HIV-1整合酶酶联免疫吸附试验及其抑制剂的研究

目的　建立一种检测人类免疫缺陷病毒 1型 (HIV - 1 )整合酶的酶联免疫吸附试验(ELISA)方法 ,用于筛选和研究HIV - 1整合酶抑制剂。方法　将质粒F1 85K C2 80SIN1 2 88转化到大肠埃希菌中 ,经IPTG诱导表达 ,柱亲和层析纯化 ,获得HIV 1整合酶融合蛋白。建立酶联免疫吸附试验方法测定其生物学活性 ,与3 2 P同位素标记方法比较 ,并用ELISA方法筛选HIV 1整合酶的抑制剂。结果　SDS PAGE电泳分析显示 ,相对分子质量 30 0 0 0上方有HIV 1整合酶融合蛋白条带出现。ELISA及3 2 P同位素标记法证实 ,此融合蛋白对于特异底物具有 3′切割和链转移活性。ELISA反应的平均P N值为 2 836± 0 1 61 ,批内及批间变异系数 (CV)分别为 4 63 %和 5 89%。检测到中药丹参提取物CEH等有抑制整合酶的活性 ,CEH的大孔树脂洗脱物CEHL活性提高。结论　ELISA法检测HIV 1整合酶活性技术简单 ,快速 ,重复性好 ,无同位素污染 ,可用于HIV 1整合酶为靶点的抑制剂的筛选及抗 HIV药物作用机理的研究。     

Discovery of HIV-1 integrase inhibitors through a novel combination of ligand and structure-based drug design

Over the past 10 years, classical computer-aided molecular design methods have not been frequently applied for the discovery of novel HIV-1 integrase (IN) inhibitors, due to the intrinsic challenges that this enzyme presents. Therefore, a novel approach that combines the chemical information of known integrase inhibitors with the enzyme's detailed 3D structure in a stepwise fashion is proposed: (I) use of a pharmacophore model (PM), which takes into account in a weighted fashion the chemical features of known ligands, in analogous manner to the to search the Maybridge and the NCI 3D databases; (II) drug-likeness optimization; (III) virtual high-throughput screening of the hits matching the PM query against 1QS4 wild-type IN structure using different Docking/Scoring combinations; (IV) visual inspection and selection of the hits in function of: binding free energies; binding mode type within the active site; retrieval among the best 20% hits in more than 6 Docking/Scoring protocols at the same time. This approach aims at a rational selection of new potential HIV-1 integrase inhibitors.     

Prospects of HIV-1 entry inhibitors as novel therapeutics

A great deal of progress has been made in understanding the mechanism of human immunodeficiency virus entry into target cells. Landmark discoveries such as the identification of viral coreceptors and the structure of a portion of the viral envelope protein (Env) bound to its receptor provided important insight into how Env mediates fusion of the viral and cellular membranes. This knowledge has been successfully applied to the development of inhibitors that target discrete steps of the entry process. Some of these compounds efficiently block HIV-1 replication in vitro and are currently being evaluated in clinical trials. In this review, we will introduce the challenges of antiviral therapy and highlight the need for novel therapeutics, such as entry inhibitors, to complement current antiviral regimens. The mechanism by which Env mediates HIV-1 entry and the therapeutic potential of small molecule inhibitors of this dynamic process will be discussed in detail.     

HIV-1 integration: an interplay between HIV-1 integrase, cellular and viral proteins

To achieve a productive infection, the reverse transcribed cDNA of the human immunodeficiency virus type 1 (HIV-1) has to be inserted in the host cell genome. The main protein required to accomplish this reaction is the virally encoded integrase. In vitro, the recombinant integrase is capable of catalyzing the two subsequent reactions of the integration process, namely the 3' processing followed by the strand transfer, without other viral and/or cellular proteins. However, a number of studies indicate that the in vivo integration process also involves cellular proteins, assisting the virus to integrate in the cellular genome. These cellular proteins can play a role during different steps of the integration process, including nuclear import, integrase catalysis, integration site selection and DNA gap repair. In this review we summarize the candidate cellular proteins involved in the HIV-1 integration process identified so far and discuss their potential roles during HIV-1 replication.     

HIV-1型整合酶蛋白的表达、纯化及复性研究

目的对HIV-1整合酶蛋白进行原核表达、纯化以及复性研究。方法从HIV-1HXB2中PCR扩增出全长的整合酶基因，连入原核表达载体pET-30a中，得到pET-30a整合酶表达质粒。再将质粒转入大肠杆菌BL21中诱导表达，经镍柱纯化后得到整合酶蛋白。纯化后蛋白在FoldIt复性液中进行稀释复性确定最佳复性液，然后蛋白在此条件下复性并用反相柱回收，最后通过对复性蛋白抽干及再溶分析复性蛋白的物理稳定性。结果HIV-1整合酶蛋白主要以包涵体形式表达，表达量占菌体总量的10％。经镍柱纯化后蛋白的总浓度为0．9mg／ml。蛋白的最佳复性液是：Tris—Cl55mrnol／L，NaCl264mmol／L，KCl11mmol／L，Gu—HCl550mmol／L，EDTA1．1mmol／L，以及附加成分中的GSH、GSSG。复性后蛋白抽干后再溶，溶液清亮透明，SDS-PAGE可见有寡聚体状态的蛋白。结论成功构建了pET-30a整合酶表达质粒。纯化后蛋白的浓度较高。确定了最佳的蛋白复性液。并且初步检测了复性蛋白的物理稳定性。这为蛋白体外活性研究和AIDS药物研究打下了基础。     

The role of lysine 186 in HIV-1 integrase multimerization

HIV-1 integrase (IN) catalyzes biochemical reactions required for viral cDNA insertion into host cell chromosomal DNA, an essential step in the HIV-1 replication cycle. In one of these reactions, the two ends of the linear viral cDNA are believed to be simultaneously ligated to chromosomal DNA by a tetrameric form of IN. The structure of the full-length IN tetramer is not known but a model consisting of the N-terminal domain and the catalytic core revealed basic residues 186 to 188 at the interface between the two IN dimers. We found that alteration of these residues, in particular changing IN lysine residue 186 to glutamate (K186Q), impairs IN oligomerization in the yeast two-hybrid system and decreases oligomeric forms of IN within virions. When expressed independently of other viral proteins in human cells, IN-K186Q did not concentrate in the nucleus as did wild-type IN. Co-expression of wild-type IN restored the multimerization defects of IN-K186Q, in both the two-hybrid system and in virions, and also rescued the nuclear targeting defects. Virions bearing IN-K186Q were not infectious in a single cycle of replication but when mixed virions containing two different IN mutants were produced, IN-K186Q was capable of complementing the catalytically inactive mutant IN-D116A. Our biochemical and functional data support the crystallographic model in which IN residue K186 lies at the interface between IN dimers and suggest that tetramerization is important, not only for concerted integration, but also for IN nuclear targeting.     

Assessment of HIV-1 entry inhibitors by MLV/HIV-1 pseudotyped vectors

Background  

Murine leukemia virus (MLV) vector particles can be pseudotyped with a truncated variant of the human immunodeficiency virus type 1 (HIV-1) envelope protein (Env) and selectively target gene transfer to human cells expressing both CD4 and an appropriate co-receptor. Vector transduction mimics the HIV-1 entry process and is therefore a safe tool to study HIV-1 entry.     

A high-throughput assay for HIV-1 integrase 3'-processing activity using time-resolved fluorescence

HIV-1 integrase (HIV-1 IN), a well-validated antiviral drug target, catalyzes multistep reactions to incorporate viral DNA into the genome of the host cell; these include both a 3'-processing (3'P) reaction and a strand transfer reaction. These enzymatic activities can be measured in vitro with short DNA oligonucleotides that mimic a single viral LTR DNA end and purified IN. A highly sensitive and reproducible time-resolved fluorescence (TRF)-based assay for HIV-1 IN 3'P activity is now reported. This assay was optimized with respect to time and concentrations of metal ions, substrate and enzyme. The assay has now been used successfully to measure HIV-1 IN 3'P activity and has been shown to detect the anti-IN activity of several known 3'P inhibition compounds accurately. This assay, which is amenable to high-throughput screening, will be useful for identification of additional HIV-1 IN 3'P inhibitors.     

A new functional role of HIV-1 integrase during uncoating of the viral core

An early and critical event of the human immunodeficiency virus type 1 (HIV-1) life cycle is uncoating of the viral core. Uncoating involves the disassembly of HIV-1 capsid (CA). The underlying mechanisms governing uncoating are poorly defined, and the role of viral and host factors in uncoating is not well understood. Cyclophilin A and TRIM5α are two cellular factors that interact with CA in exerting their effects on HIV-1 replication. Here, we review the current understanding of uncoating and the new functional role of HIV-1 IN during uncoating.