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.

项目概要组装大型双链 DNA (dsDNA) 病毒的关键步骤是包装由 ATP 驱动的运动复合体将基因组转化为预组装的衣壳。和许多噬菌体一样，包装是由末端酶催化的，该酶利用为了实现这一点，末端酶组装成不同的基因组底物。启动、运动和终止复合体，以逐步从基因组中切除单个基因组串联体，并同时将其包装到衣壳中，这需要酶循环。稳定核酸酶和动态运动中间体之间的关系。包装起始和运动易位广泛，基因组包装终止在所有病毒中仍然没有得到充分的研究和表征，主要是因为定义的实验噬菌体系统尚未开发出来，严格的生化测定是一个例外。允许对整个组装途径进行分子解剖。使用噬菌体来询问终止，这是终止过程中最后也是最不明确的步骤包装途径。解决了基因组包装的两个基本问题；易位马达如何识别基因组末端，同时也感知到足够的 DNA 长度已被包装，转变为位点特异性结合的核酸酶复合物，并且 (ii)“整理蛋白”如何促进末端成熟和终止酶从我们描述了高度集成且不丢失紧密包装的 DNA 的核衣壳。协同生化、生物物理、单分子和结构方法来表征鉴于此过程，这是病毒组装过程中保守且重要但基本上未被研究的步骤。在所有 dsDNA 病毒（原核病毒和真核病毒）中都高度保守，并且生物学中起始-易位-终止途径的共性，结果将具有广泛的意义对病毒学和细胞生物学的影响。