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

项目总结 组装大型双链DNA(DsDNA)病毒的关键步骤是包装一个 基因组通过三磷酸腺苷驱动的马达复合体预先组装成前壳蛋白。在疱疹病毒中 和许多噬菌体一样，包装是由一种终止酶催化的，这种酶利用一种 连体基因组底物。为了做到这一点，终止酶组装成不同的 起始、运动和终止复合体，从基因组中连续地切除个体基因组 合并，并同时将其包装到衣壳中。这需要酶的循环 在稳定的核酸酶和动态的运动中间体之间。虽然我们对此的理解 包装的启动和运动移位广泛，基因组包装的终止 对所有病毒的研究仍然很少，特征也很差，主要是因为定义了实验 系统还没有开发出来。噬菌体是一个例外，在这种情况下严格的生化分析 允许对整个组装途径进行分子解剖。该多PI应用程序提出了 要使用噬菌体来询问终止，最后也是最糟糕的一步 包装路径。讨论了基因组打包的两个基本问题：(I) 移位马达是如何识别基因组结束的，同时也感觉到足够的 长度的DNA已被包装，过渡到位点特异结合的核酸酶复合体，以及 (2)“终末蛋白”如何促进末端成熟和终止酶的排出 核衣壳在不丢失紧密包装的DNA的情况下。我们描述了高度集成和 协同生化、生物物理、单分子和结构方法来表征 这是病毒组装过程中保守和必要的一步，但在很大程度上还没有被研究过。鉴于这一过程 在所有dsdna病毒中都是高度保守的，无论是原核还是真核生物，而且 生物学中起始-转位-终止途径的共性，其结果将具有广泛的意义 对病毒学和细胞生物学的影响。

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.