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健康问题，全世界有2亿人感染。HCV感染者通常会发展为慢性肝炎、肝硬化和肝癌。迄今为止，还没有可用的疫苗，目前的治疗方案昂贵，与严重的副作用有关，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。 * 作为（+）-正义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必须是动态结构，其能够容易地适应RNA的不同区域的解旋、延伸和暴露于病毒和细胞蛋白以用于病毒蛋白质产生、RNA复制和包装。尽管已经发现了许多关于HCV基因组翻译和复制所需的2°结构，但这些RNA调控基序已经通过关注5'和3'非编码区加上一些内部序列来鉴定。大约85%的基因组和负链中间体仍然没有被鉴定。RNA的标志之一是存在2 '-羟基。通过引物延伸（SHAPE）分析的选择性2'羟基酰化来探测2'-羟基基团已经成为询问RNA结构的金标准。受碱基配对或其他相互作用约束的核苷酸表现出差的SHAPE反应性，而柔性或单链核苷酸更可能采样使2 '-羟基易于亲核攻击的多种构象。然后使用末端标记的引物检测所得的2 '-O-加合物作为引物延伸反应的终止物。SHAPE提高了RNA结构模型的准确性，并增加了我们对RNA调控基序的理解。最近，已经开发了SHAPE试剂，其适于询问活细胞中的RNA结构。* 该提案的主要假设是HCV基因组RNA形成对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、病毒中和活细胞中对HCV基因组进行SHAPE，以鉴定介导病毒生命周期各个阶段的RNA结构。具体目标如下：*1）使用SHAPE在体外、体内和体外对HCV基因组进行表征。虽然先前的报道表明包装信号可能存在于5' NCR中，但迄今为止尚未鉴定出病毒包装信号。为此，我们将对病毒RNA（体外）和病毒体（体内）以及脱蛋白病毒体（体外）进行SHAPE分析。我们预计，这种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)使用活细胞SHAPE表征HCV生命周期中的RNA调控基序。在这个目标中，我们将使用一组在HCV生命周期的一个或多个阶段停滞的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。将对鉴定的调控RNA基序进行诱变，并评估对翻译、复制或颗粒产生的影响。*这项研究非常重要，因为深入了解对病毒生命周期至关重要的RNA结构将提高我们对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.