RAPID FLUOROMETRIC ASSAY OF HIV-INDUCED CELL FUSION

表达HIV-1包膜糖蛋白gp120/gp41的细胞可以融合 与表达HIV受体CD4的细胞形成多核巨细胞 细胞或合胞体。这种细胞间的融合似乎是一种 病毒传播以及细胞死亡的主要原因，并可能发挥作用 在艾滋病的发展中起着重要作用。 目前测量HIV诱导的细胞融合的方法是基于 HIV感染的CD4细胞或细胞内合胞体的产生 使用瞬时表达系统表达env。这类检测通常 需要几天时间，给出不同的结果，并有一个直观的读数 这既是劳动密集型的，也是主观的。这一阶段的第一个目标 一个项目是开发一种可重复性的、无病毒的细胞融合试验 稳定表达HIV-1gp120/gp41的细胞与细胞间合胞体的形成 CD_4。第二个目标是证明这些细胞之间的融合可以 利用荧光猝灭技术快速、特异地进行检测 (FDQ)方法。这种化验方法对于膜的检测将是无价的。 实时聚变和精确测量聚变动力学。 在第二阶段，FDQ融合分析将用于基础研究 艾滋病毒诱导的细胞融合，包括分子鉴定，在 此外还有参与膜融合的CD4和gp120/gp41。通过 利用不同毒株的env基因建立新的细胞系 对于HIV-1，该检测将有助于检测融合特性 不同的菌株。这种分析的几个应用将是 调查，包括开发新的艾滋病毒临床标记物 疾病，如抗合胞抗体的存在和 合胞体诱导菌株的出现。最后，化验结果将有助于 作为病毒-细胞融合的模型，允许识别和 新型抗融合化合物的开发及单抗中和 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.