Department of Microbiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA.
Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA.
Cancers (Basel). 2014 Jul 8;6(3):1464-86. doi: 10.3390/cancers6031464.
Merkel Cell Polyomavirus (MCPyV) was recently discovered as a novel human polyomavirus that is associated with ~80% of Merkel Cell Carcinomas. The Large Tumor antigen (LT) is an early viral protein which has a variety of functions, including manipulation of the cell cycle and initiating viral DNA replication. Phosphorylation plays a critical regulatory role for polyomavirus LT proteins, but no investigation of MCPyV LT phosphorylation has been performed to date. In this report mass spectrometry analysis reveals three unique phosphorylation sites: T271, T297 and T299. In vivo replication assays confirm that phosphorylation of T271 does not play a role in viral replication, while modification at T297 and T299 have dramatic and opposing effects on LT's ability to initiate replication from the viral origin. We test these mutants for their ability to bind, unwind, and act as a functional helicase at the viral origin. These studies provide a framework for understanding how phosphorylation of LT may dynamically regulate viral replication. Although the natural host cell of MCPyV has not yet been established, this work provides a foundation for understanding how LT activity is regulated and provides tools for better exploring this regulation in both natural host cells and Merkel cells.
默克尔细胞多瘤病毒（MCPyV）最近被发现是一种新型的人类多瘤病毒，与~80%的默克尔细胞癌有关。大肿瘤抗原（LT）是一种早期病毒蛋白，具有多种功能，包括操纵细胞周期和启动病毒 DNA 复制。磷酸化在多瘤病毒 LT 蛋白的调控中起着关键作用，但迄今为止尚未对 MCPyV LT 磷酸化进行研究。在本报告中，质谱分析揭示了三个独特的磷酸化位点：T271、T297 和 T299。体内复制实验证实 T271 的磷酸化在病毒复制中不起作用，而 T297 和 T299 的修饰对 LT 从病毒原点起始复制的能力有显著的、相反的影响。我们测试了这些突变体在病毒原点处结合、解旋和作为功能解旋酶的能力。这些研究为理解 LT 的磷酸化如何动态调节病毒复制提供了一个框架。虽然 MCPyV 的天然宿主细胞尚未确定，但这项工作为理解 LT 活性如何被调控提供了基础，并为更好地探索天然宿主细胞和默克尔细胞中的这种调控提供了工具。
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