RAPID FLUOROMETRIC ASSAY OF HIV-INDUCED CELL FUSION

表达HIV-1包膜糖蛋白gp 120/gp 41的细胞可以融合 与表达HIV受体CD 4的细胞形成多核巨噬细胞， 细胞或合胞体。 这种细胞与细胞的融合似乎是一种机制， 病毒传播以及细胞死亡的主要原因，并可能发挥 在艾滋病的发展中起着重要作用。 目前用于测量HIV诱导的细胞融合的方法是基于 在感染HIV的CD 4+细胞或细胞中产生合胞体 使用瞬时表达系统表达env。 这样的测定通常 需要几天，给出可变的结果，并有一个视觉读数 这是劳动密集型和主观的。 本阶段的第一个目标 1个项目是开发一种可重复的，无病毒的细胞融合试验， 对稳定表达HIV-1 gp 120/gp 41的细胞之间合胞体形成的影响， CD4。 第二个目标是证明这些细胞之间的融合可以 使用荧光去猝灭快速且具有特异性地测定 (FDQ)法 这样的测定对于检测膜 用于真实的实时聚变和精确测量聚变动力学。 在第2阶段，FDQ融合试验将用于以下基础研究： HIV诱导的细胞融合，包括分子的鉴定， 除了CD 4和gp 120/gp 41，它们参与膜融合。 通过 用来自不同菌株的env基因建立新的细胞系 HIV-1的融合特性，该检测将是有用的， 不同的隔离物。 该测定的几种应用将是 研究，包括开发新的艾滋病毒临床标志物 疾病，如抗合胞抗体的存在和 合胞体诱导分离株的出现。 最后，分析将服务于 作为病毒-细胞融合的模型，允许鉴定和 新型抗融合化合物和单克隆中和的开发 抑制病毒进入的抗体。

Cells which express gp120/gp41, the HIV-1 envelope glycoprotein, can fuse with cells expressing CD4, the HIV receptor, to form multi-nucleated giant cells or syncytia. Such cell-to-cell fusion appears to be a mechanism for virus transmission as well as a primary cause of cell death, and may play an important role in the development of AIDS. Current methods for measuring HIV-induced cell fusion are based on the production of syncytia in CD4+ cells infected with HIV or in cells expressing env using a transient expression system. Such assays typically require several days, give variable results, and have a visual readout which is both labor intensive and subjective. The first goal of this Phase 1 project is to develop a reproducible, virus-free cell fusion assay based on syncytium formation between cells stably expressing HIV-1 gp120/gp41 and CD4. The second goal is to demonstrate that fusion between these cells can be assayed rapidly and with specificity using the fluorescence dequenching (FDQ) method. Such an assay would be invaluable for detecting membrane fusion in real time and for accurately measuring fusion kinetics. In Phase 2, the FDQ fusion assay would be used in basic research studies of HIV-induced cell fusion, including the identification of molecules, in addition to CD4 and gp120/gp41, which are involved in membrane fusion. By establishing new cell lines with env genes derived from different strains of HIV-1, the assay would be useful in examining the fusogenic properties of different isolates. Several applications of this assay would be investigated, including the development of new clinical markers for HIV disease, such as the presence of anti-syncytial antibodies and the appearance of syncytium-inducing isolates. Finally, the assay would serve as a model of virus-cell fusion, permitting the identification and development of novel anti-fusion compounds and monoclonal neutralizing antibodies which inhibit viral entry.