趋化因子受体HIV-1

β－趋化因子受体５（ＣＣＲ５）是近年来新克隆的趋化因子受体超基因家族的一个重要成员之一,由于它的被发现才使人们认识到ＨＩＶ－１ 的感染除了Ｔ 细胞上的ＣＤ４＋ 外还有另一条通过巨噬细胞表面辅助受体ＣＣＲ５ 介导的传播途径。本文就近年来有关趋化因子受体基因家族的组成成员、结构与功能、不同人种的基因型、ＨＩＶ－１ 与细胞表面辅助受体ＣＣＲ５ 的结合,进入机理及阻断ＨＩＶ－１ 感染的可能途径作一详要的综述     

趋化因子RANTES和SDF-1细胞内共表达抑制HIV-1感染的研究

目的　观察HIV 1辅受体CCR5和CXCR4的配体在细胞内共表达抑制HIV 1感染的作用。方法　应用磷酸钙沉淀法共转染HIV 1辅受体及其配体的质粒 ,制成辅受体表型剔除的靶细胞 ,与转染HIV 1膜蛋白质粒的细胞混合 ,观察合胞体形成并记数 ;脂质体介导法将含有报告基因CAT而缺失HIV包膜蛋白的质粒与HIV包膜蛋白质粒共转染 2 93细胞 ,包装成具有一次感染活性的假病毒 ,感染转化pCMV R K S K、pCMV R K、pCMV S K或pCMV的PM 1细胞 ,采用同位素薄层层析分析法检测CAT活性。结果　pCMV R K S K转染可以显著抑制M及T嗜性HIV膜蛋白诱导的合胞体形成 ;CAT检测发现与pCMV转染组相比 ,当两种嗜性重组病毒感染pCMV R K S K转染组PM 1细胞时 ,仅检测到背景水平的CAT活性。结论　HIV 1辅受体CCR5 CXCR4表型剔除可以明显抑制M和T嗜性HIV 1病毒进入靶细胞     

HIV-1病毒感染因子基因克隆、表达、纯化及其抗体制备的研究

目的制备HIV-1病毒感染因子(Vif)及其抗体。方法用PCR技术从HIV-1NL4·3cDNA质粒中扩增病毒感染因子(vif)基因,vif基因全长为579nt(核苷酸),翻译成含192个氨基酸的蛋白质。将测序鉴定过的vif基因克隆到原核表达载体pET-32a上,以包涵体的形式在大肠埃希菌BL21(DE3)中高效表达,Vif蛋白C端融合6×His标签便于纯化及鉴定。应用酶切鉴定、SDS-PAGE及WesternBlot等方法确保基因片段的正确性及表达蛋白的特异性。间接ELISA法测定兔多克隆抗体滴度。结果成功地获得了高纯度的Vif融合蛋白,纯度可达80%以上。用其制备多克隆抗体滴度可达1∶204800。结论获得高纯度的Vif融合蛋白及其高效价的抗体。     

中国HIV-1感染疾病长期不进展者CCR2-64I、SDF1-3’A和CCR5Δ32的基因变异研究

目的 探讨CCR2-641、SDF1-3’A、CCR5Δ32基因变异对中国HIV—1感染疾病长期不进展者（long-term nonprogressors，LTNPs）疾病进程的影响。方法 收集17例中国HIV-1感染LTNPs和39例中国HIV-1感染疾病典型进展者（typical progressors，TPs）的抗凝全血标本，用全自动核酸提取仪提取基因组DNA，采用PCR／RFLP技术对CCR2、SDF1、CCR5基因进行检测分析。结果 LTNP组的CCR2-64I、SDF1-3’A基因突变率分别为50％和62．5％，TP组的CCR2-64I和SDF1-3’A基因突变率分别为23．08％、33．33％，LTNP组CCR2—64I、SDF1-3’A基因突变率明显高于TP组（P〈0．05）；LTNP组有1例CCR5Δ32杂合突变，未发现纯合变异，而TP组未发现CCR5Δ32突变。结论 CCR2-64I、SDF1-3’A和CCR5Δ32基因突变可能是中国HIV-1感染者疾病长期不进展的保护性因素之一。     

中国HIV-1感染疾病长期不进展者CCR2-64I、SDF1-3′A和CCR5Δ32的基因变异研究

目的探讨CCR2-64I、SDF1-3′A、CCR5Δ32基因变异对中国HIV-1感染疾病长期不进展者(long-termnonprogressors,LTNPs)疾病进程的影响。方法收集17例中国HIV-1感染LTNPs和39例中国HIV-1感染疾病典型进展者(typical progressors,TPs)的抗凝全血标本,用全自动核酸提取仪提取基因组DNA,采用PCR/RFLP技术对CCR2、SDF1、CCR5基因进行检测分析。结果LTNP组的CCR2-64I、SDF1-3′A基因突变率分别为50%和62·5%,TP组的CCR2-64I和SDF1-3′A基因突变率分别为23·08%、33·33%,LTNP组CCR2-64I、SDF1-3′A基因突变率明显高于TP组(P<0·05);LTNP组有1例CCR5Δ32杂合突变,未发现纯合变异,而TP组未发现CCR5Δ32突变。结论CCR2-64I、SDF1-3′A和CCR5Δ32基因突变可能是中国HIV-1感染者疾病长期不进展的保护性因素之一。     

中国HIV-1感染疾病长期不进展者CCR2-64I、SDF1-3′A和CCR5△32的基因变异研究

目的探讨CCR2-64I、SDF1-3′A、CCR5△32基因变异对中国HIV-1感染疾病长期不进展者(long-term nonprogressors,LTNPs)疾病进程的影响.方法收集17例中国HIV-1感染LTNPs和39例中国HIV-1感染疾病典型进展者(typical progressors,TPs)的抗凝全血标本,用全自动核酸提取仪提取基因组DNA,采用PCR/RFLP技术对CCR2、SDF1、CCR5基因进行检测分析.结果LTNP组的CCR2-64I、SDF1-3′A基因突变率分别为50%和62.5%,TP组的CCR2-64I和SDF1-3′A基因突变率分别为23.08%、33.33%,LTNP组CCR2-64I、SDF1-3′A基因突变率明显高于TP组(P＜0.05);LTNP组有1例CCR5△32杂合突变,未发现纯合变异,而TP组未发现CCR5△32突变.结论CCR2-64I、SDF1-3′A和CCR5△32基因突变可能是中国HIV-1感染者疾病长期不进展的保护性因素之一.     

趋化因子及其受体的研究新进展

近年来 ,随着生物信息学和基础信息的发展 ,人们发现了许多新的趋化细胞因子及其受体 ,特别是趋化因子受体被发现充当HIV感染的协同受体后 ,趋化因子领域已经引起了人们的广泛关注。本文探讨了趋化因子及其受体在抗肿瘤治疗 ,抗HIV感染及抗炎方面的临床应用价值 ,就趋化因子及其受体领域的研究新进展进行综述。     

009 趋化因子及其受体的研究新进展

近年来，随着生物信息学和基础信息的发展，人们发现了许多新的趋化细胞因子及其受体，特别是趋化因子受体被发现充当HIV感染的协同受体后，趋化因子领域已经引起了人们的广泛关注。本文探讨了趋化因子及其受体在抗肿瘤治疗，抗HIV感染及抗炎方面的临床应用价值，就趋化因子及其受体领域的研究新进展进行综述。     

HIV-1辅助受体的多态性及其研究进展

HIV-1进入人体细胞不但需要CD4受体，还需要一些趋化因子受体作为辅助受体。本文就辅助受体的基因多态性，基因多态性对HIV感染和AIDS病程进展的影响和作用原理，不同的辅助受体和配体基因多态性的相互关系，以及辅助受体在艾滋病的预防和药物开发中的应用前景等相关研究进展做一综述。     

基线极高HIV-1 RNA水平对初治HIV-1感染者外周血total HIV-1 DNA的影响

目的:观察基线HIV-1 RNA > 50万copies/mL的HIV-1感染者,在高效抗反转录病毒治疗（highly active antiretroviral therapy,HAART）前后外周血totalHIV-1 DNA的变化。方法:从国家十二五科技重大专项课题中选取基线HIV-1 RNA > 50万copi...     

Phenotytic Knockout of HIV-1 Chemokine Coreceptor CXCR4 and CCR5 by Intrakines for Blocking HIV-1 Infection

Acquired immunodeficiency syndrome (AIDS) and humanimmunodeficiency virus (HIV) infection continues to bemajor global health concerns. Although highly active an-tiretroviral therapy (HAART) has led to profound and pro-longed reductions in circulating viru…     

Molecular determinants of HIV-1 subtype C coreceptor transition from R5 to R5X4

The molecular mechanism(s) underlying transition from CCR5 to CXCR4 usage of subtype C viruses remain largely unknown. We previously identified a subtype C HIV-1 infected child whose virus demonstrated CXCR4 usage along with CCR5 upon longitudinal follow-up. Here we delineated the molecular determinants of Env involved in expanded coreceptor usage. Residue changes in three positions of Env V3 domain are critical for the dual tropic phenotype. These include: substitution of arginine at position 11, MG or LG insertion between positions 13 and 14, and substitution of threonine at the position immediately downstream of the GPGQ crown. Introducing these mutations into V3 region of a heterologous R5 virus also conferred dual tropism. Molecular modeling of V3 revealed a possible structural basis for the dual tropic phenotype. Determining what defines a subtype C X4 virus will lead to a better understanding of subtype C HIV-1 pathogenesis, and will provide important information relevant to anti-retroviral therapy.     

Comparison of in vivo and in vitro evolution of CCR5 to CXCR4 coreceptor use of primary human immunodeficiency virus type 1 variants

During the course of at least 50% of HIV-1 subtype B infections, CCR5-using (R5) viruses evolve towards a CXCR4-using phenotype. To gain insight in the transition from CCR5 to CXCR4 coreceptor use, we investigated whether acquisition of CXCR4 use in vitro of R5 viruses from four patients resembled this process in vivo. R5 variants from only one patient acquired CXCR4 use in vitro. These variants had envelopes with higher V3 charge and higher number of potential N-linked glycosylation sites when compared to R5 variants that failed to gain CXCR4 use in vitro. In this patient, acquisition of CXCR4 use in vitro and in vivo was associated with multiple mutational patterns not necessarily involving the V3 region. However, changes at specific V3 positions were prerequisite for persistence of CXCR4-using variants in vivo, suggesting that positive selection targeting the V3 loop is required for emergence of CXCR4-using variants during natural disease course.     

HIV-1协同受体CXCR4、CCR5及相关趋化因子SDF-1在胎盘的表达

目的：研究HIV-1协同受体CXCR4、CCR5及CXCR4的特异性配体SDF-1在人胎盘组织的表达，探索HIV-1子宫内垂直传播的分子机制。方法：半定量RT-PCR检测早、中、晚孕期胎盘及早孕滋养细胞CXCR4、CCR5 mRNA水平；免疫组化和免疫细胞化学检测早孕胎盘及原代培养滋养细胞CXCR4、CCR5蛋白表达；原位杂交及免疫组化分析SDF-1在早孕胎盘的表达；ELISA测定滋养细胞SDF-1的动态分泌水平。结果：各孕期胎盘表达CXCR4及CCR5 mRNA；CXCR4蛋白定位于滋养细胞，而CCR5蛋白定位于绒毛基质中。滋养细胞可转录并翻译SDF-1，且能分泌可溶性SDF-1。结论：滋养细胞同时表达CXCR4及SDF-1，SDF-1可能通过降调CXCR4而拮抗X4-HIV-1感染胎儿细胞；R5-HIV-1或许能通过滋养层裂隙感染CCR5^#基质细胞和/或Hotbauer细胞，从而发生子宫内垂直传播。     

人PBMCs内表达CCR5Delta32蛋白对HIV-1辅受体抑制作用的研究

目的:在人PBMCs内表达CCR5Delta32蛋白,研究其对细胞表面HIV-1辅受体CCR5和CXCR4的抑制作用。方法:构建pLenti-CCR5Delta32慢病毒载体,包装后产生重组慢病毒。将其转染PBMCs,Western blot检测目的蛋白的表达。继续培养靶细胞,FACS分析细胞表面CCR5和CXCR4分子的变化。结果:成功构建了pLenti-CCR5Delta32慢病毒载体,包装后产生重组慢病毒。将其转染PBMCs,Western blot检测到目的蛋白的表达。FACS分析表明,靶细胞内目的蛋白的表达对靶细胞表面辅受体CCR5和CXCR4的产生起抑制作用,抑制率在转染后第6天达到高峰(CCR5的抑制率为51.69%,CXCR4的抑制率为61.05%)。结论:靶细胞内目的蛋白的成功表达及其对靶细胞表面HIV-1辅受体CCR5和CXCR4产生的抑制作用,为后续的AIDS基因治疗研究奠定了基础。     

Constrained use of CCR5 on CD4+ lymphocytes by R5X4 HIV-1: Efficiency of Env-CCR5 interactions and low CCR5 expression determine a range of restricted CCR5-mediated entry

R5X4 HIV-1 has impaired utilization of CCR5 on primary CD4+ lymphocytes but the mechanisms responsible are not well defined. Using a panel of diverse R5X4 Envs we identified a spectrum of CCR5 use on CD4+ lymphocytes. Greater lymphocyte CCR5 use correlated with relative resistance to CCR5 mAbs and small molecule antagonists. Increasing CCR5 expression on lymphocytes increased the proportion of entry mediated by CCR5 for all R5X4 isolates except 89.6. In cell lines with regulated CCR5 expression, strains with greater lymphocyte CCR5 use better exploited limiting levels of CCR5. Introduction of an R306S mutation in the 89.6 V3 domain enhanced its utilization of CCR5 at low levels and switched its preference to CCR5 for lymphocyte entry. Thus, the degree to which R5X4 HIV-1 use primary lymphocyte CCR5 is determined by low CCR5 expression coupled with variations in the efficiency of Env-CCR5 interactions, which is in part governed by V3 sequences.     

Coreceptor usage by HIV-1 and HIV-2 primary isolates: the relevance of CCR8 chemokine receptor as an alternative coreceptor

The human immunodeficiency virus replication cycle begins by sequential interactions between viral envelope glycoproteins with CD4 molecule and a member of the seven-transmembrane, G-protein-coupled, receptors' family (coreceptor).In this report we focused on the contribution of CCR8 as alternative coreceptor for HIV-1 and HIV-2 isolates. We found that this coreceptor was efficiently used not only by HIV-2 but particularly by HIV-1 isolates. We demonstrate that CXCR4 usage, either alone or together with CCR5 and/or CCR8, was more frequently observed in HIV-1 than in HIV-2 isolates. Directly related to this is the finding that the non-usage of CXCR4 is significantly more common in HIV-2 isolates; both features could be associated with the slower disease progression generally observed in HIV-2 infected patients.The ability of some viral isolates to use alternative coreceptors besides CCR5 and CXCR4 could further impact on the efficacy of entry inhibitor therapy and possibly also in HIV pathogenesis.     

Functional and genetic analysis of coreceptor usage by dualtropic HIV-1 subtype C isolates

It is widely documented that a complete switch from the predominant CCR5 (R5) to CXCR4 (X4) phenotype is less common for HIV-1 subtype C (HIV-1C) compared to other major subtypes. We investigated whether dualtropic HIV-1C isolates represented dualtropic, mixed R5 and X4 clones or both. Thirty of 35 functional HIV-1 env clones generated by bulk PCR amplification from peripheral blood mononuclear cells (PBMCs) infected with seven dualtropic HIV-1C isolates utilized CXCR4 exclusively. Five of 35 clones displayed dualtropism. Endpoint dilution of one isolate did not yield a substantial proportion of R5-monotropic env clones. Sequence-based predictive algorithms showed that env sequences from PBMCs, CXCR4 or CCR5-expressing cell lines were indistinguishable and all possessed X4/dualtropic characteristics. We describe HIV-1C CXCR4-tropic env sequence features. Our results suggest a dramatic loss of CCR5 monotropism as dualtropism emerges in HIV-1C which has important implications for the use of coreceptor antagonists in therapeutic strategies for this subtype.     

An aptamer that neutralizes R5 strains of HIV-1 binds to core residues of gp120 in the CCR5 binding site

We have previously isolated nucleic acid ligands (aptamers) that bind the surface envelope glycoprotein, gp120, of HIV-1, and neutralize infection of diverse sub-types of virus. Our earlier studies have identified the overall structure of one of these aptamers, B40, and have indicated that it binds to gp120 in a manner that competes with that of the HIV-1 coreceptor, CCR5, and select “CD4i” antibodies with epitopes overlapping this region. Here, we sought to map the B40 binding site on gp120 more precisely by analysing its interaction with a panel of alanine substitution mutants of gp120. Furthermore, we tested our hypothesis concerning the structure of the 40 nucleotide functional core of the aptamer by the solid-phase synthesis of truncated and chemically modified derivatives. The results confirm our structural predictions and demonstrate that aptamer B40 neutralizes a diverse range of HIV-1 isolates as a result of binding to relatively conserved residues on gp120 at the heart of the CCR5-binding site. These structural insights may provide the basis for the development of potential anti-viral agents with high specificity and robustness.