Cytomegalovirus UL80 Proteins:Interactions and Modifications that Impact Function

描述（由申请人提供）：我们建议回答有关分子相互作用的问题，指导感染性巨细胞病毒形成的最早步骤。我们的重点是开放阅读框UL80a编码的两个遗传相关蛋白，称为组装蛋白前体（pAP）和衣壳成熟蛋白酶前体（pPR）。这些蛋白质与自身和其他蛋白质相互作用，协调新生原衣壳的组装和成熟，这是产生传染性病毒所必需的过程。我们最近克服了一个溶解度问题，该问题使纯化蛋白在体外的工作变得复杂，并将基于试点研究的令人鼓舞的结果，应用这种新方法来寻求线索和测试模型。我们将通过三个具体目标来做到这一点：(i)测试预测磷酸化对pAP和pPR关键相互作用的影响；（ii）验证pAP与pPR自相互作用中“羧基卷曲结构域”的差异优势，并确定其生物学重要性和相关性；（iii）确定pAP和pPR中是否需要cmv特异性的第二核定位信号（NLS2）才能与衣壳门蛋白相互作用（pUL104）。从这项工作中获得的信息将有助于确定在衣壳形成和成熟过程中驱动UL80蛋白结合和解离的变化序列，并有助于开发破坏（例如，用于治疗）或利用（例如，基因/药物传递载体）疱疹病毒组装的新策略。

DESCRIPTION (provided by applicant): We propose to answer questions about molecular interactions guiding the earliest steps in formation of infectious cytomegalovirus. Our focus is on two genetically related proteins encoded by open reading frame UL80a, called the assembly protein precursor (pAP) and the capsid maturational protease precursor (pPR). Together these proteins interact with themselves and with other proteins to coordinate assembly and maturation of the nascent procapsid shell - a process essential to produce infectious virus. We have recently overcome a solubility problem that complicates working with the purified proteins in vitro, and will apply this new approach to pursue leads and test models based on encouraging results from pilot studies. We will do this through three Specific Aims: (i) test predicted effects of phosphorylation on key interactions of pAP and pPR; (ii) verify the differential dominance of the "carboxyl coiled-coil domain" in the self-interaction of pAP versus pPR self-interaction, and determine its biological importance and relevance; and (iii) establish whether a CMV-specific second nuclear localization signal (NLS2) in pAP and pPR is required for their interaction with the capsid portal protein (pUL104). Information gained from this work will help define the sequence of changes that drive UL80 protein associations and dissociations during capsid formation and maturation, and contribute to the longer-term goal of developing new strategies for disrupting (e.g., for therapeutics) or exploiting (e.g., gene/drug delivery vectors) herpesvirus assembly. PUBLIC HEALTH RELEVANCE: Cytomegalovirus is a herpes-group virus that is a threat to people with weakened immune systems, including the very young and the very old. This research seeks to understand how two genetically-related proteins guide the earliest steps in forming infectious virus. Information obtained will relate the molecular interactions driving capsid formation will the biological mechanism of virus formation, and will help identify new ways to interfere therapeutically with that process.