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

研究埃博拉病毒转录因子 VP30 的结构和功能以开发抗病毒疗法

• 批准号: 1789828
• 金额: --
• 依托单位: University of Leeds
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2016
• 资助国家: 英国
• 起止时间: 2016 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-1789828
• 关键词: Investigating; structure; function; Ebola; virus

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.

埃博拉病毒 (EBOV) 是现存最严重的人类病原体之一，可引起毁灭性的出血热，死亡率高达 70%。最近在西非爆发的埃博拉病毒已导致 10,000 多人死亡，受影响国家的基本基础设施和经济普遍崩溃。这次疫情的范围和持续时间，以及对人类福祉的巨大影响，凸显了对埃博拉病毒感染有效的暴露前和暴露后治疗的迫切需要。特别令人担忧的是埃博拉病毒对健康的长期影响及其在眼睛、睾丸和中枢神经系统等免疫特权部位的能力。病毒的持续重新出现大部分是由无症状携带者传播造成的。需要抗病毒治疗来进入并消灭这些免疫特权部位的病毒。 EBOV 是一种负链 RNA 病毒，表达 6 种蛋白质。该奖学金将研究其中之一的结构和功能，即 VP30。这项工作将增进我们对埃博拉病毒生命周期基本分子过程的理解，并为抗病毒治疗的开发提供重要信息。VP30是大型病毒编码聚合酶复合物的重要组成部分，而聚合酶复合物是破译存储在埃博拉病毒基因内的信息并从而导致疾病所需的机器。为了完成其工作，VP30 必须与其他 EBOV 蛋白以及构成 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块相匹配。当我们识别出这些相互作用界面时，我们可以设计抑制性化合物来阻止 VP30 进行这些相互作用，从而阻止其发挥作用。为了研究细胞内这些成分的关联，我们将利用由利物浦大学的合作者 Julian Hiscox 教授开发的 EBOV 复制子系统。该系统允许重建活性埃博拉病毒复制复合物，能够执行真实的复制和转录，但不需要专门的生物防护。我们将使用共焦显微镜首先定位细胞中埃博拉病毒复制复合物的成分，然后进行超分辨率成像，以更好地定义它们的亚细胞定位，并研究复合物成分相互作用的动力学。我们还有可能使用完整细胞的冷冻电镜成像来对这些细胞位置进行成像。此外，如果聚合酶复合物的成分可以重组表达和纯化，则可以通过冷冻电子显微镜来追踪复合物的高分辨率结构。实验计划将检验三个假设；首先，VP30通过其表面的特定结合位点与其他病毒成分进行关键接触。其次，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，这些抑制性化合物可阻止 VP30 在细胞内聚合酶的作用下发挥作用，从而提供潜在的抗埃博拉治疗方法。

项目成果

Structure and Function of the Human Respiratory Syncytial Virus M2-1 Protein.

人类呼吸道合胞病毒 M2-1 蛋白的结构和功能。

• DOI: 10.1007/978-981-10-8456-0_11
• 发表时间: 2018
• 期刊: Sub-cellular biochemistry
• 作者: Muniyandi S

The Structure of the Human Respiratory Syncytial Virus M2-1 Protein Bound to the Interaction Domain of the Phosphoprotein P Defines the Orientation of the Complex.

• DOI: 10.1128/mbio.01554-18
• 发表时间: 2018-11-13
• 期刊: mBio
• 影响因子: 6.4
• 作者: Selvaraj M;Yegambaram K;Todd EJAA;Richard CA;Dods RL;Pangratiou GM;Trinh CH;Moul SL;Murphy JC;Mankouri J;Éléouët JF;Barr JN;Edwards TA
• 通讯作者: Edwards TA