This is a competitive renewal application. The previous grant was for studying how the trancriptional regulatory proteins of three pathogenic viruses activate transcription, specifically, the Tat protein of human immunodeficiency virus type 1 (HIV-1), the Tax protein of human T-cell leukemia virus type 1 (HTLV-1), and the pX protein of hepatitis B virus (HBV). In this application, the applicant seeks to understand in greater detail the mechanism of action of these viral regulatory proteins and their role in viral replication and human disease. The rationale for the proposal relies heavily on two relatively new technologies. First, the chromatin immunoprecipitation (ChIP) assay, which allows for the detection of specific proteins that are physically associated with DNA in living cells. Second, the genome-wide expression analysis using high density DNA microarrays, which enables the analysis of transcription profiles of a large array of human genes.
The specific aims are: (1) To understand in greater detail how HIV-1 Tat stimulates transcription in vitro and in vivo. The investigator has identified a Tat cofactor, Tat-SF1, and has shown that it is a general transcription elongation factor. A second elongation factor has been implicated, AIEF, and remains to be identified. Tat-SF1 and AIEF and the mechanisms of Tat activation in vivo will be investigated further. (2) To study how HTLV-1 Tax and HBV pX regulates DNA binding of cellular bZIP proteins. The investigator has shown that Tax and pX dramatically increase the DNA binding activity and alter the target selectivity of a wide variety of cellular proteins that possess a basic region-leucine zipper (bZIP) DNA binding domain, through promotion of dimerization. Experiments are proposed to undersstand the basis of altered DNA binding specificity, the cellular proteins involved, and details of the DNA-protein interactions. (3) To analyze how HTLV-1 Tax and HBV pX activate transcription in vivo and transform cells. To test the prediction of different models of how these proteins activate transcription, to identify cellular proteins involved in the activation, and to identify cellular genes activated that may contribute to disease. (4) To understand the mechanism of BEF action. The investigator has identified a nuclear protein BEF (for bZIP-enhancing factor) which is required for bZIP prtoein functions, work synergistically with Tax and pX. However, BEF works by a different mechanism, as a molecular chaperone. Experiments are proposed to further study the mechanism of BEF action.
| Margolis, D M; Ostrove, J M; Straus, S E (1993) HSV-1 activation of HIV-1 transcription is augmented by a cellular protein that binds near the initiator element. Virology 192:370-4 |
