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

项目摘要 大的双链DNA（dsDNA）病毒组装的关键步骤是包装一个 通过ATP驱动的马达复合体将基因组组装成预组装的原衣壳。在疱疹病毒中 和许多噬菌体一样，包装是由一种末端酶催化的， 多联体基因组底物。为了实现这一点，末端酶组装成不同的 起始、马达和终止复合体，以从细胞中切除个体基因组， 串联体，并伴随地将其包装到衣壳中。这需要酶循环 在稳定的核酸酶和动态的马达中间体之间。尽管我们对 包装起始和运动易位广泛，基因组包装终止 在所有病毒中仍然缺乏研究和表征，主要是因为定义的实验 系统尚未开发。噬菌体抗体是一个例外，其中严格的生化测定 允许对整个组装途径进行分子解剖。这个多PI应用程序提出 使用噬菌体噬菌体抗体来询问终止，这是最后一步，也是最缺乏特征的一步。 包装途径。解决基因组包装的两个基本问题;（i） 易位马达是如何识别基因组末端的，同时又感知到足够的 一段DNA已被包装，转变为位点特异性结合的核酸酶复合物， (ii)“精整蛋白”如何促进末端成熟和末端酶从细胞中排出？ 核衣壳而不丢失紧密包装的DNA。我们描述了高度集成和 协同生物化学、生物物理、单分子和结构方法来表征 这是病毒装配中保守而重要的步骤，但在很大程度上未被研究。鉴于这一过程 在所有的dsDNA病毒中是高度保守的，包括原核和真核的， 生物学中起始-易位-终止途径的共同性，结果将具有广泛的 在病毒学和细胞生物学中的意义。

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.