THE MOLECULAR MECHANISMS OF ADULT T CELL LEUKEMIA INDUCTION BY HTLV-1 TAX

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 背景资料。人类T细胞白血病病毒1型(HTLV-1)是一种人类癌症，被称为成人T细胞白血病(ATL)。世界上约有1100-2000万人感染(HTLV-1+)，感染发生在加勒比海、中非、日本和意大利。美国的献血者通常会接受HTLV-1病毒的检测，大约0.025%的美国血液呈阳性。目前还没有针对该病毒的疫苗。该病毒感染人类CD4+T细胞并整合其前病毒DNA，经过一段潜伏期后，约3%-5%的HTLV-1+患者患上ATL，这是一种虚弱和侵袭性的癌症，目前尚无治愈方法，预后也很差。 理论基础和假设。实验室的研究重点是病毒蛋白TAX，一种40 kDa、353个氨基酸的磷酸化蛋白。自20世纪80年代初以来的大量研究表明，TAX是HTLV-1的致癌蛋白，将TAX导入正常人T细胞会导致转化。Tax启动和维持宿主细胞转化表型的机制尚不清楚。阻碍这一领域取得进展的一个因素是缺乏结构数据(二级和三级结构)和税收的其他实际特征。我们研究的主要假设是，依赖血清反应因子(SRF)的转录激活导致HTLV-1转化的启动。拟议项目的重点假设是，Tax调节SRF及其伙伴三元复合因子(TCF)的结合活性，后者结合DNA，形成Tax/SRF/TCF/DNA复合体，从而导致有税的复合体的Ka比没有税的复合体的Ka更高。此外，我们认为，该综合体的税收监管直接取决于其结构。 方法：研究方法。该项目的重点是确定单独和作为SRF/DNA复合体一部分的TAX的生物物理性质。我们使用的方法是(1)。在细菌和人类细胞中表达TAX、SRF和TCF，(2.)采用双硫酸铵沉淀、分级柱层析两步法分离纯化蛋白质。确定野生型和突变型TAX与SRF/TCF/DNA复合体的结合作用和不与SRF/TCF/DNA复合体的结合作用；用圆二色谱确定含有和不含有SRF/TCF/DNA复合体的野生型和突变型TAX的二级结构。 结果。我们已经能够表达和纯化对生物化学研究具有重要意义的单体Tax。我们也获得了SRF，尽管我们无法将全长67 kDa的SRF从第一个8月下游的第二个8月开始的截断SRF中分离出来。 讨论和解释。我们的回收率很低，我们需要增加蛋白质的产量才能开始生化实验。

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.