Transcription of human immunodeficiency virus (HIV), the etiologic agent of AIDS, is regulated by the cellular factor NF-kB, which binds to the HIV enhancer. NF-kB, originally described as a factor binding to the immunoglobulin k enhancer, is implicated in transcriptional regulation of numerous genes. The goals of this project are to determine the structure of NF-kB, its role in HIV transcription, and the transcriptional roles of related factors H2TF1, MBP-1 and KBF1 which bind to the same cognate sequence as NF-kB. To accomplish the latter goals, parallel studies of these related factors will be necessary. These factors may be present simultaneously in the nuclear milieu and it therefore remains to be established which factors are actually involved in positive or negative regulation of transcription in vivo. cDNA cloning of NF-kB and H2TF1 will be the initial step of the project. Partial protein sequence of NF-kB and H2TF1 purified from HeLa cells by DNA affinity chromatography will be used to isolate cDNA clones. A collateral approach will involve screening of cDNA expression libraries with oligonucleotide probes, a method developed in Dr. Sharp's laboratory. Functional domains of NF-kB and H2TF1 will be mapped by deletion analysis of cDNA expression constructs in transfected cells, using a reporter plasmid method. A set of antibody reagents specific for each factor will be generated from proteins overexpressed from the cDNA clones. These reagents will be used to determine whether NF-kB and H2TF1 are bound to the HIV enhancer in vivo, under a variety of physiologic conditions. The methodology will involve UV-crosslinking, followed by immunoprecipitation and Southern blot analysis of the precipitated DNA. This approach has been used to measure quantitative differences in the density of RNA polymerase II (pol II) on heat shock gene hsp70, before and after heat shock. The above reagents will also be used to comparatively study the NF-kB role in regulation of immunoglobulin gene transcription in normal B cell development. This project is part of a long term approach to understand the regulation of HIV gene expression and is ultimately directed toward the development of new approaches to the treatment and prevention of AIDS.
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