How does Chikungunya virus regulate the switch between genome translation and replication?

基孔肯雅病毒（CHIKV）是一种虫媒病毒，由埃及伊蚊和白纹伊蚊传播。自2004年重新出现以来，它在非洲、亚洲和美洲的亚热带地区和越来越多的欧洲、亚洲和北美的温带地区造成了许多流行病，地理范围不断扩大。CHIKV传播的主要风险因素是全球蚊子媒介分布的变化——这是气候变化相关的季节变化和平均气温上升、全球化以及土地利用变化的结果。伊蚊的入侵种群。白纹伊蚊已经在英国被发现，气温上升预计会增加它们的活动时间和活动范围。基孔肯雅病与持续数月或数年的严重衰弱性关节疼痛和合并症的恶化有关。在患有高血压或糖尿病等疾病的老年患者中，死亡率很高。尽管流行病的数量不断增加，地理范围不断扩大，但仍然没有特定的抗病毒治疗方法或获得许可的疫苗。CHIKV控制其基因组翻译和复制的机制和相互作用尚不清楚。我们已经证明，CHIKV需要宿主蛋白MSI2才能在人类细胞中复制其基因组。我们和其他人也表明宿主蛋白DHX9是有效的CHIKV翻译所必需的，同时抑制病毒基因组的复制。我们证明了MSI2和DHX9与CHIKV基因组的5'区特异性相互作用，并在该病毒基因组区域内鉴定了MSI2 RNA结合位点。我们之前发表的一项研究表明，病毒基因组的5'区形成了复杂的RNA结构，这是CHIKV复制所必需的。我们还获得了与这些调节MSI2与CHIKV基因组结合的RNA结构一致的初步数据。因此，该项目的科学目标是建立这些不同因素如何相互作用的详细功能和结构模型，以调节CHIKV翻译和基因组复制。我们将解决以下目标：(i)我们将确定CHIKV基因组中的MSI2和DHX9 RNA结合位点以及RNA结构在MSI2和DHX9结合中的作用。（ii）共聚焦成像将用于确定在CHIKV转译和基因组复制期间MSI2、DHX9和nsPs的定位、共定位、表达和降解的变化。使用相同的方法，我们将确定单个RNA结构对这些变化和相互作用的影响。（iii）我们先前发表的研究表明，CHIKV基因组5'区的RNA结构对病毒基因组复制至关重要。因此，我们将使用体外和细胞方法来确定与MSI2和DHX9相互作用后CHIKV基因组的RNA结构。我们还将确定与CHIKV基因组复制和病毒转译不同阶段特异性相关的RNA结构。（iv）为了完成我们对MSI2， DHX9和CHIKV基因组5'区之间相互作用的理解，我们使用低温电子显微镜生成了未结合和蛋白质结合的RNA三级结构的高分辨率3D模型。有了其他目标的结果，这将使我们能够产生一个详细的CHIKV翻译和基因组复制调控模型。除了进一步了解CHIKV如何控制其基因组的翻译和复制外，研究结果还将为相关的人类致病病毒提供一个模型。对这一基本机制的进一步了解将为新的治疗靶点和减毒疫苗设计提供见解。

Chikungunya virus (CHIKV) is an arbovirus, transmitted by Aedes aegypti and Aedes albopictus mosquitos. Since its re-emergence in 2004, it has caused numerous epidemics across expanding geographical ranges within sub-/tropical areas of Africa, Asia and the Americas and increasingly across temperate regions of Europe, Asian and North America. The main risk factor for spread of CHIKV is change in global mosquito vector distribution - as a consequence of climate change associated seasonal variations and increasing mean temperatures, globalisation and changes in land use. Invasive populations of Ae. albopictus have been identified in the UK and temperature rises are expected to increase their months of activity and range.Chikungunya disease is associated with severe debilitating joint pain for months or years and exacerbation of co-morbidities. High mortality rates are observed among elderly patients with pre-existing conditions, such as hypertension or diabetes. Despite increasing numbers of epidemics and their expanding geographical spread, there remains no specific antiviral therapy or licenced vaccine.The mechanism and interactions by which CHIKV controls translation and replication of its genome are unclear. We have shown that CHIKV requires host protein MSI2 to replicate its genome in human cells. We and others have also shown that host protein DHX9 is required for efficient CHIKV translation, while simultaneously inhibiting replication of the virus genome. We demonstrated that MSI2 and DHX9 interact specifically with the 5' region of the CHIKV genome and identified an MSI2 RNA binding site within this region of the virus genome. We previously published a study demonstrating that the 5' region of the virus genome forms complex RNA structures, essential for CHIKV replication. We have also produced preliminary data consistent these RNA structures regulating the MSI2 binding to the CHIKV genome. Therefore, the scientific objective of the project is to generate a detailed functional and structural model of how these different factors interact, to regulate CHIKV translation and genome replication.We will address the following objectives: (i) We will identify MSI2 and DHX9 RNA binding sites within the CHIKV genome and the role of RNA structure in MSI2 and DHX9 binding. (ii) Confocal imaging will be used to determine changes in localisation, co-localisation, expression and degradation of MSI2, DHX9 and nsPs during CHIKV translation and genome replication. Using the same approach, we will determine the influence of individual RNA structures on these changes and interactions. (iii) Our previously published studies demonstrate that RNA structures in the 5' region of the CHIKV genome are essential for virus genome replication. Therefore, we will use in vitro and in cell approaches to determine the RNA structure of the CHIKV genome following interaction with MSI2 and DHX9. We will also determine the RNA structure specifically associated with CHIKV genome replication and different stages of virus translation. (iv) To complete our understanding of the interactions between MSI2, DHX9 and the 5' region of CHIKV genome we use Cryo Electron Microscopy to generate high-resolution 3D models of unbound and protein-bound RNA tertiary structures. With results from the other aims, this will allow us to produce a detailed model of the regulation of CHIKV translation and genome replication.As well as furthering our understanding of how CHIKV controls translation and replication of its genome, results will provide a model for related human pathogenic viruses. An increased understanding of this fundamental mechanism will provide insight towards novel therapeutic targets and attenuated vaccine design.