Role of a new polyomavirus in Merkel cell carcinoma

描述（由申请人提供）：我们最近发现了一种单克隆整合到人默克尔细胞癌（MCC）中的新的人多瘤病毒，我们称之为默克尔细胞多瘤病毒（MCPyV）。MCC是一种罕见的神经外胚层癌，由于其不寻常的流行病学，怀疑是由病毒感染引起的。它是最具侵袭性的皮肤癌，只有50%的晚期患者存活9个月或更长时间。MCPyV具有5.4 kbase基因组，与鼠和非洲绿色猴嗜淋巴多瘤病毒（分别为MuPyV和LPyV）密切相关。MCPyV与SV 40和四种已知的人类多瘤病毒有远亲关系。人类血清调查显示，来自美国、日本和德国的15-30%的人群具有对LPV的交叉反应性抗体，其实际上可能代表对MCPyV感染的反应性。我们使用来自外周血细胞的MCPyV基因组的直接检测发现类似的MCPyV感染率。如果得到证实，这些研究结果表明，全球有超过10亿人暴露于MCPyV感染。Southern杂交结果表明，MCPyV以体细胞和单克隆的方式整合到MCC基因组的不同位点。一个细胞整合位点已被定义为受体型蛋白酪氨酸磷酸酶-γ（PTPRG）内含子1。MCPyV还在肿瘤中表达高度保守的T抗原。MCPyV的N-末端编码转化相关的DnaJ和LXCXE口袋蛋白结合结构域。然而，所有肿瘤来源的MCPyV T抗原都具有消除T抗原起点结合和/或质粒复制功能的T抗原突变。维持整合的病毒不需要这些功能，这表明MCC以至少两个步骤产生：首先，MCPyV整合到宿主基因组中;其次，出现截短突变，允许表达N-末端转化结构域，但消除有害的C-末端结构域。MCPyV可能通过插入突变、表达T抗原或两者在肿瘤发生中发挥作用。我们的建议试图通过以下方式来理解这些转化和肿瘤发生的机制：1）鉴定额外的细胞整合位点，2）分析啮齿动物细胞和细胞信号传导测定中的T抗原转化功能，3）在T抗原表达后进行细胞范围的蛋白质组学分析，4）鉴定新的细胞T抗原直接相互作用物和5）产生具有靶向默克尔机械感受器细胞的MCPyV T抗原表达的转基因小鼠。通过这种系统的方法，我们预计我们将了解这种新病毒如何有助于人类致癌。公共卫生相关性：该提案旨在了解默克尔细胞病毒（MCV）如何导致人类癌症。MCV在默克尔细胞癌中被发现为整合病毒，其中它表达T抗原，T抗原是一种已经从密切相关的病毒中充分表征的癌蛋白。本研究将探讨MCV T抗原与其他病毒T抗原的异同，以及MCV整合和T抗原表达引起的细胞蛋白失调。最后，我们试图产生一个模型系统MCV肿瘤发生，将是有用的药物筛选和预防。

DESCRIPTION (provided by applicant): We have recently discovered a new human polyomavirus monoclonally-integrated into human Merkel cell carcinomas (MCC) that we call Merkel cell polyomavirus (MCPyV). MCC is a rare neuroectodermal cancer suspected to be caused by a viral infection because of its unusual epidemiology. It is the most aggressive skin cancer and only 50% of patients with advanced disease survive 9 months or longer. MCPyV has a 5.4 kbase genome closely related to murine and African green monkey lymphotropic polyomaviruses (MuPyV and LPyV, respectively). MCPyV is distantly related to SV40 and the four known human polyomaviruses. Human serosurveys show that 15-30% of populations from the US, Japan and Germany have cross-reactive antibodies to LPV which may actually represent reactivity to MCPyV infection. We find similar MCPyV infection rates using direct detection of MCPyV genome from peripheral blood cells. If confirmed, these findings suggest that over a billion persons have been exposed to MCPyV infections worldwide. Southern blotting shows that MCPyV is integrated into MCC genome at different sites in a somatic and monoclonal pattern. One cellular integration site has been defined as the receptor-type protein tyrosine phosphatase-gamma (PTPRG) intron 1. MCPyV also expresses a highly conserved T antigen in tumors. The N-terminus of MCPyV encodes transformation-associated DnaJ and LXCXE pocket protein-binding domains. All tumor- derived MCPyV T antigens, however, possess T antigen mutations that eliminate T antigen origin binding and/or plasmid replication functions. These functions are not needed to maintain integrated virus, suggesting that MCC arises in at least two steps: first, MCPyV integrates into the host genome; second, truncation mutations arise allowing expression of N-terminal transforming domains, but eliminating deleterious C- terminal domains. MCPyV may play a role in tumorigenesis through insertional mutagenesis, expression of T antigen or both. Our proposal seeks to understand these mechanisms for transformation and oncogenesis by 1) identifying additional cell integration sites, 2) analyzing T antigen transforming functions in rodent cells and in cell signaling assays, 3) performing cell-wide proteomic analysis following T antigen expression, 4) identifying novel cellular T antigen direct interactors and 5) generating transgenic mice with MCPyV T antigen expression targeted to Merkel mechanoreceptor cells. Through this systematic approach we anticipate we will learn how this new virus contributes to human carcinogenesis. PUBLIC HEALTH RELEVANCE: This proposal seeks to understand how Merkel cell virus (MCV) contributes to human cancers. MCV has been found as an integrated virus in Merkel cell carcinoma where it expresses T antigen, an oncoprotein that has been well-characterized from closely-related viruses. This proposal will investigate similarities and differences between MCV T antigen and T antigen from other viruses as well as dysregulation of cellular proteins from MCV integration and T antigen expression. Finally, we seek to generate a model system for MCV tumorigenesis that will be useful for drug screening and prevention.