HIV-1 gene expression is regulated by a diverse set of cellular transcription factors which bind to cis-acting regulatory elements in the HIV-1 LTR. The interplay between these cellular factors and the viral transactivator protein Tat is critical for gene regulation. In addition to modulation of HIV-1 gene expression by Tat, a variety of other regulators of the signal transduction pathway can increase HIV-1 gene expression by altering the activity of cellular transcription factors. It is the interaction of upstream transcription factors, the general transcriptional machinery, and Tat which results in activation of HIV-1 gene expression. The focus of this grant proposal is to better understand the complex transcriptional machinery that results in increases in HIV-1 replication. In particular, we wish to further analyze the cis-acting regulatory elements that influence viral replication, to use biochemical fractionation to identify cellular transcription factors important for HIV-1 gene regulation, and to utilize in vitro transcription assays to dissect components of the transcriptional pathway necessary for Tat activation. Our ability to produce viruses containing site-directed mutants in each of the critical cis-acting HIV-1 LTR elements, the development of immobilized HIV-1 template assays which give Tat induced increases in in vitro transcription from HIV-1 LTR, and the purification of both novel and previously characterized general and upstream cellular transcription factors provides us with the techniques and reagents necessary to better understand Tat activation.
The specific aims of this proposal are: (l) to use immobilized HIV-1 templates bound to streptavidin beads to dissect steps in the transcriptional pathway that are altered by Tat and cellular regulatory proteins; (2) to better characterize the mechanism by which specific sequences and cellular factors in the HIV-1 TATA element influence activation of gene expression; (3) to determine how the transcriptional elongation properties of RNA polymerase II are modified by cellular factors and Tat; (4) to understand how different mutations in HIV-1 LTR regulatory regions alter viral replication and subsequent pathogenesis in the thymus reconstituted SCID-hu mouse model and (5) to determine how the in vivo binding properties of cellular factors to the HIV-1 LTR are altered in viruses containing mutations in either the tat gene or in different HIV-1 LTR regulatory regions. These studies will help to address the role of cellular factors which modulate HIV-1 gene expression.
