INTERACTIONS OF HIV-1GP120 WITH BRAIN CELLS--ROLE OF PAG

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Description (taken from application): Patients with HIV infection develop a dementing illness, the pathogenesis of which is only partially understood. The virus infects microglia and astrocytes and is accompanied by neuronal cell loss, although the neurons themselves are only rarely infected. Both glial cell types may potentially act as a reservoir for the virus and several investigators have raised concern that even if we were to successfully eradicate the virus from the periphery, virus sequestered within these glial cells could potentially re-seed the lymphoid tissue. Although significant progress has been made in our understanding of the mechanism of viral entry in microglial cells, the mechanism of viral entry into astrocytes remains undetermined. An important step to further our understanding of HIV-astrocyte interactions would be to characterize the cell surface molecules on the astrocyte cell surface that interact with the HIV envelope. The interactions of gp120 with astrocytes may be important not only for viral entry, but also for causing functional changes in astrocytes and thus indirectly in neurons. It has been shown that viral proteins, such as gp120, which are released by HIV-infected cells can act on astrocytes to produce large increases in intracellular calcium and increase extracellular levels of glutamate-like substances which can cause neurotoxicity. We present in this proposal, preliminary data showing that we have identified a unique receptor (Pag) on the surface of astrocytes that interacts with gp120 to cause changes in intracellular calcium and neuronal cell death. We have also developed a panel of monoclonal antibodies to this protein. We propose to develop a cDNA library from human fetal astrocytes using a mammalian expression vector and use the antisera to screen the library. Following several rounds of immunoselection, the isolated clones will be sequenced and transfected in Pag negative cell lines. The transfected cells will be screened for gp120 binding properties and for gp120-induced changes in intracellular calcium. The role of Pag in viral entry by itself or in conjunction with other HIV-coreceptors will also be determined.