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多组分基因组并将其包装成单个颗粒的机制。在上一个资助期间，我们将注意力集中在双链、正链RNA野田村病毒（FHV）上，它代表了一个相对简单的系统来解决这个问题。通过使用分子和细胞生物学方法的组合，我们发现特定的基因组包装严重依赖于病毒外壳蛋白和基因组RNA的确定的亚细胞位置。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，并在不同的细胞微环境中。此外，我们有证据表明ER和线粒体在这一过程中起着关键作用。我们的数据已经总结在更新的诺达病毒RNA包装模型中，我们打算在下一个资助期内确认并进一步完善该模型。我们相信，在这个建议中概述的实验不仅将阐明分子病毒学的一个重要方面，但也揭示了细胞生物学的新方面。目的1：确定病毒RNA 2（FHV外壳蛋白的信使）的细胞定位，并定位其翻译位点。在目标2中，我们将确定外壳蛋白如何从ER运输到线粒体（病毒RNA合成的位点），以及细胞因子是否参与此过程。在目标3中，我们将鉴定特异性FHV基因组包装的决定因素，特别是基因组片段是共转录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抑制剂的形成。 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协调的相互作用。一些病毒，如流感病毒，将多个基因组片段包装到单个颗粒中，并且这些片段被选择的机制尚不清楚。我们研究了一种将两个基因组片段包装到同一颗粒中的小昆虫病毒，我们发现这种病毒的衣壳蛋白独立地选择细胞不同位置的两个基因组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种机制的分子细节，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.