Transforming Mechanisms of Merkel Cell Polyoma Virus

描述(申请人提供)：推动这项研究应用的总体假设是，默克尔细胞多瘤病毒(MCPyV)与Myc同源物和Tip60/TRRAP复合体结合的小T抗原(ST)是病毒介导的默克尔细胞癌(MCC)发生所必需的。我的实验室从2个不同的MCC细胞系中大规模免疫沉淀MCPyV ST，鉴定出LMyc、NMyc、Max以及完整的15组分Tip60/TRRAP复合体。鉴于Myc在肿瘤发生中的已知作用以及Tip60/TRRAP复合体在Myc依赖基因表达中的作用，我们认为ST与Myc和Tip60/TRRAP复合体结合是MCPyV转化和肿瘤发生所必需的。具体目的：(1)评价MCPyV在人成纤维细胞和原代小鼠Merkel细胞中的细胞转化能力。(2)确定MCPyV ST如何与Myc和Tip60/TRRAP复合体结合。确定ST中与Myc和Tip60/TRRAP复合体结合所需的结构域和残基。确定与MCPyV ST结合需要Tip60/TRapp复合体的哪些组件。确定MCPyV介导的转化的维持是否依赖于完整的Tip60/TRRAP复合体。(3)评价Myc和Tip60/TRRAP复合体在MCPyV转化和肿瘤发生中的作用。确定一个显性的负性Myc物种或特定Myc同源物的缺失是否能在体外和体内抑制MCPyV的转化。确定ST是否以Myc或Tip60/TRRAP复合体依赖的方式招募到染色质。意义重大。理解MCPyV如何促进MCC发展的一个关键障碍是缺乏对MCPyV大T抗原(LT)或ST在从经典多瘤病毒、SV40和小鼠多瘤病毒转化过程中的贡献是否以及如果是这样的认识上的差异。此外，由于没有其他人类肿瘤报告含有整合的MCPyV病毒基因组，因此尚不清楚为什么Merkel细胞似乎唯一对MCPyV的转化敏感。这个拟议的项目通过建立在我的研究实验室最近的一项发现基础上解决了这些关键障碍，即MCPyV ST与Myc和Tip60/TRRAP复合体在细胞中特异性结合。

DESCRIPTION (provided by applicant): The overall hypothesis that drives this research application is that Merkel cell polyomavirus (MCPyV) small T antigen (ST) binding to Myc homologues and the Tip60/TRRAP complex is required for viral mediated oncogenesis of Merkel cell carcinoma (MCC). My laboratory identified LMyc, NMyc, Max as well as the entire, 15-component, Tip60/TRRAP complex in a large-scale immunoprecipitation for MCPyV ST from 2 different MCC cell lines. Given the known role of Myc in oncogenesis and the Tip60/TRRAP complex in Myc dependent gene expression, we propose that ST binding to Myc and the Tip60/TRRAP complex is required for MCPyV transformation and tumorigenesis. Specific Aims: (1) Assess the cellular transforming potential of MCPyV in human fibroblasts and primary mouse Merkel cells. (2) Determine how MCPyV ST binds to Myc and the Tip60/TRRAP complex. Identify domains and residues in ST required for binding to Myc and the Tip60/TRRAP complex. Determine what components of the Tip60/TRAPP complex are required for binding to MCPyV ST. Determine whether the maintenance of MCPyV-mediated transformation is dependent on an intact Tip60/TRRAP complex. (3) Assess the contribution of Myc and the Tip60/TRRAP complex to MCPyV transformation and tumorigenesis. Determine if a dominant negative Myc species or loss of specific Myc homologues can inhibit MCPyV transformation in vitro and in vivo. Determine whether ST is recruited to chromatin in a Myc or Tip60/TRRAP complex dependent manner. Significance. A critical barrier to understanding how MCPyV contributes to the development of MCC is the lack of appreciation of whether and, if so, how MCPyV large T antigen (LT) or ST differ in their contributions to transformation from the canonical polyomaviruses, SV40 and mouse polyomavirus. Furthermore, it is not clear why Merkel cells seem to be uniquely susceptible to transformation by MCPyV since no other human tumor has been reported to contain integrated MCPyV viral genome. This proposed project addresses these critical barriers by building on a recent discovery from my research laboratory that MCPyV ST binds specifically in cells to Myc and the Tip60/TRRAP complex.