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

丙型肝炎病毒感染是一个全球性的健康问题，全世界有2亿人感染。丙型肝炎病毒感染者通常会患上慢性肝炎、肝硬变和肝癌。到目前为止，还没有可用的疫苗，而且目前的治疗方案昂贵，与严重的副作用相关，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。*作为一种(+)有义的RNA病毒，丙型肝炎病毒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，能够容易地适应RNA的不同区域的解离、延长和暴露于病毒和细胞蛋白，以用于病毒蛋白的生产、RNA复制和包装。虽然关于丙型肝炎病毒基因组翻译和复制所需的2°结构已经发现了很多，但这些RNA调控基序是通过关注5‘和3’非编码区以及一些内部序列来鉴定的。大约85%的基因组和负链中间体仍未确定其特征。*RNA的特征之一是存在一个2‘-羟基。利用引物延伸分析的选择性2‘-羟基酰化反应(SHAPE)探测2’-羟基已成为询问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，而柔性或单链核苷酸更有可能采样大量的构象，使2‘-羟基易于受到亲核攻击。然后使用末端标记的引物检测得到的2‘-O-加合物作为引物延伸反应的终止。形状提高了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，已经开发出了能够询问活细胞中RNA结构的形状试剂。*这一建议的主要假设是，丙型肝炎病毒基因组RNA形成对丙型肝炎病毒生命周期至关重要的多种动态结构。为了验证这一假设，我们将在体外、病毒和活细胞中对丙型肝炎病毒基因组进行SHAPE，以确定介导病毒生命周期不同阶段的RNA结构。将探索以下特定目标：*1)使用SHAPE对体外、病毒体和病毒体中的丙型肝炎病毒基因组进行表征。虽然之前的报告表明包装信号可能存在于5‘NCR中，但到目前为止还没有发现病毒包装信号。为此，我们将对病毒RNA(体外)、病毒粒子(病毒粒子)以及脱蛋白病毒粒子(病毒粒子)进行形状分析。我们预计，这一分析将有助于定义包装信号，并揭示病毒粒子内丙型肝炎病毒基因组的复杂结构。*2)使用活细胞形状表征丙型肝炎病毒生命周期中的RNA调控基序。为此，我们将使用一组在丙型肝炎病毒生命周期的一个或多个阶段停滞不前的丙型肝炎病毒突变株来研究对病毒翻译、复制和包装至关重要的RNA调控元件。已确定的调控RNA基序将受到突变，并将评估对翻译、复制或颗粒生产的影响。*拟议的研究具有非常重要的意义，因为深入了解对病毒生命周期重要的RNA结构将增进我们对丙型肝炎病毒生物学的理解。新的RNA调控基序的发现将为基于RNA结构的抑制剂的设计、完善丙型肝炎病毒复制模型和确定丙型肝炎病毒基因组中新的RNA调控元件铺平道路。

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.