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

(1)项目目标：- 开发生物学和生物化学检测方法，用于监测HIV-1病毒介导的细胞融合和gp 120/CD 4/辅助受体之间三分子复合物的形成。- 研究HIV-1辅助受体在已知为HIV-1感染靶点的原代人细胞上的表达和功能，并研究促炎细胞因子对HIV-1辅助受体功能和原代人细胞感染性的影响。- 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：- 开发了生物学（Ca++通量、趋化性、HIV融合）和生物化学测定，以测量人细胞上的CD 4/gp 120复合物与T-嗜性和M-嗜性包膜的HIV-1共受体（CXCR 4和CCR 5）的关联。它们包括来自细胞系和来自原代人细胞的CD 4/共受体的免疫共沉淀（即，单核细胞和巨噬细胞），以及使用我们的兔抗CXCR 4和抗CCR 5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、CD 34+祖细胞、外周血T细胞、单核细胞（MO）和巨噬细胞（MDM）。- 开发新试剂用于HIV-1关键共受体（CXCR 4、CCR 5）的免疫沉淀和Western印迹分析。- 应用新的生物化学检测方法，包括“蛋白质组学”，以确定HIV-1辅助受体的翻译后修饰，影响其在原代人类细胞中的功能。 (3)主要发现：- 研究检查了促炎细胞因子对辅助受体表达和功能的影响：培养的LC上表面CXCR 4的表达被IL-4和TGF B上调，并被IFN a/B/g抑制。表面表达的变化与cLC对X4- HIV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变异体的出现和进展为AIDS。- 用IFNg或IL-6处理巨噬细胞导致X4病毒感染增强。-在对单核细胞和巨噬细胞（MO/MDM）的生物化学研究中，发现CXCR 4的翻译后修饰在MO与MDM中显著不同。在Mo中，所有的CXCR 4分子都是单体的，而MDM表面上发现了主要的高MW CXCR 4物质。CXCR 4分子量从低到高的转变与与T-嗜性（X4-）包膜融合的丧失相关。 - 辅助受体与CD 4的竞争可能改变人类细胞对T嗜性和M嗜性分离株感染的易感性。

(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.