Genome packaging in DNA viruses

描述(申请人提供)：基因组包装是包括拖尾双链DNA噬菌体和疱疹病毒在内的大型双链DNA(DsDNA)病毒形态发生的关键步骤，也是组装感染性后代病毒粒子的关键。基因组包装是一种精确协调的分子协同作用，即将来自DNA连锁体的一定量的病毒DNA插入到预先形成的前衣壳中，然后将更多的病毒蛋白结合到衣壳上以保留包装的DNA。这一DNA插入过程是由一个强大的分子机器完成的，该机器由终止酶和门户组成。该入口在衣壳的单个顶端形成一条管道，允许病毒DNA在病毒组装期间进入，在感染期间退出。终止酶复合体包含一个DNA识别亚基和一个催化亚基，前者与病毒DNA特异结合，后者为包装反应提供能量，并将基因组长度的DNA从连接体中切割出来。由于异常的复杂性和缺乏高分辨率的结构数据，这些大型病毒中基因组包装的分子机制还不是很清楚。特别是，这些基因组包装蛋白必须形成高阶分子组装才能发挥作用。门户蛋白形成嵌入衣壳的环状十二聚体。然而，关于终止酶是如何组装的，它是如何与门户组装的，以及门户是如何嵌入衣壳中的，人们知之甚少。我们的目标是通过使用结构方法分析基因组包装蛋白质的组装来了解DNA病毒中基因组包装的分子机制。我们特别感兴趣的一般问题是，许多蛋白质和其他生物分子是如何以多个副本存在的，如何分级地组装成一个复杂的系统，以完成复杂的生物过程。在本提案中，我们试图：(I)阐明完整的感染性细菌病毒的高分辨结构；(Ii)阐明终止酶组成蛋白的结构；(Iii)阐明终止酶DNA识别蛋白与DNA的结合方式；以及(Iv)探索终止酶复合体的体外组装。疱疹病毒是人类重要的病原体，会导致从水痘到各种形式的癌症等各种疾病。这项建议预计将通过确定新的抗病毒目标来控制和预防由疱疹病毒引起的感染和疾病，从而促进公共卫生。 公共卫生相关性：人类疱疹病毒与从水痘到危及生命的癌症等重要人类疾病有关。大多数人类群体中的大多数人都感染了几种疱疹病毒。我们试图研究疱疹病毒和许多其他大型双链DNA病毒在生命周期中的共同分子机制，以寻找新的措施来控制和预防由这些病毒病原体引起的感染和疾病。

DESCRIPTION (provided by applicant): Genome packaging is a key step in morphogenesis of large double-stranded DNA (dsDNA) viruses including tailed double-stranded DNA bacteriophages and herpesviruses, and is essential for assembly of infectious progeny virions. Genome packaging is a precisely coordinated molecular synergy, in which a certain amount of viral DNA from a DNA concatemer is inserted into a preformed procapsid, followed by binding of additional viral proteins to the capsid to retain the packaged DNA. This DNA insertion process is fulfilled by a powerful molecular machine consisting of the terminase and the portal. The portal forms a conduit at a single vertex of the capsid that allows viral DNA to enter during virus assembly and exit during infection. The terminase complex contains a DNA-recognition subunit that specifically binds to the viral DNA, and a catalytic subunit that provides the energy for the packaging reaction and cleaves the genome-length DNA from the concatemer. Molecular mechanisms of genome packaging in these large viruses are not well understood, owing to unusual complexity and lack of high resolution structural data. In particular, these genome-packaging proteins have to form high-order molecular assemblies in order to function. The portal protein forms a ring-like dodecamer embedded in the capsid. However, little is known about how the terminase assembles, how it assembles with the portal, and how the portal is embedded in the capsid. Our goal is to understand molecular mechanisms of genome packaging in DNA viruses by analyzing assemblies of genome-packaging proteins using structural approaches. We are particularly interested in the general question of how numerous proteins and other biological molecules, each present in multiple copies, assemble hierarchically into a sophisticated system to fulfill a complex biological process. In the present proposal, we seek to: (i) elucidate the high resolution structure of a complete, infectious bacterial virus; (ii) elucidate structures of component proteins of the terminase; (iii) elucidate the mode of DNA-binding for a terminase DNA-recognition protein; and (iv) explore the assembly of the terminase complex in vitro. Herpes viruses are important human pathogens that cause diseases ranging from chickenpox to various forms of cancer. This proposal is anticipated to contribute to public health by identification of novel targets of antiviral to control and prevent infection and diseases caused by herpes viruses. PUBLIC HEALTH RELEVANCE: Human herpes viruses are linked to important human diseases ranging from chickenpox to life-threatening cancer. The majority of individuals in most human populations are infected by several herpes viruses. We seek to investigate a common molecular mechanism in life cycles of herpes virus and many other large double- stranded DNA viruses, in search of new measures to control and prevent infection and diseases caused by these viral pathogens.