The Role of ALTO in the MCPyV Lifecycle and Tumorigenicity

ALTO 在 MCPyV 生命周期和致瘤性中的作用

• 批准号: 8653347
• 负责人: DENISE A. GALLOWAY
• 金额: $ 36.52万
• 依托单位: FRED HUTCHINSON CANCER RESEARCH CENTER
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-02-01 至 2019-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8653347
• 关键词: Adenoviruses; Affect; Affinity Chromatography; Amino Acids; Antigens; Antithymoglobulin; Apoptosis; Autophagocytosis; Binding; Biological Assay; Cell Line; Cell Proliferation; Cellular Membrane; Chiroptera; Complementary DNA; Cytoskeleton; Data; Exons; Genes; Genome; Goals; Gorilla gorilla; Hamsters; Human; Kinetics; Knock-out; Large T Antigen; Mass Spectrum Analysis; Membrane; Merkel Cells; Merkel cell carcinoma; Modeling; Molecular Chaperones; Mus; Mutate; Mutation; Nucleotides; Oncogenes; Open Reading Frames; Pan Genus; Pathway interactions; Phosphorylation; Phosphotransferases; Phosphotyrosine; Plasmids; Polyomavirus; Polyomaviruses Large T Proteins; Process; Production; Protein Phosphatase 2A Regulatory Subunit PR53; Protein phosphatase; Proteins; RNA Splicing; Raccoons; Reading Frames; Relative (related person); Retinoblastoma Protein; Role; SH2-Binding Motif; Signal Pathway; Signal Transduction; Simian virus 40; Site; Small T Antigen; Testing; Transfection; Tumor Suppressor Proteins; Tumorigenicity; Tyrosine Phosphorylation; Viral; Viral Tumor Antigens; Virion; Virus; antigen binding; cell growth; cell transformation; helicase; keratinocyte; mouse polyomavirus; novel; public health relevance; tumor; tumorigenesis

DESCRIPTION (provided by applicant): Murine and simian polyomaviruses (MCPyV and SV40) have been extraordinarily useful models to study transformation and tumorigenicity, processes that are attributable to proteins encoded in the early region (ER) of their genomes by the T antigen genes. To date eleven human polyomaviruses have been discovered but only one, Merkel Cell polyomavirus (MCPyV) is clearly associated with a human tumor, Merkel Cell Carcinoma (MCC). Little is known about the mechanism by which MCPyV contributes to tumorigenesis, nor is much known about the viral lifecycle. Like all polyomaviruses, MCPyV encodes large and small T antigens (LT and ST). Interestingly we discovered that the +1 reading frame relative to MCPyV LT exon 2 encodes another protein that we have called ALTO (Alternate to LT antigen Open reading frame). ALTO is evolutionarily related to the middle T antigen of mouse polyomavirus through a conserved hydrophobic C-terminus and defines a new clade of mammalian polyomaviruses that includes PyVs from mouse, hamster, raccoon, bat gorilla, chimp and human. Our goals are to understand the role of ALTO in the MCPyV lifecycle and how it affects tumorigenicity. Transfection of a circularized MCPyV genome results in transient amplification for several days. We have constructed WT and ALTO-mutated genomes and will compare the levels and kinetics of viral replication, expression of the T antigens, virion production and infectivity. The mechanism by which MCPyV transforms cells and contributes to tumorigenicity is not well understood. We will examine the effects of LT, ST and ALTO on cell proliferation, immortalization and transformation. Preliminary data indicates that ALTO induces markers of autophagy and apoptosis. We will determine more fully how ALTO affects these pathways. The T antigens of PyVs exert their effects through binding to cellular proteins, thus it is likely that ALTO also influences the virus lifecycle and tumorigenicity through specific partners. We will take both an unbiased and a biased approach to identify the partners. First we will use tandem affinity purification and mass spectrometry to identify ALTO associated proteins. Secondly, we will specifically test the hypothesis that ALTO is regulated by tyrosine phosphorylation.

描述（由申请人提供）：鼠和猿类多瘤病毒（MCPYV和SV40）是研究转化和肿瘤性的非常有用的模型，这些过程可归因于T抗原基因在其基因组早期（ER）中编码的蛋白质归因于蛋白质。到目前为止，已经发现了11种人类多瘤病毒，但只有一个，默克尔细胞多瘤病毒（MCPYV）显然与人类肿瘤默克尔细胞癌（MCC）有关。对于MCPYV促进肿瘤发生的机制知之甚少，对病毒生命周期的众所周知。像所有多瘤病毒一样，MCPYV编码大小的T抗原（LT和ST）。有趣的是，我们发现相对于MCPYV LT外显子2的+1阅读框编码了我们称为Alto的另一种蛋白质（替代LT抗原开放式阅读框）。 Alto通过保守的疏水性C末端与小鼠多瘤病毒的中间T抗原相关，并定义了包括小鼠，仓鼠，浣熊，蝙蝠大猩猩，bot大猩猩和人类的PYV的新的哺乳动物多瘤病毒。我们的目标是了解Alto在MCPYV生命周期中的作用及其如何影响肿瘤性。循环MCPYV基因组的转染会导致瞬时扩增几天。我们已经构建了WT和中音突变的基因组，并将比较病毒复制的水平和动力学，T抗原的表达，病毒体 生产和感染力。 MCPYV转化细胞并导致肿瘤性的机制尚不清楚。我们将研究LT，ST和ALTO对细胞增殖，永生和转化的影响。初步数据表明，中音会诱导自噬和凋亡的标记。我们将更全面地确定中音如何影响这些途径。 PYVS的T抗原通过与细胞蛋白结合发挥作用，因此中音很可能还通过特定伴侣影响病毒生命周期和肿瘤性。我们将采取一种公正和有偏见的方法来识别伴侣。首先，我们将使用串联亲和力纯化和质谱法来识别中音相关的蛋白质。其次，我们将特别检验以下假设，即ALTO受酪氨酸磷酸化调节。