Molecular biology of viral capsids

描述（由申请人提供）：病毒衣壳蛋白是病毒转译、RNA复制、RNA包装和细胞先天免疫反应调节过程的重要调节剂。在被包装成单独病毒粒子的分段基因组的病毒中，衣壳蛋白必须协调每个rna的活动。这个项目的总体目标是更好地理解RNA病毒感染的早期步骤。也就是说，衣壳是如何与病毒粒子内的病毒rna相互作用并促进进入细胞的？雀麦花叶病毒（BMV）将作为本研究的模型病毒。BMV是一种非包膜分段正链RNA病毒，在顺式和反式作用因子、复制酶复合体的形成以及病毒RNA依赖性RNA合成机制方面做出了显著贡献。初步结果表明，BMV衣壳上富含精氨酸的基序有助于BMV rna的选择性封装，并作为细胞穿透信号。后一组观察结果导致了植物病毒通过蛋白质转导机制进入细胞的假设。该项目包含两个目标，第一个目标是通过低温电子显微镜和单粒子重建的结合，RNA构象和RNA-衣壳相互作用的生化制图，以及相互作用如何影响感染过程的分子遗传学分析来了解病毒粒子的结构。第二个目的是利用荧光标记肽和病毒，共聚焦显微镜和电子显微镜，阐明细胞穿透肽和病毒衣壳进入细胞的机制。这项工作将有助于提高对病毒感染过程的理解。

DESCRIPTION (provided by applicant): Viral capsid proteins are important regulators of viral processes from translation, to RNA replication, to RNA packaging, and the modulation of cellular innate immune responses. In viruses with segmented genomes that are packaged into separate virions, the capsid protein must coordinate the activities for each of the RNAs. The overall goal of this project is to better understand the early steps in an RNA virus infection. That is, how does the capsid interact with the viral RNAs within the virion and facilitate entry into cells? Brome mosaic virus (BMV) will be the model virus for this study. BMV is a nonenveloped segmented positive-strand RNA virus that has served as a model system with notable contributions to the cis- and trans-acting factors, the formation of the replicase complex, and the mechanism of viral RNA-dependent RNA synthesis. Preliminary results showed that an arginine-rich motif of the BMV capsid contributes to the selective encapsidation of BMV RNAs and acts as a cell-penetration signal. The latter set of observations led to the hypothesis that plant viruses gain entry into cells through a protein transduction mechanism. This project contains two aims, the first will seek to understand the architecture of the virion through a combination of cryoelectron microscopy and single particle reconstruction, biochemical mapping of RNA conformation and RNA-capsid interaction, and a molecular genetic analysis of how the interactions will influence the infection process. The second aim seeks to elucidate the mechanism of cell entry of the cell-penetration peptides and the viral capsid using fluorescently-labeled peptides and viruses, confocal microscopy and electron microscopy. The work will lead to improved understanding of the viral infection process.