Investigating Merkel Cell Polyomavirus Small T Antigen-Host Interactions

研究默克尔细胞多瘤病毒小 T 抗原-宿主相互作用

• 批准号: 8899465
• 负责人: Christian Jose Berrios
• 金额: $ 3.06万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-01 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8899465
• 关键词: Accounting; Affect; Affinity; Attention; Binding; Binding Proteins; Biological Assay; Cell Line; Cell Proliferation; Cells; Chimera organism; Chromatin; Complex; Fibroblasts; Growth; Health; Human; Human Virus; Immunocompromised Host; In Vitro; Infection; Integration Host Factors; Large T Antigen; Left; Malignant Epithelial Cell; Malignant Neoplasms; Measures; Mediating; Merkel Cells; Merkel cell carcinoma; Methylation; Modeling; Mutate; Mutation; N-terminal; NF-kappa B; Normal Cell; Nuclear Receptors; Oncogene Proteins; Pathway interactions; Play; Point Mutation; Polyomavirus; Property; Protein Binding; Protein Phosphatase 2A Regulatory Subunit PR53; Protein phosphatase; Proteins; Proteomics; Publications; RNA Interference; Rattus; Role; S Phase; SET Domain; ST7 gene; Signal Transduction; Simian virus 40; Small T Antigen; Testing; Translations; Validation; Viral; Viral Genome; Viral Proteins; Viral Tumor Antigens; Zinc; antigen binding; cell growth; cell transformation; histone methyltransferase; human disease; in vitro Assay; metaplastic cell transformation; mutant; novel; older patient; synthetic construct; tumorigenesis

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.

描述（由申请方提供）：虽然多瘤病毒（PyV）通常会导致健康人终身和无症状感染，但它们可能会导致免疫功能低下或老年患者的严重疾病。在默克尔细胞癌（MCC）中发现的默克尔细胞多瘤病毒（MCV）重新聚焦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与细胞蛋白结合的大T（LT）和小T（ST）抗原。PyV T抗原通过与关键宿主因子结合并诱导重要细胞网络中的显著扰动来诱导细胞生长控制。例如，SV 40 ST通过结合蛋白磷酸酶PP 2A的A和C亚基同时取代PP 2A的B亚基来促进细胞转化。最近显示MCV-ST可以以不依赖于PP 2A的方式转化大鼠成纤维细胞。值得注意的是，在正常人类细胞中对病毒蛋白进行的大规模和系统的蛋白质组学筛选揭示了MCV-ST与核受体SET结构域蛋白1（NSD 1）之间高度富集的新型相互作用。NSD 1通过直接甲基化激活NF-κ B; NF-κ B的组成性激活是大多数癌症的标志。本申请的重点是确定MCV-ST的转化能力是否取决于宿主细胞因子，除了PP 2A结合，包括NSD 1。本申请将检验MCV和SV 40 ST可以以PP 2A独立方式促进细胞转化的假设。为了检验假设，将进行以下3个特定目的：（SA 1）确定MCV-ST和SV 40-ST的独特结构域是否以PP 2A非依赖性方式促进细胞转化。我将比较SV 40-ST与MCV-ST在BJ-hTERT-RasV 12-SV 40 LT细胞中的转化活性，所述细胞通过加入SV 40-ST而转化。我将确定不能与PP 2A结合但保持其他性质完整的SV 40和MCV ST突变体是否可以诱导细胞转化。我将测试交换SV 40-ST和MCV-ST的N-末端J和独特结构域的嵌合体的转化活性。（SA 2）确定MCV-ST是否以PP 2A非依赖性方式与NSD 1相互作用，以及这种相互作用是否有助于MCV-ST介导的转化。将在正常和MCC细胞系中使用各种MCV-ST构建体在体外进行这种相互作用的验证。我将确定与NSD 1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PP 2A结合。将进行NSD 1的RNAi敲低以确定MCV-ST介导的转化中对NSD 1的需求。NSD 1的组蛋白甲基转移酶活性将在体外并与MCV-ST复合进行测定。（SA 3）确定MCV-ST和NSD 1是否对NF-κ B活化有影响。将评价MCV-ST-NSD 1复合物形成对NSD 1直接靶点NF-κ B活化的潜在影响。如果成功，该应用将提供证据表明PyV ST可以以PP 2A依赖和独立的方式转化细胞。