Investigating Merkel Cell Polyomavirus Small T Antigen-Host Interactions

描述(申请人提供)：尽管多瘤病毒(PYV)通常会在健康人中造成终生和无症状的感染，但它们也可以在免疫功能低下或老年患者中导致严重疾病。在默克尔细胞癌(MCC)中发现了默克尔细胞多瘤病毒(MCV)，重新关注了PYV在人类疾病中的重要作用。PYV的细胞转化和致癌依赖于大T(LT)和小T(ST)抗原与细胞蛋白的结合。PYV T抗原通过与关键宿主因子结合并在重要的细胞网络中诱导显著的扰动来诱导细胞生长控制。例如，SV40ST通过与蛋白磷酸酶PP2A的A和C亚基结合，同时取代PP2A的B亚基，促进细胞转化。最近的研究表明，MCV-ST能够以PP2A非依赖性的方式转化大鼠成纤维细胞。值得注意的是，对正常人类细胞中的病毒蛋白进行的大规模和系统的蛋白质组筛选显示，MCV-ST和核受体集结构域包含蛋白1(NSD1)之间存在高度丰富的新的相互作用。NSD1通过直接甲基化激活核因子-kB；在大多数癌症中，核因子-kB的结构性激活是一个标志。这一应用的重点是确定MCV-ST的转化能力是否取决于宿主细胞因素，除了PP2A结合，包括NSD1。这一应用将检验MCV和SV40ST能够以PP2A独立的方式促进细胞转化的假设。为了验证这一假设，将执行以下三个特定目标：(SA1)确定MCV-ST和SV40-ST的独特结构域是否以PP2A非依赖性的方式促进细胞转化。比较SV40-ST和MCV-ST在加入SV40-ST后转化的BJ-hTERT-RasV12-SV40LT细胞的转化活性。我将确定不能与PP2A结合但保持其他特性不变的SV40和MCV ST突变体是否可以诱导细胞转化。我将测试交换N端J和SV40-ST和MCV-ST独特结构域的嵌合体的转化活性。(SA2)确定MCV-ST是否以PP2A非依赖的方式与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各种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。对NSD1进行RNAi敲除，以确定在MCV-ST介导的转化中对NSD1的需求。NSD1的组蛋白甲基转移酶活性将在体外和与MCV-ST的络合物中进行测定。(SA3)确定MCV-ST和NSD1是否对核因子-kB的激活有影响。将评估MCV-ST-NSD1复合体的形成对NSD1的直接靶标-NF-kB激活的潜在影响。如果成功，这项应用将提供证据，证明PYV ST可以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.