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

在许多传染性病毒，如巨细胞病毒、爱泼斯坦-巴尔病毒和其他疱疹病毒的组装过程中，DNA包装马达将病毒基因组插入保护性蛋白质衣壳中。大端酶（LT）蛋白是马达的关键组成部分，代表了药物开发的可行靶点。该博士项目将结合蛋白质工程、结构生物学（包括x射线和冷冻电镜）、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dsDNA病毒的DNA包装过程，我们将在分析atp驱动的DNA易位机制和控制病毒衣壳内DNA紧密包装的物理原理的同时，解决BBSRC促进世界级基础生物科学的职责。此外，该系统将允许有效筛选潜在的DNA易位抑制剂和包装化合物文库，作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，将包括学生在Inspiralis有限公司为期3个月的实习，利用Inspiralis在atp酶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性生化分析。学生将受益于学术/行业合作伙伴关系的培训，该合作伙伴关系为培养“高技能研究人员”提供了一个平台，而研究本身将为英国“在细胞、分子和结构生物学等核心基础学科的实力”做出贡献，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LT蛋白和化学抑制剂，这将增强我们对机制的理解，并产生稳定的核酸酶和三磷酸腺苷酶反应中间体复合物；（ii）利用生物物理、生化和高分辨率结构方法表征这些复合物；（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。目前尚不清楚LT蛋白如何与DNA相互作用，其atp酶和核酸酶活性如何开启和关闭，以及这些如何被小分子抑制。这项研究有望产生一个反应机制的高分辨率结构“快照”库。最近的进展有助于进一步了解与RNAse H-like和atp酶折叠相关的酶的反应机制和新的抑制剂的开发。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宝贵的。实验方法本项目将使用巨细胞病毒、eb病毒和其他疱疹病毒的LT蛋白。如果证明疱疹LT蛋白在结构和机制方面具有挑战性（即溶解度或稳定性问题），该项目将重新关注来自嗜中温细菌病毒SPP1和HK97的可处理同源LT蛋白，其纯化程序已经在Antson实验室建立。目标将通过生物化学、生物物理和结构方法的结合来解决。突变LT蛋白转运和切割DNA的能力将通过体外和体内试验进行监测。

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.