HIV-1 MEDIATED MEMBRANE FUSION AS TARGET OF ANTI-VIRAL THERAPY

(1)项目目标：开发用于监测HIV-1的敏感检测方法 酵母介导的细胞融合 为了识别表面受体（在 除CD 4受体外，其他受体参与HIV-1融合和细胞进入。 将获得的知识应用于开发能够 阻断无细胞或细胞相关的HIV-1感染。 （二） 实验方法：开发了生物和生化测定法 为了测量人细胞上的CD 4/gp 120复合物与 T细胞系嗜性共受体。 其中包括下调 用可溶性gp 120处理细胞后的无尾CD 4分子， PMA，和含有gp 120的三分子复合物的共沉淀， 在37 ℃下用sgp 120处理的人CD 4 +-细胞的CD 4和CXCr-4（融合蛋白） C. 我们还用Western blotting与抗融合素兔血清产生 在实验室里。 我们测试了一系列抗CD 4-gp 120的mAb，这些mAb在 Jonny Gershoni的实验室。 (3)主要发现：我们 鉴定了HIV-1融合所需的第二种膜成分 并且可以被PKC激活剂佛波酯（PMA）下调。 它 还发现gp 120与CD 4的相互作用诱导其缔合， 与第二组分形成三分子复合物（gp 120-X-CD 4） 其可以稳定融合过程，并聚集在网格蛋白包被的 坑 生物化学分析表明，在gp 120处理后， 表面生物素化的人CD 4+细胞（但不是非人细胞）， 可以使CD 4/gp 120与45 kDa蛋白质共免疫沉淀 显示为HIV-1辅助受体融合蛋白（CXCR-4）。 我们已经确定 稳定gp 120/CD 4受体/附件的单克隆抗体 分子复合物，并增加三分子复合物的数量 沉淀。 类似的生化分析正在进行中， 嗜巨噬细胞的辅助成分。

(1) Goals of project: To develop sensitive assays for monitoring HIV-1 envelope-mediated cell fusion. To identify surface receptors (in addition to the CD4 receptors) involved in HIV-1 fusion and cell entry. To apply the knowledge gained towards development of agents capable of blocking infection by cell-free or cell-associated HIV-1. (2) Experimental approach: Biological and biochemical assays were developed to measure the association of the CD4/gp120 complex on human cells with the T-cell-line tropic co-receptor. They included downmodulation of tailless CD4 molecules after treatment of cells with soluble gp120 and PMA, and co-precipitation of a tri-molecular complex containing gp120, CD4, and CXCr-4 (Fusin) from human CD4+- cells treated with sgp120 at 37 C. We also used Western blotting with anti-fusin rabbit sera generated in the lab. We tested a series of mAbs against CD4-gp120 generated in the laboratory of Jonny Gershoni. (3) Major Findings: We have identified a second membrane compenent which is required for HIV-1 fusion and can be down-modulated by the PKC activator, phorbol ester (PMA). It was also found that gp120 interaction with CD4 induces its association with the second components forming a tri-molecular complex (gp120-X-CD4) which may stabilize the fusion process, and aggregates in clathrin coated pits. Biochemical assays have shown that after gp120 treatment of surface biotinylated human CD4+ cells (but not of non-human cells), it is possible to co-immunoprecipitate CD4 /gp120 with a 45 kDa protein shown to be the HIV-1 coreceptor fusin (CXCR-4). We have identified monoclonal anibodies which stabilize the gp120/CD4 receptor/accessory molecule complex., and increase the amount of tri-molecuular comples precipitated. Similar biochemical assays are under way with the macrophage-tropic accessory component.