Functional characterisation of the molecular interactome of emerging viral haemorrhagic fever arenaviruses

最近的埃博拉和拉沙热流行凸显了人类病毒性出血热对人类健康和发展中世界社会经济地位的巨大影响。沙粒病毒是引起甚高频病毒的最大家族；它们分布在世界各地，在南美洲和西非流行，主要在塞拉利昂、利比里亚和尼日利亚。目前最常见的沙病毒拉沙病毒在尼日利亚暴发，这使得必须了解病毒发病机制和免疫逃避的分子基础，确定驱动病毒出现的因素，并确定药物靶点。根据其系统发育、地理分布和血清学交叉反应性，沙粒病毒科可分为旧大陆病毒（西非流行的OW病毒）和新世界病毒（南美洲流行的NW病毒）。沙粒病毒在其天然啮齿动物宿主中引起持续感染，并通过直接接触传染性物质或接触啮齿动物的尿液/粪便将病毒传播给人类。死亡率极高；目前还没有疫苗，仅有的几种治疗方法也无效。总体目标是绘制负责增强和拮抗沙粒病毒感染的复杂途径，重要的是，探索宿主适应的分子基础。尽管沙粒病毒的基因组很小，只编码4种蛋白质，但它已经发展出了维持高水平复制和避免宿主免疫反应的策略，这意味着多种病毒和宿主蛋白质相互作用。到目前为止，很少有宿主细胞蛋白相互作用被确定。目前对免疫因子调节的了解仅限于病毒核蛋白（NP）和基质蛋白(Z)及其对I型干扰素（IFN）诱导的抑制。鉴于这些蛋白参与病毒生命周期的早期到晚期，它们的免疫抑制功能不太可能仅限于这一机制。此外，它们具有的结构特征可能是它们调节不同病毒过程的能力的基础，这些过程对病毒传播至关重要。为了揭示沙粒病毒相互作用组以及OW和NW病毒之间的差异机制，学生将致力于：1)开发蛋白质组学筛选，以鉴定NP和z的新相互作用伙伴。开发这些方法将揭示定义致病性差异的新辅助因子。NP和Z的突变分析将为实验提供信息，可以描述致病性和非致病性病毒株使用的不同机制。鉴定的蛋白质相互作用物的验证将涉及通过马尔堡合作进行的CRISPR/Cas9敲除和CAT4病毒分析。与此相关的将是使用结构方法，即x射线晶体学和核磁共振，以2)阐明NP和Z与宿主因子相互作用的分子细节；利用这些相互作用是将本研究应用于治疗方法设计的关键。结构信息将定义新的宿主相互作用的分子细节，并将为未来有效治疗的设计提供信息。NP和Z非常适合进行结构研究，Aim 1中揭示的相互作用将被用来获得蛋白质复合物结构。了解更多信息的参考资料：萨帕塔,J.C.;Salvato， M.S.（2013）宿主特异性适应导致的沙粒病毒变异。病毒5,241 -278.2。Yun, N.E.;Walker， D.H.（2012）拉沙热的发病机制。病毒4,2031-2048

The recent Ebola and Lassa fever epidemics highlight the immense impact human viral haemorrhagic fevers (VHFs) have on human health and on the socio-economic status of the developing world. Arenaviruses are the largest family of VHF-causing viruses; they have worldwide distribution and are endemic in South America and West Africa, mainly in Sierra Leone, Liberia and Nigeria. The current outbreak by the most common arenavirus, Lassa, in Nigeria, makes it imperative to understand the molecular basis of viral pathogenesis and immune evasion, to identify factors that drive viral emergence and to identify drug targets.The Arenaviridae family is divided into Old World (OW - endemic in West Africa) and New World (NW - endemic in South America) viruses based on their phylogeny, geographical distribution and serological cross reactivity. Arenaviruses cause persistent infections in their natural rodent hosts and viral transmission to humans occurs through direct contact with infectious materials or exposure to rodent urine/faeces. Fatality rates are extremely high; there is no vaccine and the few therapeutic options are ineffective. The overarching aim is to map the complex pathways that are responsible for potentiating and antagonising arenavirus infection and, importantly, explore the molecular basis of host adaptation.Despite possessing a small genome and encoding for only 4 proteins, arenaviruses have developed strategies to maintain high levels of replication and avoid host immune responses, implying multiple viral and host protein interactions. Thus far, few host cell protein interactions have been identified.Current knowledge of the modulation of immune factors is restricted to the viral nucleoprotein (NP) and matrix protein (Z) and their inhibition of type I interferon (IFN) induction. Given, the involvement of these proteins in early to late steps of the viral life cycle, it is unlikely that their immunosuppressive function is exclusively limited to this mechanism. Furthermore, they possess structural characteristics that could be fundamental to their ability to modulate different viral processes central to viral spread.In order to unravel the arenavirus interactome and the differential mechanisms between OW and NW viruses, the student will aim to:1) develop proteomic screens for the identification of novel interacting partners of NP and Z. Developing these methods will reveal novel co-factors that define pathogenicity differences. Mutational analysis of NP and Z will inform experiments that can delineate differential mechanisms used by pathogenic and non-pathogenic viral strains. Validation of identified protein interactors will involve CRISPR/Cas9 knockout and CAT4 virus assays conducted via collaboration in Marburg. Linked to this will be the use of structural methods, i.e. X-ray crystallography and NMR, to 2) elucidate the molecular details of the interactions of NP and Z with host factors; exploiting these interactions is key in the application of this research to the design of therapeutics. Structural information will define the molecular details of novel host interactions and will inform the future design of effective therapeutics. NP and Z are highly amenable for structural studies and interactions revealed in Aim 1 will be exploited to obtain protein complex structures.References to learn more:1. Zapata, J.C.; Salvato, M.S. (2013) Arenavirus Variations Due to Host-Specific Adaptation. Viruses 5, 241-278.2. Yun, N.E.; Walker, D.H. (2012) Pathogenesis of Lassa Fever. Viruses 4, 2031-2048