Administrative Supplement for GM100888

项目摘要 病毒基因组包装是一个复杂的，非自发的，多步骤的酶反应，在尾随 噬菌体和疱疹病毒通过形成空的前体衣壳(或前衣壳)而进行 通过两种蛋白质的作用充满遗传物质，称为大终末酶和小终末酶。尽管 通常作为单个亚基研究，病毒末端结合、泵和裂解组装成病毒DNA 结构、功能和组成特征不佳的大分子复合体。最近的 人巨细胞病毒小末端酶亚基(PUL56)特异性抗病毒药物的发现 对病毒包装马达产生了进一步的兴趣。 在这项赠款中，结合了结构生物学的混合方法(即X-结晶学、冷冻电子显微镜、 氢/氢交换质谱学)与现代生物化学方法(即构象- 特定的合成纤维、定点突变、酵母1-杂交)，我们试图理解其原理 通过比较分析来自不同DNA病毒的发动机来管理病毒基因组包装。我们 对破译大分子集合体的原子结构特别感兴趣 包装反应中的终止酶亚基及功能保守的S终止酶的作用 从细菌病毒到疱疹病毒。这项研究试图填补一个显著的、不断扩大的知识空白 在基因组包装的酶学之间，由于单分子的存在，人们对基因组包装的了解越来越多 生物物理学研究，以及催化包装的分子机器。建立在 在上一个资金周期，我们寻求：1.)阐明病毒过程中形成的终止酶组件的结构 基因组包装；2)人巨细胞病毒小末端酶保守结构的确定 (PUL56)及其与病毒DNA和抗病毒药物letermovir的相互作用。

Project Summary Viral genome packaging is a complex, non-spontaneous, multi-step enzymatic reaction that in tailed bacteriophages and herpesviruses proceeds via formation of an empty precursor capsid (or procapsid) that is filled with genetic material by the action of two proteins, known as large and small terminase. Though commonly studied as individual subunits, viral terminases bind, pump and cleave viral DNA assembled into large macromolecular complexes of poorly characterized structure, function and composition. The recent discovery of a potent antiviral agent specific to Human Cytomegalovirus small terminase subunit (pUL56) has grown further interest in viral packaging motors. In this grant, combining hybrid methods in structural biology (i.e. X-crystallography, cryo-electron microscopy, hydrogen/deuterium exchange mass spectrometry) with modern biochemical approaches (i.e. conformation- specific synthetic Fabs, site directed mutagenesis, yeast 1-hybrid), we seek to understand the principles governing viral genome packaging through the comparative analysis of motors from different DNA viruses. We are particularly interested in deciphering the atomic structure of macromolecular assemblies formed by terminase subunits during the packaging reaction and the role of S-terminase that is functionally conserved from bacterial viruses to herpesviruses. This research tries to fill a significant and growing knowledge gap between the enzymology of genome packaging, which is increasing well-understood thanks to single molecule biophysical studies, and the molecular machines catalyzing packaging. Building upon the work initiated in the previous funding cycle, we seek to: 1.) Elucidate the architecture of terminase assemblies formed during viral genome packaging; 2.) Determine the conserved architecture of Human Cytomegalovirus small terminase (pUL56) and its interaction with viral DNA and the antiviral drug letermovir.