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、多步酶促反应， 噬菌体和疱疹病毒通过形成空的前体衣壳（或原衣壳）来进行， 在两种蛋白质的作用下充满了遗传物质，这两种蛋白质被称为大小末端酶。虽然 病毒末端酶通常作为单独的亚基进行研究，它们结合、泵送和切割组装成 结构、功能和组成特征不明确的大分子复合物。近期 一种对人巨细胞病毒小末端酶亚单位（pUL 56）特异的有效抗病毒剂的发现， 对病毒包装发动机产生了更大的兴趣。 在这项资助中，结合结构生物学中的混合方法（即X晶体学，冷冻电子显微镜， 氢/氘交换质谱法）与现代生物化学方法（即构象- 特异性合成Fab，定点诱变，酵母单杂交），我们试图了解这些原理 通过对来自不同DNA病毒的马达的比较分析来控制病毒基因组包装。我们 特别感兴趣的是破译大分子组装体的原子结构， 包装反应中的S-末端酶亚基和功能保守的S-末端酶的作用 从细菌病毒到疱疹病毒这项研究试图填补一个重大的和不断增长的知识差距 基因组包装的酶学，这是越来越好地理解感谢单分子， 生物物理研究和催化包装的分子机器。在2004年开始的工作的基础上， 上一个融资周期，我们寻求：1。阐明了病毒感染过程中形成的末端酶组装体的结构 基因组包装; 2.）人巨细胞病毒小端酶保守结构的确定 （pUL 56）及其与病毒DNA和抗病毒药物莱特莫韦的相互作用。

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.