The Role of ALTO in the MCPyV Lifecycle and Tumorigenicity

描述(申请人提供)：小鼠和猿猴多瘤病毒(MCPyV和SV40)是研究转化和致瘤性的非常有用的模型，这些过程可归因于由T抗原基因编码在其基因组早期区域(ER)的蛋白质。到目前为止，已经发现了11种人类多瘤病毒，但只有一种，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。像所有多瘤病毒一样，MCPyV编码大小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(替代LT抗原开放阅读框)。ALTO通过一个保守的疏水C末端与小鼠多瘤病毒的中T抗原进化相关，并定义了一个新的哺乳动物多瘤病毒分支，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是如何影响致瘤性的。环状MCPyV基因组的瞬时扩增可持续数天。我们已经构建了WT和ALTO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、病毒粒子 产生性和传染性。MCPyV转化细胞并促进肿瘤发生的机制尚不清楚。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可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而发挥作用，因此ALTO也可能通过特定的伙伴影响病毒的生命周期和致瘤性。我们将采取不偏不倚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受酪氨酸磷酸化调控的假设。

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.