度达到90%以上,收集蛋白酶峰稀释复性并通过超滤进行浓缩。经检测,纯化的蛋白酶具有较高的活性。用荧光标记的蛋白酶底物检测不同浓度indinavir对蛋白酶活性的影响,表明该方法可以用于蛋白酶抑制剂的筛选。     

RANTES衍生物与HIV-1进入抑制剂

HIV-1-1进入抑制剂的研究是近年来艾滋病药物研发领域的新热点,其中最受关注的是以CCR5为靶点的新药研发。CCR5是病毒进入细胞的主要辅助受体,在HIV-1进入宿主细胞过程中起着非常重要的作用。作为CCR5的天然配体,CC类的趋化因子RANTES、MIP-1α和MIP-1β都是极具潜力的HIV-1抑制剂,特别是有关对RANTES的定向设计的研究尤为引人关注,其目的是设计出一种既有很强的抗病毒能力而又不引发炎症反应的HIV-1拮抗剂。就RANTES衍生物应用于抑制HIV进入细胞方面的研究进行了综述。     

HIV-1 Tat蛋白神经毒作用的研究进展

HIV-1感染者常并发神经系统损伤,患者主要表现为运动功能障碍,认知和行为受损等。研究表明,HIV-1多种蛋白参与上述病理过程,其中Tat蛋白发挥了极为重要的作用。外周Tat蛋白可透过血脑屏障进入脑组织,与脑内感染细胞分泌或释放至胞外的Tat共同作用于NMDAR、mGluR1、多巴胺转运蛋白等,损伤神经系统。该文主要对Tat蛋白的神经毒作用作一综述。     

HIV-1Tat蛋白抑制DNA修复和增强细胞辐射敏感性

近年来临床研究发现,艾滋病合并肿瘤患者放疗后产生的正常组织和皮肤毒性反应明显高于普通肿瘤患者.本研究将探讨HIV-1Tat蛋白是否影响细胞对电离辐射敏感性及机理. 两个表达Tat蛋白的细胞系TT2和TE671-Tat均来源于人的横纹肌肉瘤细胞（TE671）并已转染了不同来源的tat基因.使用细胞辐射后克隆形成率检测辐射敏感性,RT-PCR和Western 印迹检测基因表达,彗星电泳和γ-H2AX位点检测DNA双链断裂和修复. TT2和TE671-Tat细胞的辐射敏感性与转染空载体及对照细胞相比明显增加.彗星电泳和γ-H2AX位点检测表明,在表达Tat蛋白的细胞中,辐射诱导DNA双链断裂的修复水平明显降低.通过RT-PCR和Western 印迹检测进一步证实,表达Tat蛋白的细胞中DNA修复蛋白DNA-PKcs的表达被抑制. HIV-1Tat蛋白抑制DNA-PKcs的表达,降低DNA双链断裂的修复,使细胞的电离辐射敏感性增高.本研究为了解AIDS合并肿瘤患者对放射治疗敏感性变化提供了重要信息.     

结合分子相似性、药效团和分子对接筛选新的HIV-1蛋白酶抑制剂

结合分子相似性、药效团和分子对接建立兼顾计算效率和预测准确度的HIV-1蛋白酶抑制剂筛选方法。首先通过对现有HIV-1蛋白酶抑制剂分子进行相似性分析,选取代表性的HIV-1蛋白酶抑制剂作为模板分子,构建和优化药效团模型,并从1万个化合物中优先筛选出500个化合物。而后采用分子对接方法进一步考察化合物与HIV-1蛋白酶结合情况,得到4个新的活性候选化合物,并进行其结合自由能计算和抗突变性分析。结果表明新候选化合物ST025723和HIV-1蛋白酶表现出较好的相互作用和抗突变性,具有深入研究的价值,同时也证明分子相似性、药效团和分子对接相结合能够快速有效地发现新颖活性候选化合物。     

Role of Glycosphingolipids in HIV-1 Entry: Requirement of Globotriosylceramide (Gb3) in CD4/CXCR4-dependent Fusion

We have recently shown that addition of human erythrocyte glycosphingolipids (GSL) to non-human CD4⁺ or GSL-depleted human CD4⁺ cells rendered those cells susceptible to gp120-gp41-mediated cell fusion (Puri et al., BBRC, 1998). One GSL fraction (Fraction 3) isolated from human erythrocyte GSL mixture exhibited the highest recovery of fusion following incorporation into CD4⁺ non-human and GSL-depleted HeLa-CD4 cells (HeLa-CD4/GSL^–). Structural analysis of Fraction 3 showed that this GSL had identical head group as the known GSL, Gal(14)Gal(1 4)Glc-Ceramide (Gb3) (Puri et al., PNAS, 1998). Here we report that presence of Gb3 in CD4⁺/CXCR4⁺ cells but not CD4⁺/CXCR4^– cells allows fusion with HIV-1_Lai-envelope glycoprotein expressing cells (TF228). Therefore, Gb3 functions in conjunction with HIV-1 co-receptor, CXCR4 to promote fusion. We propose that Gb3 functions by recruiting CD4 and/or CXCR4 at the fusion site through structurally specific interactions.     

Identification of membrane-active regions of the HIV-1 envelope glycoprotein gp41 using a 15-mer gp41-peptide scan

The identification of membrane-active regions of the ectodomain of the HIV-1 envelope glycoprotein gp41 has been made by determining the effect on membrane integrity of a 15-mer gp41-derived peptide library. By monitoring the effect of this peptide library on membrane leakage, we have identified three regions on the gp41 ectodomain with membrane-interacting capabilities: Region 1, which would roughly correspond to the polar sequence which follows the fusion domain and extends to the N-terminal heptad repeat region; Region 2, which would correspond to the immunodominant loop; and Region 3, which would correspond to the pre-transmembrane region of gp41. The identification of these three regions supports their direct role in membrane fusion as well as facilitating the future development of HIV-1 entry inhibitors.     

Sphingolipids, cholesterol, and HIV-1: A paradigm in viral fusion

Our previous studies show that the depletion of cholesterol or sphingolipids (raft-associated lipids) from receptor-bearing adherent cell lines blocks HIV-1 entry and HIV-1 Env-mediated membrane fusion. Here we have evaluated the mechanism(s) by which these lipids contribute to the HIV-1 Env-mediated membrane fusion. We report the following: (1) GSL depletion from a suspension T lymphocyte cell line (Sup-T1) reduced subsequent fusion with HIV-1IIIB-expressing cells by 70%. (2) Cholesterol depletion from NIH3T3 cells bearing HIV-1 receptors (NIH3T3CD4R5/NIH3T3CD4X4) did not impair subsequent fusion with HeLa cells expressing the corresponding HIV-1 Envs. In contrast GSL depletion from these targets reduced fusion by 50% suggesting that GSL facilitate fusion in different ways. (3) GSL-deficient GM95 cells bearing high receptors fused with HIV-1 Env-expressing cells at 37°C with kinetics similar to that of GSL + NIH3T3 targets. Based on these observations, we propose that the plasma membrane cholesterol is required to maintain the integrity of receptor pools whereas GSLs are involved in stabilizing the coupling of inter-receptor pools.     

The dominant-negative action of a fusion protein containing the cytoplasmic domain of human immunodeficiency virus type 1 transmembrane protein gp41 in virus replication

We previously described a novel mode of downregulation of human immunodeficiency virus type 1 (HIV-1) Gag expression by a cytoplasmic domain fusion protein of the envelope (Env) transmembrane protein, β-galactosidase (β-gal)/706–856, which contains the cytoplasmic tail of gp41 fused at the C terminus of Escherichia coli β-gal. In the present study, we showed that this mediator conferred a dose-dependent dominant interference with virus infectivity. In the context of an HIV-1 provirus, this inhibitor downregulated steady-state Env expression. Paradoxically, Env overexpression suppressed β-gal/706–856-mediatd Gag downregulation. Sucrose gradient ultracentrifugation and confocal microscopy revealed that Gag, Env, and β-gal/706–856 had stable interactions and formed aggregated complexes in perinuclear regions. Moreover, Env overexpression hindered colocalization of Gag with β-gal/706–856 in the perinuclear region. Further cytoplasmic domain mapping analyses showed a correlation between the ability of cytoplasmic subdomains to downregulate Gag expression and the ability of these subdomains to stably interact with Gag. These studies show that redirection of Gag from its cytoplasmic synthesis site to a perinuclear compartment is a prerequisite for β-gal/706–856-mediated Gag downregulation. The results also illustrate that the dynamic interplay among Gag, Env, and β-gal/706–856 can modulate Gag and Env expression, thus controlling HIV-1 infection.     

Molecular dynamics simulations and conductance studies of the interaction of VP1 N-terminus from Polio virus and gp41 fusion peptide from HIV-1 with lipid membranes

Abstract

The icosahedral Polio virus capsid consists of 60 copies of each of the coat proteins VP1, VP2, VP3 and myristolyated VP4 (myrVP4). Catalyzed by the host cell receptor the Polio virus enters the host cell via externalization of myrVP4 and the N terminal part of VP1. There are several assumptions about the individual role of both of the proteins in the mechanism of membrane attachment and genome injection. We use the first 32 N terminal amino acids of VP1 and applied molecular dynamics simulations to assess its mechanism of function when attached and inserted into hydrated lipid membranes (POPC). Helical models are placed in various positions in regard to the lipid membrane to start with. As a comparison, the first 33 amino acids of the fusion peptide of gp41 of HIV-1 are simulated under identical conditions. Computational data support the idea that VP1 is not penetrating into the membrane to form a pore; it rather lays on the membrane surface and only perturbs the membrane. Furthermore, this idea is strengthened by channel recordings of both peptides showing irregular openings.     

The strand transfer oligonucleotide inhibitors of HIV-integrase

Retroviral integrase participates in two catalytic reactions, which require interactions with the two ends of the viral DNA in the 3′processing reaction, and with a targeted host DNA in the strand transfer reaction. The 3′-hydroxyl group of 2′-deoxyadenosine resulting from the specific removing of GT dinucleotide from the viral DNA in the processing reaction provides the attachment site for the host DNA in a transesterification reaction. We synthesized oligonucleotides (ONs) of various lengths that mimic the processed HIV-1 U5 terminus of the proviral long terminal repeat (LTR) and are ended by 2′-deoxyadenosine containing a 3′-O-phosphonomethyl group. The duplex stability of phosphonomethyl ONs was increased by covalent linkage of the modified strand with its complementary strand by a triethylene glycol loop (TEG). Modified ONs containing up to 10 bases inhibited in vitro the strand transfer reaction catalyzed by HIV-1 integrase at nanomolar concentrations.     

化学趋化因子受体在HIV感染中的作用及其在AIDS治疗中的应用价值

化学趋化因子受体作为协同受体,为人免疫缺陷病毒（ＨＩＶ－１）进入细胞所必需。其中ＣＸＣＲ４被亲Ｔ细胞的病毒株利用,而ＣＣＲ５被亲巨噬细胞的病毒株利用,它们是大多数病毒株利用的协同受体。协同受体和ＣＤ４一起形成复合受体,ｇｐ１２０与之结合后发生构象改变,使ｇｐ４１暴露出来,引起膜的融合。ＨＩＶ协同受体发现为治疗艾滋病开辟了新的途径。利用趋化因子拮抗剂、单克隆抗体和天然配体封闭趋化因子受体可阻止ＨＩＶ