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

最近的埃博拉和拉沙热流行突出了人类病毒性出血热对人类健康和发展中国家社会经济状况的巨大影响。沙粒病毒是引起VHF的病毒的最大家族;它们在世界范围内分布，并且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。目前在尼日利亚爆发的最常见的沙粒病毒--拉沙病毒，使得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和确定药物靶点成为当务之急。（OW -西非地方性）和新世界（NW -南美洲地方性）病毒的血清学交叉反应性。沙粒病毒在其天然啮齿动物宿主中引起持续感染，病毒通过直接接触传染性物质或暴露于啮齿动物尿液/粪便传播给人类。死亡率极高;没有疫苗，少数治疗方案无效。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。尽管拥有一个小的基因组，仅编码4种蛋白质，沙粒病毒已开发出策略，以保持高水平的复制和避免宿主免疫反应，这意味着多种病毒和宿主蛋白质的相互作用。迄今为止，很少有宿主细胞蛋白质相互作用已被确定，目前的知识的调节免疫因子仅限于病毒核蛋白（NP）和基质蛋白（Z）和它们的抑制I型干扰素（IFN）的诱导。鉴于这些蛋白质参与病毒生命周期的早期到晚期步骤，它们的免疫抑制功能不太可能仅限于这种机制。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，这些过程对病毒的传播至关重要。为了解开沙粒病毒相互作用组和OW与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的新型相互作用伴侣。开发这些方法将揭示定义致病性差异的新辅因子。NP和Z的突变分析将为实验提供信息，这些实验可以描述致病性和非致病性病毒株使用的差异机制。已鉴定的蛋白质相互作用物的验证将涉及通过在马尔堡合作进行的CRISPR/Cas9敲除和CAT 4病毒测定。与此相关的将是使用结构方法，即X射线晶体学和NMR，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本研究应用于治疗设计的关键。结构信息将定义新的宿主相互作用的分子细节，并将为未来设计有效的治疗方法提供信息。NP和Z非常适合于结构研究，目标1中揭示的相互作用将被用于获得蛋白质复合物的结构。参考文献：1. Zapata，J.C.; Salvato，M.S.（2013）沙粒病毒变异由于主机特异性适应。病毒5，241-278.2. Yun，N.E.;步行者，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