Hacking at the cellular level; How do viruses subvert intracellular networks for viral RNA genome trafficking within infected cells?

奈罗病毒是一组昆虫传播的RNA病毒，其中包括极端人类病原体克里米亚-刚果出血热病毒（CCHFV），该病毒被世卫组织“研究与发展蓝图”列为重点病原体。目前没有疫苗或疗法可用于预防或治疗CCHFV疾病，这是一个迫切的未得到满足的需求。奈罗病毒是一种被包膜的病毒，内化到核内体内的细胞中，并在核内体逃逸后，其RNA基因组转运到特定的目的地。这种转运不是通过随机扩散发生的——相反，纳米病毒破坏细胞过程，将它们的基因组移动到一个被称为病毒工厂的位置。顾名思义，这个工厂是剧烈合成代谢活动的场所，在这里病毒成分被大量生产，注定要组装成新的病毒。病毒工厂的形成是一个多步骤的过程，对感染和疾病至关重要。然而，目前还没有关于奈罗病毒基因组如何转移到工厂位点或工厂结构的详细信息。该项目将填补这一知识空白，为未来的抗病毒治疗提供潜在的细胞和病毒靶点，并提供关键宿主-病原体相互作用的详细信息。利用密切相关但非致病性的哈扎拉奈罗病毒（HAZV）作为CCHFV的模型，该项目将确定被奈罗病毒劫持的细胞途径和成分，使它们能够从核内体到达病毒工厂，并确定工厂本身的位置和超微结构。为了实现这些目标，我们将首先使用反向遗传学在HAZV RNA基因组中引入位点特异性突变，以开发带有遗传编码荧光和表位标签的工程感染性HAZV变异的“工具包”，这将允许可视化和跟踪细胞内进入途径上的所有HAZV RNA和蛋白质成分。接下来，利用我们之前生成的基于RNAi和crispr的基因敲除/敲除筛选数据（Fuller等人，2020），我们将通过可视化由于基因消融或使用特定抑制剂或显性阴性表达而导致的蛋白质功能中断的细胞中受损的RNA基因组转运来确定细胞因子的参与。最后，我们将使用电子显微镜观察切片细胞，使用最先进的低温聚焦离子束，以及高分辨率光学显微镜技术，如STED，来可视化病毒工厂组织。我们最近首次使用光学和电子显微镜对汉坦病毒复制工厂的位置和内容进行了表征（Davies et al, 2020），这里将采用类似的技术。

Nairoviruses are a group of insect-borne RNA viruses that include the extreme human pathogen Crimean-Congo haemorrhagic fever virus (CCHFV), which is listed as a priority pathogen in the WHO 'Research and Development Blueprint'. No vaccines or therapies are currently available to prevent or treat CCHFV disease, and this represents an urgent unmet need. Nairoviruses are enveloped viruses that internalise into cells within endosomes, and following endosome-escape, their RNA genomes transit to a specific destination. This transit does not occur by random diffusion - instead nairoviruses subvert cellular process to move their genomes to a site known as a viral factory. As its name suggests, the factory is the site of intense anabolic activity, where viral components are mass-produced, destined for assembly into new viruses. Formation of the virus factory is a multi-step process, critical for infection and disease. However, no detailed information of how the nairovirus genome transits to the factory site, or factory structure, is currently available. This project will fill this knowledge gap, providing potential cellular and viral targets for future anti-viral therapies, as well as providing detailed information of critical host-pathogen interactions.Using the closely related yet non-pathogenic Hazara nairovirus (HAZV) as a model for CCHFV, this project will identify cellular pathways and components that are hijacked by nairoviruses to allow their journey from the endosome to the virus factory, as well as determine the location and ultrastructure of the factory itself. To achieve these aims, we will first use reverse genetics to introduce site-specific mutations within the HAZV RNA genome to develop a 'tool-kit' of engineered infectious HAZV variants bearing genetically encoded fluorescent and epitope tags, which will allow visualization and tracking of all HAZV RNA and protein components within cells on the entry pathway. Next, using our previously generated RNAi and CRISPR-based gene knock-down/knock-out screen data (Fuller et al, 2020), we will determine cell factor involvement by visualizing impaired RNA genome transit in cells with disrupted protein function, resulting from genetic ablation or use of specific inhibitors or expression of dominant-negatives. Finally, we will visualize virus factory organisation using electron microscopy observation of sectioned cells, using state-of-the-art cryo-focused ion beam, alongside high-resolution light microscopy techniques such as STED. We recently characterised for the first time the location and content of the hantavirus replication factory using light and electron microscopy (Davies et al, 2020) and similar techniques will be adopted here.