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）：鼠类和猿类多瘤病毒（MCPyV 和 SV40）已成为研究转化和致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的早期区域 (ER) 编码的蛋白质。迄今为止，已发现 11 种人类多瘤病毒，但只有一种默克尔细胞多瘤病毒 (MCPyV) 与人类肿瘤默克尔细胞癌 (MCC) 明确相关。人们对 MCPyV 促进肿瘤发生的机制知之甚少，对病毒生命周期也知之甚少。与所有多瘤病毒一样，MCPyV 编码大 T 抗原和小 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 阅读框编码另一种蛋白质，我们称之为 ALTO（LT 抗原开放阅读框的替代）。 ALTO 通过保守的疏水性 C 末端与小鼠多瘤病毒的中间 T 抗原在进化上相关，并定义了哺乳动物多瘤病毒的新分支，其中包括来自小鼠、仓鼠、浣熊、蝙蝠大猩猩、黑猩猩和人类的 PyV。我们的目标是了解 ALTO 在 MCPyV 生命周期中的作用以及它如何影响致瘤性。转染环状 MCPyV 基因组会导致数天的瞬时扩增。我们构建了 WT 和 ALTO 突变基因组，并将比较病毒复制、T 抗原表达、病毒粒子的水平和动力学 产量和传染性。 MCPyV 转化细胞并导致致瘤性的机制尚不清楚。我们将研究 LT、ST 和 ALTO 对细胞增殖、永生化和转化的影响。初步数据表明 ALTO 诱导自噬和细胞凋亡的标志物。我们将更全面地确定 ALTO 如何影响这些途径。 PyV 的 T 抗原通过与细胞蛋白结合发挥作用，因此 ALTO 很可能还通过特定伙伴影响病毒生命周期和致瘤性。我们将采取公正和有偏见的方法来确定合作伙伴。首先，我们将使用串联亲和纯化和质谱法来鉴定 ALTO 相关蛋白。其次，我们将具体检验ALTO受酪氨酸磷酸化调节的假设。