RAPID FLUOROMETRIC ASSAY OF HIV-INDUCED CELL FUSION

HIV 诱导的细胞融合的快速荧光测定

• 批准号: 3489609
• 负责人: PAUL J MADDON
• 金额: $ 5万
• 依托单位: PROGENICS PHARMACEUTICALS, INC.
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-04-01 至 1993-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3489609
• 关键词: AIDS; CD4 molecule; CHO cells; HIV envelope protein gp120; HIV envelope protein gp41; HeLa cells; acidity /alkalinity; antireceptor antibody; antiviral antibody; cell fusion; clone cells; fluorescent dye /probe; fluorimetry; gene expression; helper T lymphocyte; human immunodeficiency virus 1; immunologic assay /test; membrane fusion; method development; mixed tissue /cell culture; neoplastic cell culture for noncancer research; pathologic process; rapid diagnosis; receptor expression; temperature; western blottings

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.

表达 HIV-1 包膜糖蛋白 gp120/gp41 的细胞可以融合 与表达CD4（HIV受体）的细胞形成多核巨细胞 细胞或合胞体。 这种细胞间融合似乎是一种机制 病毒传播以及细胞死亡的主要原因，并且可能发挥作用 在艾滋病的发生发展中发挥着重要作用。 目前测量 HIV 诱导的细胞融合的方法基于 感染 HIV 的 CD4+ 细胞或细胞内产生合胞体 使用瞬时表达系统表达 env。 此类测定通常 需要几天的时间，给出不同的结果，并有直观的读数 这既是劳动密集型的又是主观的。 本阶段第一个目标 1 项目是开发一种可重复的、无病毒的细胞融合测定法 稳定表达 HIV-1 gp120/gp41 的细胞之间合胞体的形成 CD4。 第二个目标是证明这些细胞之间的融合可以 使用荧光去猝灭进行快速且特异性的测定 （FDQ）方法。 这种测定对于检测膜具有无价的价值。 实时融合并准确测量融合动力学。 在第二阶段，FDQ融合测定将用于基础研究 HIV 诱导的细胞融合，包括分子的识别， 除了参与膜融合的 CD4 和 gp120/gp41 之外。 经过 使用来自不同菌株的 env 基因建立新的细胞系 HIV-1 的检测将有助于检查融合特性 不同的分离株。 该测定的几个应用是 进行了调查，包括开发新的艾滋病毒临床标志物 疾病，例如抗合胞体抗体的存在和 合胞体诱导分离株的出现。 最后，该测定将用于 作为病毒-细胞融合的模型，允许识别和 新型抗融合化合物和单克隆中和剂的开发 抑制病毒进入的抗体。