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

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.