MECHANISTIC STUDIES ON VIRAL ASSEMBLY

本提案的目的是在分子水平上审查 病毒前体组装成感染性病毒的机制 粒子病毒组装的最后步骤之一是包装病毒， 病毒的基因组变成一个保护性的蛋白质外壳，称为衣壳，或头部。 对于所有的DNA包装，已经提出了类似的机制。 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。终止酶是所有酶共同的酶 这些病毒，并负责包装一个单一的基因组， 多联体前体。λ噬菌体已经被广泛地 多年来的研究，代表了一个理想的系统， 病毒DNA包装。因此，我们建议使用噬菌体λ末端酶作为 研究DNA包装和病毒装配的模型酶。 本项目的重点是λ的催化活性 终止酶及其在病毒DNA包装中的作用。这种酶 具有位点特异性内切酶活性，DNA刺激的ATP酶 活性和DNA解旋酶活性，所有这些都协同工作， 影响基因组包装。λ末端酶的DNA包装起始于 将稳定的多蛋白复合物（termasome）组装到 在一些实施方案中，双链体的多联体包装基底和双链体的位点特异性切口可以是多联体包装基底和双链体的位点特异性切口。 在通过酶进行链分离之前或之后立即， 前体与二元蛋白质、DNA中间体和端体结合 从装配现场释放。可能是末端酶转位 由ATP的水解提供动力，DNA被积极地包装到 病毒性前体 本提案中描述的实验系统地探讨了 蛋白质-蛋白质和蛋白质-DNA相互作用需要组装一个稳定 酶。DNA中间体，它使DNA双链体产生切口，随后 使复合体脱离所需的相互作用， 随之而来三种催化活性之间的相互作用， 组装-释放过程以及ATP和ATP水解的作用 在这些功能进行了详细检查。遗传学研究表明 在λ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，以进一步 探讨包装过程的催化机理。生物物理 和动力学技术被用来详细检查每个步骤， 参与基因组包装的启动。 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链，并可能包括组装在真核腺病毒 和疱疹病毒组。与其他DNA操纵机制的相似性 酶如大肠杆菌的recBCD核酸酶/解旋酶。大肠杆菌，限制 核酸内切酶，特别是IIS和III型，以及 在转录起始期间的开放启动子复合物进一步 这表明，了解这种催化剂的性质， 包装机可能会让我们深入了解DNA的一般机制 通过多蛋白酶复合物操纵。

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.