Mechanistic analysis of RNA-replication elements involved in early stages of chikungunya virus replication.

基孔肯雅病毒(CHIKV)是一种蚊子传播的正链RNA病毒，会导致人类丧失能力的慢性关节疼痛。该病毒于2004年在印度洋周围重新成为一种流行病，目前已被输入许多国家，到2015年，美洲和欧洲报告了113.5万例病例。目前仍没有疫苗或特定的抗病毒疗法，由于缺乏对其生物学的了解，开发受到阻碍。鉴于这些暴发的规模和不断扩大的地理分布，迫切需要对这种病毒进行更多的研究。在许多正链RNA病毒中，调控早期复制事件的基本机制涉及病毒基因组中的茎环。这种结构可以作为RNA复制元件(RRE)，并通过与宿主和病毒蛋白的相互作用来调节一系列必要的过程，如病毒翻译和基因组复制。在初步研究中，我们使用结构和反向遗传分析相结合的方法来研究在CHIKV基因组的蛋白质编码区和非编码区保守的RNA茎环元件。在导致这一应用的初步研究中，我们证明了这些茎环在CHIKV复制的早期阶段作为RRE发挥作用。每个元素中的沉默突变抑制了人类细胞培养中的CHIKV复制，而增强了蚊子细胞中的CHIKV复制。在这两种情况下，野生型复制水平在进一步诱变以恢复茎环结构后得到恢复。这一点以及我们初步研究的进一步证据使我们提出CHIKV RRES在早期病毒复制事件中发挥作用，通过宿主特异性与反式激活蛋白的相互作用来调节初始翻译和基因组复制。我寻求支持来确定RRE功能的宿主特异性遗传和结构决定因素、早期病毒复制过程中的确切角色以及特定的蛋白质反式激活剂相互作用。使用亚基因组复制子和传染性病毒比较人类和蚊子细胞系统的结果，将有助于对相关机制的详细剖析。目标1：通过分析一系列互补系统中的工程突变体，我们将在CHIKV复制的早期阶段确定每个RRE中的精确功能结构域，并产生有关RNA-RNA/RNA-病毒蛋白局部或远程功能相互作用的信息。RRE在CHIKV翻译和基因组复制中的作用将在人类和蚊子细胞培养中确定。目的：将使用各种成熟的和创新的形状分析方法进行比较RNA结构分析，产生关于RRE结构的补充定量信息。通过对突变体和野生型在不同生理相关条件下以及感染细胞复制复合体内的比较分析，可以直接了解RNA构象和功能之间的关系。目的：蛋白质组学分析将使用一系列体外和细胞内技术，包括SILAC质谱学和PAR-CLIP。剖析人类和蚊子细胞以及天然和突变元素之间RRE-蛋白质组相互作用的差异，将准确定位与单个RNA结构和茎环区功能相关的相互作用。通过这些互补的体外和生理方法，我们将定义在CHIKV复制过程中RRE功能所必需的结构和反式激活相互作用。在翻译和基因组复制中的角色将被定义，并通过对比宿主特定的和突变的表型的比较，我们将剖析功能机制。我们希望这些结果能直接应用于识别新的治疗靶点。此外，从机理上理解RRE突变的衰减将直接应用于基因稳定的减毒疫苗的研究。

Chikungunya virus (CHIKV) is a mosquito-transmitted positive-stranded RNA virus that causes incapacitating chronic joint pain in humans. Having re-emerged as an epidemic in 2004 around the Indian Ocean the virus has been imported into many countries, with >1,135,000 cases reported in the Americas and Europe by 2015. There remains no vaccine or specific antiviral therapy, with development hampered by lack of insight into its biology. Given the scale and expanding geographic distribution of these outbreaks, more research on this virus is urgently needed.In many positive-strand RNA viruses essential mechanisms for regulating of early replication events involve stem-loops within the virus genome. Such structures can act as RNA-replication elements (RRE) and through interaction with host and viral proteins regulate a range of essential processes such as virus translation and genome replication. In preliminary studies we used a combination of structural and reverse genetic analysis to investigate RNA stem-loop elements conserved in protein coding and non-coding regions of the CHIKV genome. In preliminary studies leading to this application we demonstrate that these stem-loops function as RREs during the early stages of CHIKV replication. Silent mutations within each element inhibited CHIKV replication in human cell culture while enhancing it in mosquito cells. In both instances wild-type levels of replication were restored following further mutagenesis to reinstate stem-loop structure. This and further evidence from our preliminary studies lead us to propose that the CHIKV RREs function during early virus replication events, regulating initial translation and genome replication via host specific interaction with trans-activating proteins. I seek support to define host-specific genetic and structural determinants of RRE function, precise roles during early virus replication and specific protein trans-activator interactions. Comparison of results between human and mosquito cell systems, using sub-genomic replicons and infectious virus, will enhance detailed dissection of mechanisms involved. OBJECTIVE-1: Through analysis of engineered mutants across a range of complimentary systems, we will determine precise functional domains within each RRE during early stages of CHIKV replication and generate information on functional local or long-range RNA-RNA/RNA-viral protein interactions. RRE function during CHIKV translation and genome replication will be defined in human and mosquito cell culture.OBJECTIVE-2: Comparative RNA structural analysis will be undertaken using a variety of established and innovative SHAPE methods, producing complimentary quantitative information on RRE structure. Direct insight into the relationship between RNA conformation and function will be provided by comparative analysis of mutants and wild-type under different physiologically relevant conditions and within the replication complex of infected cells.OBJECTIVE-3: Proteomic analysis will be undertaken using a range of in vitro and intracellular techniques, including SILAC mass spectroscopy and PAR-CLIP. Dissecting differences between RRE-proteome interactions in human vs. mosquito cells and native vs. mutated elements will pinpoint interactions relevant to the function of individual RNA structures and stem-loop regions.Through these complimentary in vitro and physiological approaches we will define structural and trans-activating interactions essential to RRE function during CHIKV replication. Roles in translation and genome replication will be defined and through comparison of contrasting host-specific and mutant phenotypes we will dissect functional mechanisms. We expect these results to have direct application in identification of novel therapeutic targets. Furthermore, mechanistic understanding of RRE-mutant attenuation will have direct application in studies towards a genetically stable attenuated vaccine.