The proposed study is intended to localize the Nef gene product of HIV- 1 during the process of virion-morphologenesis in infected cells. This gene product appears to modify the virus during its assembly in producer cells such that it is mor infectious to recipient cells. nef is known to be a myristolated, membrane-associated protein but its distribution within cells is not known at the ultrastructural level. We intend to determine by immunoelectron microscopy whether Nef is found in close prosimity to assembling virions or if it is actually incorporated into virions. First, primary antibodies will be screened by immunofluorescence for their ability to detect Nef. Three cell types will be tested: uninfected cells, cells acutely infected with HIV-1 and cells constituitively expressing Nef. After identification of conditions that allow detection of Nef at the level of confocal fluorescence microscopy, these studies will be extended to the electron microscopic level, using the facilities at the NCMIR.
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