Transforming Mechanisms of Merkel Cell Polyoma Virus

描述（由申请人提供）：驱动本研究申请的总体假设是，默克尔细胞多瘤病毒（MCPyV）小T抗原（ST）与Myc同源物和Tip 60/TRRAP复合物的结合是病毒介导的默克尔细胞癌（MCC）肿瘤发生所必需的。我的实验室在来自2种不同MCC细胞系的MCPyV ST的大规模免疫沉淀中鉴定了LMyc、NMyc、Max以及整个15组分Tip 60/TRRAP复合物。鉴于Myc在肿瘤发生中的已知作用和Tip 60/TRRAP复合物在Myc依赖性基因表达中的作用，我们提出ST与Myc和Tip 60/TRRAP复合物的结合是MCPyV转化和肿瘤发生所需的。 具体目的：（1）评估MCPyV在人成纤维细胞和原代小鼠默克尔细胞中的细胞转化潜力。(2)确定MCPyV ST如何与Myc和Tip 60/TRRAP复合物结合。鉴定ST中与Myc和Tip 60/TRRAP复合物结合所需的结构域和残基。确定Tip 60/TRAPP复合物的哪些组分是与MCPyV ST结合所必需的。确定MCPyV介导的转化的维持是否依赖于完整的Tip 60/TRRAP复合物。(3)评估Myc和Tip 60/TRRAP复合物对MCPyV转化和肿瘤发生的贡献。确定显性阴性Myc种类或特定Myc同源物的丢失是否可以在体外和体内抑制MCPyV转化。确定ST是否以Myc或Tip 60/TRRAP复合物依赖性方式募集到染色质。意义理解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在其对典型多瘤病毒（SV 40和小鼠多瘤病毒）转化的贡献方面是否不同以及如果是的话如何不同的认识。此外，尚不清楚为什么默克尔细胞似乎唯一易受MCPyV转化的影响，因为没有其他人肿瘤被报道含有整合的MCPyV病毒基因组。这个拟议的项目通过建立在我的研究实验室最近发现的MCPyV ST在细胞中特异性结合Myc和Tip 60/TRRAP复合物来解决这些关键障碍。

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.