Structural and mechanistic characterisation of DNA packaging motors from human Cytomegalovirus and related viruses

在许多传染性病毒的组装过程中，如巨细胞病毒、爱泼斯坦-巴尔病毒和其他疱疹病毒，DNA包装马达将病毒基因组插入到保护性蛋白衣壳中。大终末酶(LT)蛋白是发动机的关键组成部分，代表着药物开发的一个可行的靶点。这个博士项目将结合蛋白质工程、结构生物学(X射线和低温EM)、生物物理学和生物化学来定义这些大分子机器的组装状态、结构和功能。通过设计一个多用途的体外系统来研究大型双链DNA病毒的DNA包装过程，我们将解决BBSRC促进世界级支撑生物科学的任务，因为我们剖析了ATP驱动的DNA易位的机制和支配病毒衣壳内DNA紧密包装的物理原理。此外，该系统将允许有效地筛选潜在的DNA易位抑制剂的化合物文库，并将其包装为抗病毒药物发现的一种方法。这个博士项目是与一个工业合作伙伴合作的，将包括为Inspiralis有限公司的学生安排为期3个月的实习，在那里将使用Inspiralis提供的ATPase和解旋酶活性分析方面的专业知识，对野生型和突变蛋白进行几种功能生化分析。学生将从学术/行业伙伴关系的培训中受益，该合作伙伴关系提供了一个平台，以发展为“高技能研究人员”，而研究本身将有助于英国“在细胞、分子和结构生物学等核心支撑学科方面的实力”，同时建立“使研究人员能够突破界限的新工具和技术”。目标(I)定义突变的LT蛋白质和化学抑制剂，它们将增强我们的机制理解，并产生稳定的核酸酶和ATPase反应中间体复合体；(Ii)使用生物物理、生化和高分辨率结构方法表征这些复合体；(Iii)利用这些信息来模拟反应机制，设计新的突变体和/或抑制剂，并分析体内扰动的后果。新奇的是，LT蛋白如何与DNA相互作用，其ATPase和核酸酶活性如何开启和关闭，以及小分子如何抑制这些都是未知的。这项研究有望产生一个反应机制的高分辨率结构快照的文库。时间最近的进展有助于进一步了解反应机制和开发具有RNaseH-like和ATPase折叠的相关酶的新型抑制剂。将这些信息应用于病毒LT蛋白对于开发对抗疱疹病毒引起的感染的新方法是非常有价值的。实验方法本项目将使用巨细胞病毒、Epstein Barr和其他疱疹病毒的LT蛋白。如果证明疱疹病毒LT蛋白对结构和机械特性太具挑战性(即溶解性或稳定性问题)，该项目将重新专注于来自中嗜性细菌病毒SPP1和HK97的易处理的同源LT蛋白，安生实验室已经为这两种病毒建立了纯化程序。目标将通过生化、生物物理和结构方法的组合来解决。突变的LT蛋白移位和切割DNA的能力将通过建立的体外和体内检测来监测。

During the assembly of many infectious viruses, such as Cytomegalovirus, Epstein-Barr virus and other herpes viruses, a DNA packaging motor inserts the viral genome into a protective protein capsid. The large terminase (LT) protein is a key component of the motor, representing a viable target for drug development. This PhD project will use a combination of protein engineering, structural biology (both X-ray and Cryo-EM), biophysics and biochemistry to define the assembly state, structure and function of these large molecular machines. By engineering a multipurpose in vitro system in which to study the DNA packaging processes of large dsDNA viruses, we will address the BBSRC remit for promoting World Class Underpinning Bioscience as we dissect the mechanism of ATP-powered DNA translocation and the physical principles that govern the tight packaging of DNA within the viral capsid. In addition, this system will allow efficient screening of compound libraries for potential inhibitors of DNA translocation and packaging as a method of anti-viral drug discovery. This PhD project is a collaboration with an industrial partner and will include a 3-month placement for the student at Inspiralis Ltd where several functional biochemical assays will be carried out with wild type and mutant proteins, using expertise in ATPase and helicase activity assays available at Inspiralis. The student will benefit from training from an academic / industry partnership providing a platform to develop as a "highly skilled researcher" while the research itself will contribute to UK "strength in core underpinning disciplines such as cellular, molecular and structural biology" whilst building "new tools and technologies that enable researchers to push the boundaries".Objectives(i) define mutant LT proteins and chemical inhibitors that will enhance our mechanistic understanding and produce stable complexes of nuclease and ATPase reaction intermediates; (ii) characterize these complexes using biophysical, biochemical and high-resolution structural approaches; (iii) use this information to model reaction mechanism and design new mutants and/or inhibitors and to assay the consequences of perturbation in vivo.NoveltyIt is unknown how LT protein interacts with DNA, how its ATPase and nuclease activities are switched on and off, and how these could be inhibited by small molecules. The research is expected to produce a library of high-resolution structural 'snapshots' of the reaction mechanism.TimelinessRecent advances have contributed to further understanding of the reaction mechanisms and novel inhibitor development in related enzymes with RNAse H-like and ATPase folds. Application of this information to viral LT proteins is invaluable for the development of novel approaches for fighting infections caused by herpesviruses.Experimental ApproachesThis project will use LT protein from cytomegalovirus, Epstein Barr, and other herpes viruses. If herpes LT proteins prove too challenging for structural and mechanistic stadues (i.e. solubility or stability issues) the project will refocus on tractable homologous LT proteins from mesophilic bacterial viruses SPP1 and HK97, for which purification procedure has already been established in Antson lab. Objectives will be addressed via a combination of biochemical, biophysical, and structural approaches. The ability of mutant LT proteins to translocate and cut DNA will be monitored using established in vitro and in vivo assays.