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

 描述(申请人提供)：许多病毒使用非编码的RNA元件来操纵细胞机制，以实现有效的感染和复制。较少的情况下，在病毒RNA的编码区中发现元件。一个例子是包括柯萨奇病毒和脊髓灰质炎病毒在内的C组肠道病毒C3蛋白酶编码区内的竞争性抑制物RNA(CiRNA)。这种RNA303核苷酸元件竞争性地抑制核糖核酸酶L，核糖核酸酶是一种核糖核酸酶，在细胞质中存在双链核糖核酸时被激活。一旦被激活，核糖核酸酶L迅速降解核糖核酸，因此是一种强大的抗病毒酶。CiRNA可以抑制这种酶，这表明有很强的选择压力来维持ciRNA序列，以抑制抗病毒反应，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。这些分子事件对于柯萨奇病毒A21作为治疗黑色素瘤、乳腺癌和前列腺癌的临床试验的新疗法也可能是重要的。抑制核糖核酸酶L的ciRNA的分子、生物物理和体内特征尚不清楚；了解核糖核酸酶L和ciRNA之间的相互作用对于了解RNA如何能够直接抑制为确保其被破坏而产生的酶具有重要意义。目的验证CRNA与核糖核酸酶L结合时折叠成独特结构，从而抑制核糖核酸酶活性的假说，确定核糖核酸酶L与核糖核酸酶的结合界面，阐明核糖核酸酶L与核糖核酸酶L复合体内抑制核糖核酸酶活性的分子机制。为了确定这个复合体的特征，我将通过化学足迹探测、结合分析以及与RNaseL野生型和突变型ciRNA的功能分析来表征不同二级结构区的重要性。由于这个复合体的大小，我将重点用X射线结晶学来表征RNase L-ciRNA复合体的结构关系。CRNA与核糖核酸酶L之间的复合物的结构将阐明抑制是直接包裹活性位点的结果，还是导致R核酸酶L活性位点几何构象变化的另一种机制。这项建议的第二个目的是追踪核糖核酸酶L和CRNA在感染过程中的定位。我计划使用单核细胞、上皮细胞和癌细胞的细胞培养，并用脊髓灰质炎病毒和柯萨奇病毒株感染这些细胞，以证明核糖核酸酶L-ciRNA复合体的生物学意义。核糖核酸酶、L和CRNA将被荧光标记，跟踪它们在发明过程中的运动，以更好地了解它们在感染中的作用。列出的细胞系正在由CRIPR-CAS9系统进行遗传编辑，以使GPF标记核糖核酸酶L，并将使用RNA FISH实验来追踪ciRNA元件。结合这些实验将可以确定核糖核酸酶L和ciRNA共定位的时间和地点。

 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.