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

描述（由申请人提供）：正链RNA病毒是严重的病原体，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。全面了解病毒复制的一个主要障碍是病毒基因组的大尺寸，这使得将RNA结构与RNA功能联系起来的工作变得复杂。我们发现一个新的构象开关激活了与芜菁皱病毒（TCV; 4054 nt）相关的satC （356 nt）中的（-）链合成。由于satC包含TCV RdRp复制所需的所有序列和结构，过去对其复制的研究提供了重要的信息，随后发现这些信息适用于更大的病毒基因组，包括那些导致人类和动物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。在本提案中，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，这些相互作用表征了wt和突变体satC的satC前活性结构和结构转变。全长和选定的satC片段将通过温度梯度凝胶电泳、紫外熔融曲线、寡核苷酸可达性和紫外交联进行分析。位点特异性突变和体内遗传选择（selex）将有助于确定单个元件和元件之间的关系。我们还将使用诱变方法结合RdRp结合分析来探索TCV序列，这些序列对（-）链合成非常重要，尽管也在satC中发现。最后，我们将研究RdRp与特定的satC和TCV发夹的结合，并确定一个satC发夹侧面的元件是否影响satC复制，同时通过两种病毒rna之间的相互作用干扰TCV复制并抑制病毒粒子积累功能。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病毒启动（-）链合成的步骤的最详细的了解。

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.