MECHANISTIC STUDIES ON VIRAL ASSEMBLY

这项提议的目的是在分子水平上研究 病毒前体组装成传染性病毒的机制 粒子。病毒组装的最后一步是包装 病毒基因组进入被称为衣壳或头部的保护性蛋白质外壳。 类似的DNA包装机制已经被提出用于所有 双链DNA噬菌体，也可能适用于哺乳动物病毒 如腺病毒和疱疹病毒。终止酶是所有人都共有的酶 并负责从这些病毒中打包单个基因组 连续体的前驱。噬菌体lambda已经被广泛地 经过多年的研究，代表了一种理想的学习系统 病毒DNA包装。因此，我们建议使用噬菌体lambda终止酶作为 用来研究DNA包装和病毒组装的模型酶。 本项目的重点是Lambda的催化活性。 终止酶及其在病毒DNA包装中的作用。这种酶 具有位点特异性核酸内切酶活性，一种DNA刺激的ATPase 活性和DNA解旋酶活性，所有这些都协同工作以 影响基因组包装。Lambda终止酶的DNA包装起始于 稳定的多蛋白复合体(终端酶)在细胞表面的组装 串联包装衬底和位置特定的双面划痕。 在酶分离链之前或之后，空的 Prohead与二元蛋白、DNA中间体和终端酶结合 从装配现场释放。随之而来的可能是终止酶的易位 由三磷酸腺苷的水解提供动力，DNA被主动包装到 病毒式传播。 本方案中描述的实验系统地探索了 蛋白质-蛋白质和蛋白质-DNA相互作用需要组装一个稳定的 酶：破坏DNA双链的DNA中间体，以及随后的 脱离复合体所需的相互作用，以便包装可以 接踵而至。这三种催化活性之间的相互作用 装配释放过程以及三磷酸腺苷和三磷酸腺苷水解酶的作用 在这些函数中都有详细的介绍。遗传学研究已经确定 在lambda的特定方面缺乏的几种突变酶 组装和这些改变的蛋白质将被用作工具来进一步 探索包装过程的催化机理。两者都是生物物理 并利用动力学技术详细检查每个步骤 参与了基因组打包的启动。 虽然机械细节不同，但从这些数据中得出的数据 实验可以用来模拟DNA包装的所有双- 链DNA噬菌体，并可能包括在真核腺病毒中组装 和疱疹病毒组。与其他DNA操作的机械相似之处 如大肠杆菌的RecBCD核酸酶/解旋酶等酶，限制了 内切酶，特别是IIS和III型，以及组装-释放 转录起始过程中的开放启动子复合体 建议对这一催化特性的理解 包装机器可能会深入了解DNA的一般机制 多蛋白酶复合体的操控。

The objective of this proposal is to examine, at the molecular level, the mechanism of assembly of viral precursors into an infectious virus particle. One of the final steps in viral assembly is the packaging of the viral genome into a protective protein coat known as the capsid, or head. Similar mechanisms for DNA packaging have been proposed for all of the double-stranded DNA bacteriophages and may also apply to mammalian viruses such as adenovirus and herpesvirus. Terminases are enzymes common to all of these viruses and are responsible for packaging of a single genome from a concatameric precursor. Bacteriophage lambda has been extensively studied over the years and represents an ideal system in which to study viral DNA packaging. We therefore propose to use phage lambda terminase as a model enzyme with which to study DNA packaging and virus assembly. The present project focuses on the catalytic activities of lambda terminase and their role in the packaging of viral DNA. This enzyme possesses a site-specific endonuclease activity, a DNA-stimulated ATPase activity and a DNA helicase activity, all of which work in concert to effect genome packaging. DNA packaging by lambda terminase initiates with the assembly of a stable multiprotein complex (termasome) onto the concatameric packaging substrate and site-specific nicking of the duplex. Prior to or immediately after strand separation by the enzyme, an empty prohead binds to the binary protein.DNA intermediate and the termasome releases from the assembly site. Translocation of terminase ensues, likely powered by the hydrolysis of ATP, and DNA is actively packaged into the viral prohead. The experiments described in this proposal systematically probe the protein-protein and protein-DNA interactions required to assemble a stable enzyme.DNA intermediate which nicks the DNA duplex, and the subsequent interactions required to disengage the complex so that packaging may ensue. The interplay between the three catalytic activities in the assembly-release processes as well as the role of ATP and ATP hydrolysis in these functions are examined in detail. Genetic studies have identified several mutant enzymes which are deficient in specific aspects of lambda assembly and these altered proteins will be utilized as tools to further probe the catalytic mechanisms of the packaging process. Both biophysical and kinetic techniques are utilized to examine in detail each of the steps involved in the initiation of genome packaging. While the mechanistic details differ, the data derived from these experiments may be used to model DNA packaging by all of the double- stranded DNA phages, and may include assembly in the eucaryotic adenovirus and herpesvirus groups. Mechanistic similarities to other DNA manipulating enzymes such as the recBCD nuclease/helicase of E. coli, the restriction endonucleases, particularly types IIS and III, and the assembly-release of an open-promoter complex during the initiation of transcription further suggest that an understanding of the catalytic properties of this packaging machine may yield insight into the general mechanisms of DNA manipulation by multiprotein enzyme complexes.