Role of a new polyomavirus in Merkel cell carcinoma

描述(申请人提供)：我们最近发现了一种新的人类多瘤病毒，它是单克隆性整合到人类默克尔细胞癌(MCC)中的，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症，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，晚期疾病患者中只有50%能存活9个月或更长时间。MCPyV基因组全长5.4Kbase，与小鼠和非洲绿猴嗜淋巴多瘤病毒(分别为MuPyV和LPyV)关系密切。MCPyV与SV40和四种已知的人类多瘤病毒有较远的亲缘关系。人类血清调查显示，来自美国、日本和德国的15%-30%的人口对LPV具有交叉反应抗体，这可能实际上代表了对MCPyV感染的反应性。通过从外周血细胞中直接检测MCPyV基因组，我们发现了相似的MCPyV感染率。如果得到证实，这些发现表明全世界有超过10亿人接触过MCPyV感染。Southern杂交结果表明，MCPyV以体细胞和单克隆性方式整合到MCC基因组的不同部位。一个细胞整合位点被定义为受体类型蛋白酪氨酸磷酸酶-伽马(PTPRG)内含子1。MCPyV在肿瘤中也表达高度保守的T抗原。MCPyV的N末端编码与转化相关的DNAJ和LXCXE口袋蛋白结合域。然而，所有肿瘤来源的MCPyV T抗原都存在T抗原突变，这些突变消除了T抗原起源结合和/或质粒复制功能。这些功能不是维持整合病毒所必需的，这表明MCC至少经历了两个步骤：第一，MCPyV整合到宿主基因组中；第二，出现截断突变，允许表达N-末端转化结构域，但消除了有害的C-末端结构域。MCPyV可能通过插入突变、表达T抗原或两者兼而有之在肿瘤发生中发挥作用。我们的建议试图通过1)确定额外的细胞整合位点，2)分析啮齿动物细胞中T抗原的转化功能和细胞信号分析，3)在T抗原表达后进行全细胞蛋白质组学分析，4)寻找新的细胞T抗原直接相互作用因子，5)建立针对Merkel机械感受器细胞的MCPyV T抗原表达的转基因小鼠，以了解这些转化和肿瘤发生的机制。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促进人类癌症的发生。公共卫生相关性：这项提案试图了解默克尔细胞病毒(MCV)如何导致人类癌症。在默克尔细胞癌中发现MCV是一种整合病毒，它表达T抗原，T抗原是一种癌蛋白，与密切相关的病毒有很好的特征。这项建议将调查MCV T抗原和其他病毒T抗原的异同，以及MCV整合和T抗原表达对细胞蛋白的失调。最后，我们试图建立一个MCV肿瘤发生的模型系统，这将对药物筛选和预防有用。

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.