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

描述(申请人提供)：这项拟议的研究旨在破译病毒RNA复制的机制，这是生产后代病毒并将其传播到未感染细胞的关键步骤。对这些机制的进一步了解将有助于我们设计新的抗病毒疗法。我们在诺达病毒NoDamura病毒(NOV)中研究这一过程，以便我们能够利用它在广泛的宿主细胞中复制其正链RNA基因组到极高水平的能力，NOV为研究更复杂的对人类致病的正链RNA病毒提供了一个很好的模型。尽管这种模式病毒尚未与人类疾病密切相关，但最近的研究已将新的NOV样病毒与人类腹泻或麻痹性疾病联系起来。因此，我们认为NOV可以作为理解人类柯萨奇病毒引起的疾病的模型，新设计的实验将NOV复制复合体组装与肠道病毒柯萨奇A病毒21(CVA21)的组装进行了比较。NOV和CVA21 RNA的复制是通过合成互补的负链复制中间体来进行的，这些中间体被用作合成额外正链的模板。这两个反应都是由病毒编码的RNA依赖的RNA聚合酶(RdRp)催化的。在宿主细胞中启动病毒RNA复制需要RdRp识别RNA模板并组装膜结合的RNA复制复合体(RC)，这是RNA在细胞中复制的位置。拟议的研究集中在影响RC组装的两个主要领域：1)RdRp识别模板和随后的RC模板招募的结构要求；2)RdRp翻译后修饰在RC组装中的作用。我们假设，所需成分的招募是由RNA模板与病毒(和/或细胞)蛋白之间的结构依赖的RNA-蛋白质相互作用以及RdRp的翻译后修饰控制的。我们将使用一种结合遗传和生化的方法来使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，用RNA结合研究来测量RNA模板与RdRp之间的相互作用，用显微镜研究RdRp在诱变后亚细胞定位的变化，以及用LC-MS/MS(LC-MS/MS)来鉴定RdRp中被跨国修饰的氨基酸。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基因组中的远程碱基配对相互作用。我们提出了以下具体目标：1)确定RNA结构元件在RdRp识别和RNA模板招募到线粒体RC中的作用；2)确定NodaVirus RdRp翻译后修饰在形成和招募其他蛋白质到膜结合RC中的作用。这些研究结果将为深入了解病毒RNA复制机制和确定抗病毒治疗的新靶点提供依据。

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.