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

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(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.