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提供对病毒选择多部分基因组并将其打包成单个颗粒的机制的更深入理解。在之前的资助期间，我们将注意力集中在两部分、正链RNA诺达病毒群屋病毒(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的亚细胞定位。具体地说，我们的数据表明，这两个基因组片段是独立包装的，并且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发挥着关键作用。我们的数据已汇总到NodaVirus RNA包装的更新模型中，我们打算在下一个资助期确认并进一步完善这一模型。我们相信，这项提议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，并绘制其翻译位置。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合成的位置，以及细胞因素是否参与这一过程。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体的形成。 公共卫生相关性：病毒颗粒的组装需要病毒衣壳蛋白和病毒基因组之间的协调相互作用。一些病毒，如流感病毒，将多个基因组片段包装成单个颗粒，而选择这些片段的机制尚不清楚。我们研究了一种将两个基因组片段包装成同一颗粒的小型昆虫病毒，我们发现该病毒的衣壳蛋白在细胞的不同位置独立地选择两个基因组RNA。这些研究为病毒如何调节分段基因组的包装提供了第一条线索。在这项提案中，我们计划进一步研究这一机制的分子细节，希望为其他病毒如何处理类似问题提供见解。

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. PUBLIC HEALTH RELEVANCE: Assembly of virus particles requires coordinated interactions between viral capsid proteins and the viral genome. Some viruses, such as the influenza viruses, package multiple genome segments into a single particle and the mechanisms by which these segments are selected are not understood. We study a small insect virus that packages two genome segments into the same particle and we have discovered that the capsid protein of this virus selects the two genomic RNAs independently and in different locations of the cell. These studies have provided the first clues as to how viruses regulate packaging of segmented genomes. In this proposal we plan to further investigate the molecular details of this mechanism in hopes of providing insights as to how other viruses deal with a similar problem.