Role of RNA Structural Switches in the Replication of a (+)-Strand RNA Virus

描述（申请人提供）：正链RNA病毒是一种严重的病原体，可引起人类和动物的脑炎、出血热和肝炎，并对植物造成毁灭性的农作物损失。尽管进行了广泛的研究，但对这些病毒的复制仍然知之甚少。全面理解病毒复制的一个主要障碍是病毒基因组的尺寸很大，这使得将RNA结构与RNA功能联系起来的工作变得复杂。我们已经发现，一种新的构象开关激活（-）-链合成satC（356 nt）与模型病毒芜菁皱（TCV; 4054 nt）。由于satC包含TCV RdRp复制所需的所有序列和结构，因此过去对其复制的研究提供了重要信息，随后发现这些信息适用于更大的病毒基因组，包括那些在人类和动物中引起重大疾病的病毒基因组。对satC和TCV复制元件的分析揭示了satC和TCV如何使用几乎相同的序列复制其基因组的惊人复杂性和差异，这对使用亚病毒RNA复制子解释结果具有重要意义。在这项提案中，我们将使用生物物理和遗传方法来定义二级和三级相互作用，其特征在于野生型和突变型satC的satC前活性结构和结构转变。将通过温度梯度凝胶电泳、UV熔解曲线、寡核苷酸可及性和UV交联分析全长和选定的satC片段。定点诱变和体内遗传选择（selex）将有助于确定单个元件和元件之间的关系。我们还将使用结合RdRp结合分析的诱变方法来探索TCV序列，这些序列对（-）-链合成具有独特的重要性，尽管也在satC中发现。最后，我们将检查RdRp结合特定的satC和TCV发夹，并确定是否元件侧翼一个satC发夹影响satC复制，同时干扰TCV复制和抑制病毒粒子积累功能，通过两个病毒RNA之间的相互作用。这些实验的成功完成将为病毒复制和辅助病毒与相关亚病毒RNA之间的相互作用提供新的范例，并提供对RNA构象开关和导致任何RNA病毒（-）-链合成起始的步骤的最详细的理解。

DESCRIPTION (provided by applicant): Positive-strand RNA viruses are serious pathogens causing encephalitis, hemorrhagic fever and hepatitis in humans and animals and devastating crop losses in plants. Despite extensive studies, replication of these viruses remains poorly understood. A major stumbling block in efforts to fully understand virus replication is the large size of viral genomes, which complicates efforts to link RNA structure with RNA function. We have discovered that a novel conformational switch activates (-)-strand synthesis in satC (356 nt) associated with the model virus Turnip crinkle (TCV; 4054 nt). Since satC contains all sequences and structures necessary for replication by the TCV RdRp, studying its replication in the past has provided significant information subsequently found to be applicable to much larger viral genomes, including those that cause significant diseases in humans and animals. Analyses of satC and TCV replication elements has revealed astonishing complexities and differences in how satC and TCV use nearly identical sequences to replicate their genomes, which has important implications for interpretation of results using subviral RNA replicons. In this proposal, we will use biophysical and genetic approaches to define secondary and tertiary interactions that characterize the satC pre-active structure and structural transitions of wt and mutant satC. Full length and selected satC fragments will be analyzed by temperature gradient gel electrophoresis, UV melting curves, oligonucleotide accessibility and UV cross-linking. Site-specific mutagenesis and in vivo genetic selection (selex) will help define individual elements and the relationship between elements. We will also use mutagenesis approaches combined with RdRp binding analyses to explore TCV sequences that are uniquely important for (-)-strand synthesis although also found in satC. Finally, we will examine RdRp binding to specific satC and TCV hairpins and determine if elements that flank one satC hairpin affect satC replication while simultaneously interfering with TCV replication and repressing virion accumulation function through an interaction between the two viral RNAs. Successful completion of these experiments will provide new paradigms for virus replication and interactions between helper viruses and associated subviral RNAs and provide the most detailed understanding of RNA conformational switches and the steps that lead to initiation of (-)-strand synthesis for any RNA virus.