The human immunodeficiency virus type 2 (HIV-2) is found in West Africa and, increasingly, in other parts of the world. Following an asymptomatic period that is often longer than that following HIV-1 infection, HIV-2 can cause AIDS. Genetically, HIV-2 is closer to certain strains of simian immunodeficiency virus (SIV) than it is to HIV-1, and work from our laboratory has demonstrated differential regulation of the transcriptional enhancers of HIV-2 and HIV-1 by cellular factors. This may have considerable effect on pathogenicity, as the interaction between cellular proteins and the enhancer modulates transcription, and hence replication, of the virus in individual cells. Unlike HIV-1, in which the two kappaB sites play the dominant role in regulating inducible enhancer function in activated T cells, enhancer activation is regulated by at least four distinct cis-acting elements in the HIV-2 enhancer: two purine-rich sites (PuB1 and PuB2), which bind the ets proto-oncogene family member Elf-1; the peri-ets, or pets, site, which binds an as yet unidentified protein; and a single kappaB site, which binds the well described components of NF-kappaB. As the pets factor plays a significant role in regulating the HIV-2 enhancer in both T cells and monocytes, and as s similar or identical factor appears to be important in the regulation of the human T cell leukemia virus type I (HTLV-1), we have begun to characterize the pets factor biochemically. We here propose to definitively identify and clone the pets factor, examine its role in mediating transcription through the HIV-2 enhancer and in replication of the virus, and to define how this factor interacts with other proteins which bind upstream regulatory elements of HIV-2. Specifically, we propose to complete the biochemical purification of the pets binding factor and then clone the factor from a cDNA library. As evidence indicates that the pets factor may interact with Elf-1, we will then use the cloned and.or purified pets factor to investigate its binding interaction with Elf-1 and the ability of these two upstream activators to interact with the basal transcription machinery. The cloned pets factor will be used to test its ability to activate HIV-2 transcription in co-transfection assays, alone or in combination with Elf-1 or other factors. In vitro transcription assays will be performed to define more exactly the role of pets and other activating factors in stimulating HIV-2 transcription and to test possible post-translational modifications which may affect the functional activity of pets. As previous studies concerning interactions of cellular proteins with the HIV-2 enhancer have been done in vitro, we propose in vivo footprinting studies to compare protein binding to the enhancer in resting and stimulated T cells. We will also examine the role of the pets element in the replication of HIV-2 in tissue culture and begin to examine whether the pets site might influence species-specific viral pathogenesis. Elucidation of the nature of the pets factor and its interaction with Elf-1 and other cellular factors will lead to a better understanding of HIV-2 transcription and replication and should suggest novel therapeutic approaches.
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