RNA that prevents RNase activity: Biochemical and biophysical studies of the ciRNA-RNase L complex

 描述（由申请人提供）：许多病毒使用非编码RNA元件来操纵细胞机制以进行有效感染和复制。在病毒RNA的编码区发现的元件较少。一个例子是C组肠道病毒（包括柯萨奇病毒和脊髓灰质炎病毒）的C3蛋白酶编码区内的竞争性抑制剂RNA（ciRNA）。该RNA 303核苷酸元件竞争性抑制RNA酶L，RNA酶L是一种通过细胞质中双链RNA的存在而被激活的RNA酶。一旦被激活，RNase L迅速降解RNA，因此是一种强大的抗病毒酶。ciRNA可以用来抑制这种酶，这表明存在强大的选择压力来维持ciRNA序列以抑制抗病毒反应并促进成功感染。了解感染过程中发生的详细分子事件对于开发针对脊髓灰质炎病毒和柯萨奇病毒感染的新疗法非常重要。这些分子事件对于一种新的治疗方法也很重要，柯萨奇病毒A21正在临床试验中作为一种治疗黑色素瘤、乳腺癌和前列腺癌的方法。抑制RNase L的ciRNA的分子、生物物理和体内特征尚不清楚;理解RNase L和ciRNA之间的相互作用对于理解RNA如何能够直接抑制确保其破坏的酶非常重要。目的之一是验证ciRNA在结合时折叠成独特结构使其能够抑制RNase L的核糖核酸酶活性的假设，确定RNase L和ciRNA之间的结合界面，并通过阐明结构相互作用来确定ciRNA和RNase L复合物内抑制的分子机制。为了确定这种复合物的特征，我将通过化学足迹探测、结合试验和功能试验来表征不同二级结构区域的重要性，这些试验使用具有RNase L的野生型和突变形式的ciRNA。由于该复合物的大小，我将重点通过X射线晶体学来表征RNase L-ciRNA复合物的结构关系。ciRNA和RNA酶L之间的复合物的结构将阐明抑制是否是封装活性位点的结果，直接诱导RNA酶L的活性位点几何结构的构象变化，或另一种机制。该提案的第二个目的是追踪感染期间RNase L和ciRNA的定位。我计划使用单核细胞、上皮细胞和癌细胞的细胞培养物，并用脊髓灰质炎病毒和柯萨奇病毒株感染这些细胞，以证明RNase L-ciRNA复合物的生物学意义。RNase L和ciRNA将被荧光标记，在本发明期间跟踪它们的运动，以更好地理解它们在感染中的作用。列出的细胞系正在通过CRIPR-Cas9系统进行基因编辑，以制备GPF标记的RNase L，RNA FISH实验将用于跟踪ciRNA元件。这些实验的组合将允许确定RNase L和ciRNA共定位的时间和位置。

 DESCRIPTION (provided by applicant): Many viruses use non-coding RNA elements to manipulate cellular machinery for effective infection and replication. Less frequently, elements are found in the coding region of a viral RNA. An example is the competitive inhibitor RNA (ciRNA) within the coding region of the C3 protease of group C enteroviruses, including coxsackievirus and poliovirus. This RNA 303 nucleotides element competitively inhibits RNase L, an RNase that becomes activated by the presence of double-stranded RNA in the cytoplasm. Once activated, RNase L rapidly degrades RNA and thus is a powerful antiviral enzyme. The ciRNA can serve to inhibit this enzyme, and this suggests there is strong selective pressure to maintain the ciRNA sequence to depress the antiviral response and facilitate successful infection. Understanding the detailed molecular events that occur during infection is important for the development of new therapies against poliovirus and coxsackievirus infection. These molecular event could also be important for a novel therapy were coxsackievirus A21 is in clinical trials as a therapy against melanoma, breast, and prostate cancers. The molecular, biophysical, and in vivo features of the ciRNA that inhibits RNase L are unknown; understanding the interactions between RNase L and ciRNA is important in understanding how an RNA is able to directly inhibit an enzyme that is made to ensure its destruction. Aim one of this proposal is t test the hypothesis that ciRNA folds into a unique structure when it binds making it able to inhibi RNAse L's ribonuclease activity, determine the binding interface between RNase L and ciRNA, and determine the molecular mechanism of inhibition within the ciRNA and RNase L complex by elucidating the structural interactions. To determine the features of this complex, I will characterize the importance of different secondary structural regions by chemical footprint probing, binding assays, and functional assays with wild-type and mutant forms of ciRNA with RNase L. Due the size of this complex I will focus on to characterize the structural relationship of the RNase L-ciRNA complex by X-ray crystallography. A structure of a complex between ciRNA and RNase L would illuminate whether inhibition is the result of encapsulating the active site, inducing a conformational change in the active site geometry of R Nase L directly, or another mechanism. The second aim of this proposal is to track the localization of RNase L and ciRNA during infection. I plan to use cell culture of monocytes, epithelial, and cancer cells and infect these cells with poliovirus and coxsackievirus strains to demonstrate the biological significance of the RNase L-ciRNA complex. RNase L and ciRNA will be fluorescently labeled track their movements during to invention to better understand their role within infection. The cell lines listed are being genetically edited by the CRIPR-Cas9 system to make GPF tagged RNase L and RNA FISH experiments will be used to track the ciRNA element. The combination of these experiments will allow to determine when and where RNase L and ciRNA co-localize.