Virus-host interactions during nodaviral infection

描述（由申请人提供）：本提案中概述的实验旨在更深入地了解病毒选择和包装多部分基因组为单个颗粒的机制。在之前的资助期间，我们将注意力集中在双链正链RNA诺达病毒禽舍病毒（FHV）上，它代表了解决这一问题的相对简单的系统。通过结合使用分子和细胞生物学方法，我们发现特定的基因组包装在很大程度上依赖于病毒外壳蛋白和基因组rna的定义亚细胞位置。具体来说，我们的数据表明，这两个基因组片段是独立包装的，在不同的细胞微环境中。此外，我们有证据表明内质网和线粒体在这一过程中都起着关键作用。我们的数据已经总结在一个更新的nodaviral RNA包装模型中，我们打算在下一个资助期确认并进一步完善这个模型。我们相信，本提案中概述的实验不仅将阐明分子病毒学的一个重要方面，而且还将揭示细胞生物学的新方面。在目标1中，我们将确定病毒RNA2的细胞位置，FHV外壳蛋白的信息，并绘制其翻译位点。在目标2中，我们将确定外壳蛋白如何从内质网运输到线粒体（病毒RNA合成的部位），以及细胞因子是否参与了这一过程。在目标3中，我们将确定特定FHV基因组包装的决定因素，特别是基因组片段是共翻译包装还是共转录包装，以及这些机制如何决定混合感染期间重组的形成。

DESCRIPTION (provided by applicant): The experiments outlined in this proposal are designed to provide a deeper understanding of the mechanisms by which viruses select and package multipartite genomes into a single particle. During the previous funding period we have focused our attention on the bipartite, positive-strand RNA nodavirus Flock House virus (FHV), which represents a relatively simple system to address this issue. By using a combination of molecular and cell biological approaches we discovered that specific genome packaging relies heavily on defined subcellular location of the viral coat protein and the genomic RNAs. Specifically, our data suggest that the two genomic segments are packaged independently and in different cellular micro-environments. In addition, we have evidence that both ER and mitochondria play a critical role in this process. Our data have been summarized in an updated model for nodaviral RNA packaging and we intend to confirm and further refine this model in the next funding period. We believe that the experiments outlined in this proposal will not only illuminate an important aspect of molecular virology but also reveal new facets of cell biology. In aim 1, we will determine the cellular location of viral RNA2, the message of FHV coat protein, and map its site of translation. In aim 2, we will determine how coat protein trafficks from the ER to mitochondria, the site of viral RNA synthesis, and whether cellular factors are involved in this process. In aim 3, we will identify determinants of specific FHV genome packaging, specifically whether genome segments are packaged co-translationally or co- transcriptionally and how these mechanisms determine formation of reassortants during mixed infections.