Investigating Merkel Cell Polyomavirus Small T Antigen-Host Interactions

描述（由申请人提供）：虽然多瘤病毒（PyV）通常在健康人中引起终身和无症状感染，但它们可在免疫功能低下或老年患者中引起严重疾病。默克尔细胞多瘤病毒（MCV）在默克尔细胞癌（MCC）中的发现重新聚焦了PyV在人类疾病中的重要作用。PyV的细胞转化和肿瘤发生依赖于大T （LT）和小T （ST）抗原与细胞蛋白的结合。PyV T抗原通过结合关键宿主因子和诱导重要细胞网络的显著扰动来诱导细胞生长控制。例如，SV40 ST通过结合蛋白磷酸酶PP2A的A和C亚基而取代PP2A的B亚基来促进细胞转化。最近有研究表明，MCV-ST能够以不依赖pp2a的方式转化大鼠成纤维细胞。值得注意的是，对正常人类细胞中病毒蛋白进行的大规模系统蛋白质组学筛选显示，MCV-ST与核受体SET结构域蛋白1 （NSD1）之间存在高度富集的新型相互作用。NSD1通过直接甲基化激活NF-kB；NF-kB的组成激活是大多数癌症的标志。本应用的重点是确定MCV-ST的转化能力是否依赖于宿主细胞因子，除了PP2A结合，包括NSD1。该应用程序将测试假设MCV和SV40 ST可以以PP2A独立的方式促进细胞转化。为了验证这一假设，将执行以下3个特定目的：（SA1）确定MCV-ST和SV40-ST的独特结构域是否以不依赖于PP2A的方式促进细胞转化。我将比较SV40-ST和MCV-ST在BJ-hTERT-RasV12- SV40LT细胞中的转化活性，加入SV40-ST后，SV40-ST发生转化。我将确定SV40和MCV ST突变体是否不能与PP2A结合，但保留其他特性完好无损，可以诱导细胞转化。我将测试交换SV40-ST和MCV-ST的n端J和独特结构域的嵌合体的转化活性。（SA2）确定MCV-ST是否以不依赖于pp2a的方式与NSD1相互作用，以及这种相互作用是否有助于MCV-ST介导的转化。这种相互作用的验证将在体外使用不同的MCV-ST结构在正常和MCC细胞系中进行。我将确定与NSD1结合所需的MCV-ST的特定残基，以及这种相互作用是否依赖于PP2A结合。我们将对NSD1进行RNAi敲低，以确定mcv - st介导的转化是否需要NSD1。NSD1的组蛋白甲基转移酶活性将在体外和MCV-ST联合检测。（SA3）确定MCV-ST和NSD1是否对NF-kB激活有影响。我们将评估MCV-ST-NSD1复合物形成对NSD1直接靶点NF-kB活化的潜在影响。如果成功，该应用将提供证据，证明PyV ST可以以PP2A依赖和独立的方式转化细胞。

DESCRIPTION (provided by applicant): Although polyomaviruses (PyV) typically cause lifelong and asymptomatic infections in healthy humans, they can cause severe illness in immunocompromised or elderly patients. The discovery of Merkel cell polyomavirus (MCV) in Merkel cell carcinomas (MCC) has refocused the significant role of PyV in human disease. Cellular transformation and oncogenesis by PyV is dependent on large T (LT) and small T (ST) antigens binding to cellular proteins. The PyV T antigens induce cell growth control by binding to key host factors and inducing significant perturbations in important cellular networks. For example, the SV40 ST contributes to cellular transformation by binding to the A and C subunits of protein phosphatase PP2A while displacing the B subunit of PP2A. It was recently shown that MCV-ST could transform rat fibroblasts in a PP2A-independent manner. Notably, a large-scale and systematic proteomic screen of viral proteins in normal human cells revealed a highly enriched and novel interaction between MCV-ST and the nuclear receptor SET domain-containing protein 1 (NSD1). NSD1 activates NF-kB through direct methylation; constitutive activation of NF-kB is a hallmark in most cancers. This application is focused on determining whether the transforming ability of MCV-ST depends on host cell factors, in addition to PP2A binding, including NSD1. This application will test the hypothesis that MCV and SV40 ST can contribute to cellular transformation in a PP2A independent manner. To test the hypothesis, the following 3 Specific Aims will be performed: (SA1) Determine if the unique domain of MCV-ST and SV40-ST contributes to cellular transformation in a PP2A- independent manner. I will compare the transforming activity of SV40-ST with MCV-ST in BJ-hTERT-RasV12- SV40LT cells, which become transformed with the addition of SV40-ST. I will determine if SV40 and MCV ST mutants that fail to bind to PP2A but leave other properties intact can induce cellular transformation. I will test the transforming activity of chimeras that swap the N-terminal J and unique domains of SV40-ST and MCV-ST. (SA2) Determine if MCV-ST interacts with NSD1 in a PP2A-independent manner and if this interaction contributes to MCV-ST mediated transformation. Validation of this interaction will be pursued in vitro using various MCV-ST constructs in normal and MCC cell lines. I will determine the specific residues of MCV-ST that are required for binding to NSD1, and if this interaction is dependent on PP2A binding. RNAi knockdown of NSD1 will be performed to determine the requirement for NSD1 in MCV-ST-mediated transformation. The histone methyltransferase activity of NSD1 will be assayed in vitro and in complex with MCV-ST. (SA3) Determine if MCV-ST and NSD1 have an effect on NF-kB activation. The potential impact of MCV-ST-NSD1 complex formation on activation of NF-kB, a direct target of NSD1, will be evaluated. If successful, this application will provide evidence tha PyV ST can transform cells in a PP2A dependent and independent manner.