Mechanisms in Viral RNA Replication Complex Assembly: Novel Targets for Antivira

描述（由申请人提供）：拟议的研究旨在破译病毒RNA复制的机制，这是产生子代病毒及其传播到未感染细胞的关键步骤。更好地理解这些机制将有助于我们设计新的抗病毒疗法。我们在野田村病毒（NoV）中研究了这一过程，以便我们能够利用其在广泛的宿主细胞中将其正链RNA基因组复制到极高水平的能力，NoV提供了一个很好的模型来研究对人类致病的更复杂的正链RNA病毒。虽然这种模式病毒尚未与人类疾病紧密相关，但最近的研究已将新型NovV样病毒与人类脑炎或麻痹性疾病联系起来。因此，我们建议，NoV可以作为一个模型，以了解人类科萨基病毒引起的疾病和新设计的实验比较NoV复制复合物组装与肠道病毒科萨基A病毒21（CVA 21）。NoV和CVA 21 RNA的复制通过互补负链复制中间体的合成发生，所述中间体用作合成另外的正链的模板。这两种反应都由病毒编码的RNA依赖性RNA聚合酶（RdRp）催化。宿主细胞中病毒RNA复制的起始需要RdRp识别RNA模板并组装膜结合的RNA复制复合物（RC），即细胞中RNA复制的位点。拟议的研究集中在影响RC组装的两个主要领域：1）RdRp识别模板的结构要求和随后的RC模板招募和2）RdRp翻译后修饰在RC组装中的作用。我们假设所需组分的募集受RNA模板和病毒（和/或细胞）蛋白之间的结构依赖性RNA-蛋白相互作用以及RdRp的翻译后修饰控制。我们将使用遗传学和生物化学相结合的方法进行突变，使RdRp与RNA模板结合或阻止RdRp的修饰，并测试它们对RNA复制的影响，RNA结合研究以测量RNA模板和RdRp之间的相互作用，显微镜研究以研究响应诱变的RdRp亚细胞定位的变化，和液相色谱-串联质谱法（LC-MS/MS）以鉴定RdRp中被跨国修饰后的氨基酸。我们还将设计一种新的RNA结构预测工具，以便于识别更复杂的病毒RNA基因组中的长距离碱基配对相互作用。我们提出了以下具体目标：1）定义RNA结构元件在RdRp识别和RNA模板募集到线粒体RC中的作用，以及2）确定野田村病毒RdRp的翻译后修饰在其他蛋白质形成和募集到膜结合RC中的作用。这些研究的结果将提供深入了解病毒RNA复制机制，并确定抗病毒治疗的新靶点。

DESCRIPTION (provided by applicant): The proposed research seeks to decipher the mechanism of viral RNA replication, a key step in production of progeny viruses and their spread to uninfected cells. A greater understanding of these mechanisms will facilitate our design of novel antiviral therapies. We study this process in the nodavirus Nodamura virus (NoV) so that we can harness its ability to replicate its positive-strand RNA genome to tremendously high levels in a wide range of host cells NoV provides an excellent model to study more complex, positive-strand RNA viruses pathogenic to humans. Although this model virus has not yet been associated firmly with human disease, recent studies have linked novel NoV-like viruses to human diarrheal or paralytic illnesses. Thus, we propose that NoV could serve as a model to understand the diseases caused by human Coxsackie viruses and newly designed experiments compare NoV replication complex assembly with that of the enterovirus Coxsackie A virus 21 (CVA21). Replication of NoV and CVA21 RNA occurs via the synthesis of complementary negative strand replication intermediates, which are used as templates for synthesis of additional positive strands. Both reactions are catalyzed by a virus-encoded RNA-dependent RNA polymerase (RdRp). Initiation of viral RNA replication in a host cell requires recognition of the RNA template by the RdRp and assembly of membrane- bound RNA replication complexes (RC's), the sites of RNA replication in the cell. The proposed studies focus on two major areas that impact RC assembly: 1) structural requirements of template recognition by the RdRp and subsequent template recruitment to the RC and 2) the role of RdRp post-translational modification in RC assembly. We hypothesize that recruitment of the required components is controlled by structure-dependent RNA-protein interactions between the RNA template and viral (and/or cellular) proteins and by post- translational modification of the RdRp. We will use a combined genetic and biochemical approach to make mutations that effect RdRp-binding to RNA templates or prevent modification of the RdRp and test their effects on RNA replication, RNA binding studies to measure interactions between the RNA template and the RdRp, microscopy studies to study changes to subcellular localization of the RdRp in response to mutagenesis, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify the amino acids in the RdRp that are post-transnationally modified. We will also design a new RNA structure prediction tool to facilitate identification of long-range base pairing interactions within more complex viral RNA genomes. We propose the following Specific Aims: 1) Define the role of RNA structural elements in recognition by the RdRp and recruitment of RNA templates into mitochondrial RC's and 2) Determine the role of post-translational modification of the nodavirus RdRp in the formation of and recruitment of other proteins to membrane-bound RC's. The results of these studies will provide insight into viral RNA replication mechanisms and identify new targets for antiviral therapies.