Using Live Cell Selective 2' Hydroxyl Acylation Analyzed by Primer Extension (SHAPE) to Investigate the Dynamic Structure of Hepatitis C Virus (HCV) RNA during the Viral Life Cycle

丙型肝炎病毒（HCV）感染是一个全球性的卫生问题，全世界有2亿人感染。感染丙型肝炎病毒的人通常会发展为慢性肝炎、肝硬化和肝癌。迄今为止，没有可用的疫苗，目前的治疗方案价格昂贵，伴有严重的副作用，只有50-70%的患者对治疗有反应。因此，迫切需要新的抗病毒策略。然而，在合理设计新的治疗策略之前，需要更好地了解病毒的生物学特性。*作为一种（+）感RNA病毒，HCV基因组本身必须作为翻译、复制和包装的模板。因此，病毒RNA必须是一种动态结构，能够很容易地适应RNA的不同区域的解绕、延伸和暴露于病毒和细胞蛋白，以进行病毒蛋白的生产、RNA复制和包装。尽管关于翻译和复制所需的HCV基因组的2°结构已经发现了很多，但这些RNA调控基序已经通过关注5‘和3’非编码区以及一些内部序列来确定。大约85%的基因组和负链中间体仍未被表征。* RNA的特征之一是2'-羟基的存在。利用引物延伸（SHAPE）分析的选择性2′羟基酰化探测2′-羟基已成为探究RNA结构的金标准。受碱基配对或其他相互作用限制的核苷酸表现出较差的形状反应性，而灵活的或单链的核苷酸更有可能具有多种构象，使2'-羟基易于受到亲核攻击。由此产生的2'- o加合物随后被检测为使用末端标记引物的引物延伸反应的停止物。SHAPE提高了RNA结构模型的准确性，增加了我们对RNA调控基序的理解。最近，SHAPE试剂已被开发出来，可用于询问活细胞中的RNA结构。*本提案的主要假设是HCV基因组RNA形成了对HCV生命周期至关重要的多个动态结构。为了验证这一假设，我们将在体外、体内和活细胞中对HCV基因组进行SHAPE检测，以鉴定介导病毒生命周期各个阶段的RNA结构。以下具体目标将探讨：*1)利用SHAPE在体外、体内和离体中表征HCV基因组。虽然以前的报告表明包装信号可能存在于5' NCR中，但迄今为止尚未发现病毒包装信号。为此，我们将对病毒RNA（体外）和病毒粒子（体内）以及去蛋白病毒粒子（体外）进行SHAPE分析。我们预计这一分析将有助于定义包装信号并揭示病毒粒子内HCV基因组的复杂结构。*2)利用活细胞SHAPE表征HCV生命周期中的RNA调控基序。为此，我们将使用一组停滞在HCV生命周期的一个或多个阶段的HCV突变体来研究对病毒翻译、复制和包装重要的RNA调控元件。已确定的调控RNA基序将被诱变，对翻译、复制或颗粒产生的影响将被评估。*拟议的研究非常重要，因为获得对病毒生命周期重要的RNA结构的见解将提高我们对HCV生物学的理解。新的RNA调控基序的鉴定将为设计基于RNA结构的抑制剂铺平道路，完善HCV复制模型，并在HCV基因组中定义新的RNA调控元件。

Hepatitis C virus (HCV) infection is a global health problem with 200 million people infected worldwide. HCV-infected individuals typically develop chronic hepatitis, cirrhosis and liver cancer. To date, there is no vaccine available, and current treatment protocols are expensive, associated with severe side effects, and only 50-70% of patients respond to therapy. Thus, there is a pressing need for novel antiviral strategies. However, before novel treatment strategies can be rationally designed, a better understanding of the biology of the virus is required. * As a (+)-sense RNA virus, the HCV genome itself must serve as a template for translation, replication, and packaging. The viral RNA must therefore be a dynamic structure that is able to readily accommodate unwinding, elongation and exposure of different regions of the RNA to viral and cellular proteins for viral protein production, RNA replication, and packaging. Although much has been uncovered about the 2° structures of the HCV genome required for translation and replication, these RNA regulatory motifs have been identified by focusing on 5' and 3' noncoding regions plus a few internal sequences. Approximately 85% of the genome and the negative-strand intermediate remain uncharacterized.* One of the hallmarks of RNA is the presence of a 2'-hydroxyl group. Probing of 2'-hydroxl groups using Selective 2' Hydroxyl Acylation analyzed by Primer Extension (SHAPE) has become the gold standard in interrogating RNA structure. Nucleotides constrained by base-pairing or other interactions exhibit poor SHAPE reactivity, while flexible or single-stranded nucleotides are more likely to sample numerous conformations that make the 2'-hydroxyl amenable to nucleophilic attack. The resultant 2'-O-adducts are then detected as stops to primer extension reactions using end-labeled primers. SHAPE has improved the accuracy of RNA structure models and increased our understanding of RNA regulatory motifs. Recently, SHAPE reagents have been developed that are amenable to interrogating RNA structure in live cells. * The main hypothesis of this proposal is that the HCV genomic RNA forms multiple dynamic structures that are critical to the HCV life cycle. To test this hypothesis, we will perform SHAPE on the HCV genome in vitro, in virio and in live cells to identify the RNA structures that mediate the various stages of the viral life cycle. The following specific aims will be explored:*1) Characterization of the HCV genome in vitro, in virio and ex virio using SHAPE. While previous reports suggest that packaging signals may be present in the 5' NCR, to date no viral packaging signals have been identified. To this end, we will carry out SHAPE analysis of viral RNA (in vitro) and in virions (in virio), as well as in deproteinized virions (ex virio). We anticipate that this analysis will help define packaging signals and reveal the complex structure of the HCV genome inside virions.*2) Characterization of the RNA regulatory motifs in the HCV life cycle using live cell SHAPE. In this aim, we will use a panel of HCV mutants that are stalled in one or more stages of the HCV life cycle to investigate RNA regulatory elements important for viral translation, replication and packaging. Identified regulatory RNA motifs will be subject to mutagenesis and the effects on translation, replication or particle production will be assessed.* The proposed research is highly significant as gaining insight into RNA structures important for the viral life cycle will improve our understanding of HCV biology. The identification of novel RNA regulatory motifs will pave the way for the design of RNA structure-based inhibitors, refine models for HCV replication, and define new RNA regulatory elements in the HCV genome.