Investigating the structure and function of the Ebola virus transcription factor VP30 in order to develop anti-viral therapeutics

埃博拉病毒（EBOV）是现存的最严重的人类病原体之一，导致毁灭性的出血热，致死率高达70%。西非最近爆发的埃博拉病毒已导致10，000多人死亡，受影响国家的重要基础设施和经济普遍崩溃。这次爆发的范围和持续时间，以及对人类福祉的巨大影响，突出了对EBOV感染的有效暴露前和暴露后治疗的迫切需要。特别令人关注的是EBOV的长期健康影响及其在免疫特权部位如眼睛、睾丸和中枢神经系统中的能力。大多数病毒的持续重新出现是由无症状携带者的传播引起的。需要抗病毒治疗来接近和破坏这些免疫特权部位的病毒。EBOV是一种负链RNA病毒，表达6种蛋白。本研究将研究其中之一，即VP30的结构和功能。VP30是病毒编码的聚合酶复合体的重要组成部分，它是破译EBOV基因中储存的信息从而致病的机器。为了完成这项工作，VP30必须与其他EBOV蛋白质以及组成EBOV基因组的RNA相互作用。我们的目标是使用晶体学确定VP30的三维结构，并使用X射线晶体学和EM确定它如何与其他构建模块相匹配。当我们确定了这些相互作用的界面，我们就可以设计出抑制性化合物来阻止VP30产生这些相互作用，从而阻止它发挥作用。为了研究这些成分在细胞内的联系，我们将利用我们的合作者利物浦大学的Julian Hiscox教授开发的EBOV复制子系统。该系统允许重组能够进行真实复制和转录的活性EBOV复制复合物，但不需要专门的生物遏制。我们将使用共聚焦显微镜首先定位细胞中EBOV复制复合物的组成部分，然后进行超分辨率成像，以更好地确定它们的亚细胞定位，并研究复合物组成部分相互作用的动力学。我们也有可能使用完整细胞的冷冻EM成像来成像这些细胞位置。此外，如果聚合酶复合物的组分可以重组表达和纯化，那么复合物的高分辨率结构将通过冷冻电子显微镜进行研究。实验计划将检验三个假设：第一，VP30通过其表面的特异性结合位点与其他病毒成分进行关键接触。第二，VP30与这些其他成分的相互作用可以被专门设计的分子破坏。第三，这些抑制性化合物在细胞中聚合酶起作用的情况下阻止VP30的功能，提供了一种潜在的抗埃博拉治疗剂。

Ebola virus (EBOV) is one of the most serious human pathogens in existence, responsible fordevastating hemorrhagic fevers with fatality rates of up to 70%. The recent outbreak of EBOV in WestAfrica has resulted in over 10,000 human deaths, as well as widespread collapse of essentialinfrastructure and economies of affected countries. The extent and duration of this outbreak, as wellas the massive impact on human well-being, have highlighted the urgent need for effective pre- andpost exposure therapies for EBOV infection. Particularly concerning is the long term healthimplications of EBOV and its ability to be harboured in immune privileged sites such as the eye, thetestes and the central nervous system. Most of the sustained re-emergence of the virus has beencaused by transmission from asymptomatic carriers. Anti-viral therapy is needed to access anddestroy the virus in these immune privileged sites. EBOV is a negative strand RNA virus, whichexpresses 6 proteins. This studentship will investigate the structure and function of one of these,namely VP30. This work will increase our understanding of the basic molecular processes of theEBOV life cycle as well as provide essential information for development of anti-viral treatments.VP30 is an essential building block of the large virus-encoded polymerase complex, which is amachine needed to decipher the information stored within EBOV genes and thus cause disease. Todo its job, VP30 must interact with other EBOV proteins, and also RNA that makes up the EBOVgenome. Our aims are to determine the three-dimensional structure of VP30 using crystallography,and determine how it fits with these other building blocks using X-ray crystallography and EM. Whenwe identify these interaction interfaces, we can design inhibitory compounds that will prevent VP30from making these interactions and thus prevent it from working. To investigate the association ofthese components within cells, we will make use of an EBOV replicon system developed by ourcollaborator Professor Julian Hiscox at The University of Liverpool. This system allows thereconstitution of active EBOV replication complexes able to perform authentic replication andtranscription, but does not require specialized biological containment. We will use confocalmicroscopy to first locate components of the EBOV replication complex in cells, progressing towardssuper resolution imaging to better define their sub-cellular localisation and to investigate the kineticsof the interactions made by components of the complex. We also have the potential to image thesecellular locations using cryo-EM imaging of intact cells. Furthermore, if components of the polymerasecomplex can be expressed recombinantly and purified, high-resolution structures of the complex willbe pursued by cryo-electron microscopy. The experimental plan will test three hypotheses; Firstly,that VP30 makes critical contacts with other virus components though specific binding sites on itssurface. Secondly, that interaction of VP30 with these other components can be disrupted byspecifically designed molecules. Thirdly, that these inhibitory compounds prevent VP30 function in thecontext of a working polymerase in cells, providing a potential anti-Ebola therapeutic.