HIV-1 mediated membrane fusion as target for anti-viral therapy

(1)项目目标：-建立监测HIV-1包膜介导的细胞融合和gp120/CD4/共受体三分子复合物形成的生物学和生化检测方法。-研究HIV-1共受体在已知HIV-1感染靶点的人原代细胞上的表达和功能，研究促炎因子对HIV-1共受体功能和人原代细胞感染性的影响。-开发能够阻断无细胞或细胞相关HIV-1感染的药物。(2)实验方法：采用生物（Ca++通量、趋化性、hiv融合）和生化方法检测人细胞上CD4/gp120复合物与HIV-1共受体（促t型和促m型包膜）（CXCR4和CCR5）的关联。它们包括来自细胞系和原代人细胞（即单核细胞和巨噬细胞）的CD4/共受体的共免疫沉淀，以及使用我们的兔抗CXCR4和抗ccr5试剂的全细胞和膜提取物的Western印迹。-对多种细胞类型进行了研究：朗格汉斯细胞（LC）、树突状细胞（DC）、胸腺细胞亚群、CD34+祖细胞、外周血T细胞、单核细胞（MO）和巨噬细胞（MDM）。-开发用于免疫沉淀和Western blot分析关键HIV-1共受体（CXCR4, CCR5）的新试剂。-应用新的生化分析，包括“蛋白质组学”，以确定HIV-1共受体的翻译后修饰，影响其在原代人细胞中的功能。(3)主要发现：-研究考察了促炎细胞因子对共受体表达和功能的影响：培养LC表面CXCR4的表达可被IL-4和TGFb上调，并被IFN a/b/g抑制，表面表达的变化与clc对X4- HIV毒株感染的易感性相关。这些发现表明，细胞因子失调可能有助于X4-HIV变异的出现和艾滋病的进展。-用IFNg或IL-6处理巨噬细胞导致X4病毒感染增强-在单核细胞和巨噬细胞（MO/MDM）的生化研究中发现，CXCR4的翻译后修饰在MO和MDM中有显著差异。在Mo中，所有的CXCR4分子都以单体形式存在，而在MDM的表面则主要发现了高分子量的CXCR4分子。从低到高CXCR4 MW物种的转变与t -型（X4-）包膜融合的丧失有关。-与CD4相关的共受体竞争可能改变人类细胞对嗜t型和嗜m型分离株感染的敏感性。

(1) Goals of project: - To develop biological and biochemical assays for monitoring HIV-1 envelope-mediated cell fusion and formation of the tri- molecular complex between gp120/CD4/co-receptor. - To study the expression and function of HIV-1 co-receptors on primary human cells known to be targets for HIV-1 infection, and to study the effects of pro-inflammatory cytokines on the function of the HIV-1 co- receptors and infectivity of primary human cells. - Development of agents capable of blocking infection by cell-free or cell-associated HIV-1. (2) Experimental approach: - Biological (Ca++ flux, chemotaxis, HIV-fusion) and biochemical assays were developed to measure the association of the CD4/gp120 complex on human cells with the HIV-1 co-receptors for T-tropic and M-tropic envelopes (CXCR4 and CCR5). They included co-immunoprecipitations of CD4/co-receptors from cell lines and from primary human cells (i.e., monocytes and macrophages), and Western blots of whole cell and membrane extracts using our rabbit anti- CXCR4 and anti-CCR5 reagents. - Studies were conducted on multiple cell types: Langerhans cells (LC), dendritic cells (DC), Thymocyte subsets, CD34+ progenitors, peripheral blood T cells, monocytes (MO) and macrophages (MDM). - Develop new reagents for immunoprecipitation and Western blot analyses of the key HIV-1 co-receptors (CXCR4, CCR5). - Apply new biochemical assays including "Proteomics" to identifying the post-translational modifications of HIV-1 co-receptors that affect their function in primary human cells. (3) Major Findings: - Studies examine the effects of proinflammatory cytokines on co-receptor expression and function: Expression of surface CXCR4 on cultured LC was upregulated by IL-4 and TGFb and inhibited by IFN a/b/g The changes in surface expression correlated with susceptibility of cLCs to infection with X4- HIV strains. These findings suggest that cytokine dysregulation may contribute to the emergence of X4-HIV variants and progression to AIDS. -Treatment of macrophages with IFNg or IL-6 resulted in enhanced infection with X4 viruses - In biochemical studies on monocyts and macrophages (MO/MDM) it was found that the post translational modification of CXCR4 is significantly different in MO vs. MDM. In Mo all the CXCR4 molecules appeared monomeric, while predominantly high MW species of CXCR4 were found on the surface of MDM. The transition from low to high CXCR4 MW species correlated with loss of fusion with T-tropic (X4-) envelopes. -Coreceptor competition for association with CD4 may change the susceptibility of human cells to infection with T-tropic and M-tropic isolates.