Structure and Function of Essential Nucleoprotein Complexes Along a Viral Genome Packaging Pathway

病毒基因组包装途径中必需核蛋白复合物的结构和功能

基本信息

批准号：
10660775
负责人：
Carlos Enrique Catalano
金额：
$ 45.17万
依托单位：
UNIVERSITY OF COLORADO DENVER
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-05-01 至 2027-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10660775
关键词：
ATP Hydrolysis Address Antibiotic Resistance Antiviral Agents Bacteriophages Binding Biochemical Biological Assay Biology Biophysics Capsid Catalytic Domain Cell physiology Cellular biology Complement Complex DNA DNA Packaging DNA biosynthesis Dissection Double Stranded DNA Virus Ecology Enzyme Kinetics Enzymes Event Evolution Genetic Transcription Genome Health Herpesviridae Human Human Microbiome Individual Length Mediating Modeling Molecular Molecular Conformation Monitor Morbidity - disease rate Motor Nucleocapsid Nucleoproteins Nucleotides Pathway interactions Play Process Protein Biosynthesis Proteins Reaction Research Research Design Role Series Signal Transduction Site Structure System Tail Testing Viral Genome Viral Packaging Virus Virus Assembly biophysical techniques density design experimental study molecular dynamics mortality novel nuclease sensor single molecule terminase translocase viral DNA virology virus development

项目摘要

PROJECT SUMMARY A key step in the assembly of the large double-stranded DNA (dsDNA) viruses is packaging of a genome into a pre-assembled procapsid by an ATP-driven motor complex. In the herpesviruses and many bacteriophages, packaging is catalyzed by a terminase enzyme that utilizes a concatemeric genome substrate. To accomplish this, terminase enzymes assemble into distinct initiation, motor and termination complexes to processively excise an individual genome from the concatemer, and concomitantly package it into the capsid. This requires that the enzymes cycle between stable nuclease and dynamic motor intermediates. While our understanding of packaging initiation and motor translocation is extensive, termination of genome packaging remains ill-studied and poorly characterized in all viruses, primarily because defined experimental systems have not been developed. Phage  is an exception wherein rigorous biochemical assays allow molecular dissection of the entire assembly pathway. This multi-PI application proposes to use phage  to interrogate termination, the final and most poorly characterized step in the packaging pathway. Two fundamental questions central to genome packaging are addressed; (i) how does the translocating motor recognize the genome end while also sensing that a sufficient length of DNA has been packaged, transition to a site-specifically bound nuclease complex, and (ii) how do “finishing proteins” promote end maturation and terminase ejection from the nucleocapsid without loss of the tightly packaged DNA. We describe highly integrated and synergistic biochemical, biophysical, single-molecule and structural approaches to characterize this conserved and essential, yet largely unstudied step in virus assembly. Given that this process is strongly conserved in all of the dsDNA viruses, both prokaryotic and eukaryotic, and the commonality of initiation-translocation-termination pathways in biology, the results will have broad implications in virology and cell biology.

项目摘要大型双链DNA（DSDNA）病毒组装的关键步骤是包装通过ATP驱动的电动机复合物进入预组装的Procapsid。在疱疹病毒中和许多细菌噬细胞，包装是由利用A的末端酶催化的串联基因组底物。为此，末端酶聚集成不同的开始，运动和终止复合物从辅助器，并同时将其包装到帽子中。这需要酶循环在稳定的核酸酶和动态运动中间体之间。而我们对包装计划和运动易位是广泛的，基因组包装的终止在所有病毒中保持不当研究，并且在所有病毒中的特征都很差，主要是因为定义的实验系统尚未开发。噬菌体是一个严格的生化测定的例外允许整个组件途径的分子解剖。此多PI应用程序建议要使用噬菌体询问终止，最终和最不良表征的步骤包装途径。解决了基因组包装中心的两个基本问题；（我）易位电动机如何识别基因组末端，同时也感觉到足够 DNA的长度已包装，过渡到特定于特定结合的核酸酶复合物，并且（ii）“完成蛋白质”如何促进终端成熟和末端酶弹出核素无丢失，不丢失紧密包装的DNA。我们描述了高度整合的协同生化，生物物理，单分子和结构方法的表征在病毒组装中，这一保守，必不可少的但在很大程度上未被研究的步骤。鉴于这个过程在原核生物和真核的所有DSDNA病毒中都很坚强，启动 - 转换 - 终止途径在生物学中的共同点，结果将具有广泛的病毒学和细胞生物学的影响。