This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Background. Human T cell leukemia virus type 1 (HTLV-1) is causes a human cancer known as adult T cell leukemia (ATL). Approximately 11-20 million people are infected (HTLV-1+) in the world with pockets of infection occurring in the Caribbean, central Africa, Japan, and Italy. United States blood donations are routinely assayed for the presence of HTLV-1, and about 0.025% of the U.S. blood supply is positive for the virus. There is no vaccine for the virus. The virus infects and integrates its proviral DNA in human CD4+ T cells, and after a latency period, about 3-5% of HTLV-1+ patients develop ATL, a debilitating and aggressive cancer with no known cure and a poor prognosis. Rationale and Hypothesis. The research in the laboratory is focused on the viral protein Tax, a 40 kDa, 353 amino acid, phosphorylated protein. Numerous studies since the early 1980s have demonstrated that Tax is the oncogenic protein of HTLV-1, and introduction of tax into normal human T cells results in transformation. The mechanism by which Tax initiates and maintains the transformed phenotype of the host cell is not yet known. One factor that has impeded the progress in this area is the lack of structural data (secondary and tertiary structure) and other physical characteristics for Tax. The main hypothesis of our research is that Tax activation of serum-response-factor (SRF)-dependent transcription leads to the initiation of HTLV-1 transformation. The focused hypothesis of the proposed project is that Tax regulates the binding activities of SRF and its partner ternary complex factor (TCF) -- which bind the DNA, forming a Tax/SRF/TCF/DNA complex ?which results in a higher Ka of the complex with Tax than without Tax. Furthermore, we believe that Tax regulation of the complex is directly dependent on its structure. Methods. The focus of the project is to characterize the biophysical properties of Tax alone and as part of the SRF/DNA complex. The methods we use are (1.) to express Tax, SRF, and TCF in bacterial and human cells, (2.) to purify the proteins by a two-step process of double ammonium sulfate precipitation, followed by size fractionation chromatography, (3.) to determine the binding interactions of wild-type and mutant Tax with and without the SRF/TCF/DNA complexes, and (4.) to use circular dichroism spectroscopy to determine the secondary structure of wild-type and mutant Tax with and without the SRF/TCF/DNA complexes. Results. We have been able to express and purify Tax as a monomer which is important for the biochemical studies. We have obtained SRF as well although we are unable to separate the full-length 67 kDa SRF from the truncated SRF which initiates from a second AUG downstream of the first AUG. Discussion and Interpretation. Our percent recovery is low and we will need to increase our production of the proteins in order to begin the biochemical experiments.
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