Transcriptional activation of the human immunodeficiency virus (HIV) is dependent on host cell proteins which activate viral gene expression in latently infected cells. In cells which contain integrated HIV, these initiating steps are mediated by the cis-acting regulatory, or enhancer, region located in the long terminal repeat (LTR) of the retrovirus. We have shown previously that an inducible transcription factor, NF-kappaB, activates the HIV enhancer following T cell activation. Activation of the HIV enhancer, induction of NF-kappaB, and replication of HIV are stimulated by pharmacologic agents, including phorbol myristate acetate (PMA), and cytokines, tumor necrosis factor-alpha cologic agents, include phorbol myristate acetate (PMA),and interleukin-1. In the monocyte lineage, NF-kappaB binding activity is associated with cellular differentiation, which provides one signal to activate HIV expression. Because mutations in the HIV enhancer abrogate NF-kappaB binding and abolish transcriptional activation, inhibition of NF-kappaB function represents one approach to prolong the latent phase of HIV infection. To selectively regulate NF-kappaB and repress HIV expression, it is essential to known which cellular genes are regulated by NF-kappaB and to define the heterogeneity and specificity of kappaB binding proteins. Several potential cellular targets of NF-kappaB have been identified: kappaB-like sequences are associated with several genes, including class I and II major histocompatibility genes, Beta-2 microglobulin, and the interleukin 2 receptor alpha (IL-2Ralpha) chain. Our previous studies have shown that a site related to kappaB is present upstream of the IL- 2Ralpha gene, which may be recognized by a distinct kappaB binding protein. In this proposal, we present preliminary data describing the isolation of a cDNA, and clone encoding this DNA binding protein. We will define this kappaB binding protein and its cDNA, and determine its relation to other kappaB binding proteins. A full length clone will be characterized, its binding activities determined, and its functional role in the activation of HIv and IL-2Ralpha transcription investigated. Induction of this transcription factor by PMA, cytokines and other agents will be examined. This clone will be used to isolate related kappaB binding proteins by low stringency hybridization. Strategies will be developed to differentially regulate the expression of NF-kappaB and the IL-2Ralpha kappaB binding proteins using cytokines and pharmacologic agents. In addition, the activity of these transcription factors will be altered through the use of DNA binding site analogues which interfere with kappaB binding, or by inactivating RNA transcripts through the use of antisense agents or ribozymes. The goal of this research is to understand the heterogeneity of kappaB binding proteins, to define cDNAs encoding them, and to selectively inhibit their action on the HIV enhancer without affecting cell viability and function.
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