Administrative Supplement for GM100888

GM100888 行政补充

基本信息

批准号：
9902027
负责人：
Gino Cingolani
金额：
$ 6.73万
依托单位：
THOMAS JEFFERSON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-08-01 至 2021-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9902027
关键词：
ATP phosphohydrolase Adenoviruses Administrative Supplement Affinity Antiviral Agents Architecture Award Bacteriophage P22 Bacteriophages Binding Binding Sites Biochemical Biochemical Reaction Biological Assay Capsid Catalytic Domain Cell Nucleus Chemicals Cleaved cell Complex Cryoelectron Microscopy Crystallography Cytomegalovirus DNA DNA Viruses Data Dependovirus Deuterium Enzymatic Biochemistry Fill-It Funding Genetic Materials Genome Goals Grant HIV-1 Herpesviridae Human Hybrids Hydrogen Importins Individual Knowledge Link Macromolecular Complexes Maps Mass Spectrum Analysis Methods Modernization Molecular Molecular Conformation Molecular Machines Molecular Structure Motor Nature Nuclear Pore Complex Parvovirus Pathogenicity Planet Earth Poxviridae Protein Biochemistry Proteins Protomer Pump RNA Viruses Reaction Research Role Salmonella Phages Site Site-Directed Mutagenesis Structure Surface Tail Techniques Testing Tobacco Mosaic Virus Viral Viral Genome Viral Packaging Virion Virus Virus Replication Work Yeasts base biophysical analysis comparative flexibility human disease in vivo interest macromolecular assembly novel nuclease pathogen single molecule stoichiometry structural biology synthetic antibodies terminase viral DNA

项目摘要

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.

项目概要病毒基因组包装是一种复杂的、非自发的、多步骤的酶促反应，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，通过两种蛋白质（称为大终止酶和小终止酶）的作用充满遗传物质。尽管通常作为单个亚基进行研究，病毒末端酶结合、泵送和切割组装成的病毒 DNA 结构、功能和组成特征较差的大分子复合物。最近的发现一种针对人巨细胞病毒小终止酶亚基 (pUL56) 的有效抗病毒剂人们对病毒包装电机的兴趣进一步加深。在这项资助中，结合了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）与现代生化方法（即构象- 特定的合成 Fab、定点诱变、酵母 1-杂交），我们寻求了解其原理通过对不同 DNA 病毒的马达进行比较分析来控制病毒基因组包装。我们对破译由以下物质形成的大分子组装体的原子结构特别感兴趣包装反应过程中的终止酶亚基以及功能保守的 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重大且不断扩大的知识空白基因组包装的酶学之间的关系，由于单分子的存在而越来越被人们所理解生物物理学研究和催化包装的分子机器。在已开展的工作的基础上在上一个资助周期中，我们寻求： 1.) 阐明病毒传播过程中形成的终止酶组装体的结构基因组包装； 2.) 确定人巨细胞病毒小终止酶的保守结构 (pUL56) 及其与病毒 DNA 和抗病毒药物莱特莫韦的相互作用。