NF-KB/REL AS AN ACTIVATOR OR INHIBITOR OF HIV-1 GROWTH

点击翻译按钮获取中文摘要

Primary infection with the type 1 human immunodeficiency virus (HIV-1), the etiologic agent of the Acquired Immune Deficiency Syndrome (AIDS), is typically followed by an extended asymptomatic period before the emergence of overt disease. the replication dynamics of HIV-1 throughout this clinical continuum appear critically dependent on the action of specific host trans-regulatory factors that are normally induced in response to immunologic stimuli. In addition to its high affinity for functional enhancer sequences present in the retroviral 5' long terminal repeat (LTR), the NF-kappaB transcription factor is rapidly induced during T cell activation and likely represents a major host regulatory pathway governing the onset of productive HIV-1 expression in persistently infected CD4+ T lymphocytes. Molecular cloning studies have revealed that the 50 kD (p50) and 65 kD (p65) subunits of NF-kappaB both share striking N-terminal sequence homology with the v-Rel oncoprotein, its normal cellular counterpart (c-Rel), and dorsal, a ventral morphogen in Drosophila. In particular, p65 is a potent transcriptional activator of the HIV-1 LTR and also serves as a "receptor" for IkappaB, a cytoplasmic inhibitor of NF-kappaB function. In sharp contrast, c-Rel acts as a repressor of HIV-1 LTR-directed transcription and may thus correspond to a counter-regulatory host cell factor that promotes viral attenuation. the principal goal of these proposed studies is to explore the physiological regulation, mechanism of action, and biological role of these various Rel polypeptides in the modulation of HIV-1 gene expression. To approach this overall objective, a comprehensive mutational analysis will be undertaken to define and completely dissect the functional domains present in p65 that are required for transactivation, HIV-1 enhancer binding, IkappaB-mediated inhibition, and multimerization. In turn, selected phenotypic variants will be used in an "interaction cloning" strategy to identify novel NF-kappaB signaling components involved in mediating HIV-1 transcriptional activation or repression via protein/protein interactions. to explore the role of NF- kappaB in transcriptional regulatory mechanisms controlling HIV-1 expression in vivo, monospecific anti-peptide antibodies will be employed to biochemically define the spectrum of Rel polypeptides present in primary CD4+ expressing cells that comprise the principal targets for HIV-1 infection. In conjunction with in vitro transcription and in vivo footprinting analyses, these antibodies will also be used to define the biochemical basis for attenuated HIV-1 expression in established cell culture models of viral latency. Finally, stably transfected human T cell lines will be generated that conditionally express either p65 or c- Rel by fusion to the ligand binding domains of a variety of steroid hormone receptors in order to directly determine their relative transcriptional effects following HIV-1 infection. Together, these proposed experimental approaches should yield fundamental mechanistic insights into two integral components of the NF-kappaB/Rel transcription factor family an their potentially divergent roles in the regulation of HIV-1 gene expression. As such, this information may be key to understanding the precise involvement of NF-kappaB in the transcriptional programs controlling HIV-1 replication in persistently and productively infected cells.