NUCLEIC ACID INTERACTIONS IN FLOCK HOUSE VIRUS

本实验主要研究禽舍病毒（FHV）的RNA-RNA相互作用，FHV是一种非包膜二十面体昆虫病毒。FHV的基因组分裂为两个不同的单链信使感RNA分子，这些分子被认为被封装在一个病毒粒子中。每种基因组RNA的一个拷贝被选择包装成单个颗粒的机制尚不清楚。我们实验室的初步研究表明，对纯化病毒粒子进行温和加热，可诱导颗粒内形成稳定的异二聚体RNA复合物，这表明被封装的核酸经历了重大的结构重排。此外，FHV产品的轻度解离表明，该复合物可能在加热之前就已经存在，尽管是以更不稳定的形式存在。假设异源二聚体的形成提供了一种在病毒颗粒组装期间将两种rna共包装的方法。在这项应用中，我们打算从纯化的病毒粒子中分离出不稳定的异二聚体和/或在体外重建它，并计划使用各种生化，物理和分子遗传学方法对其进行表征。此外，与John E. Johnson博士的实验室合作，一种新开发的低分辨率x射线晶体学技术将用于观察FHV粒子加热引起的RNA结构变化。这些研究结果将为研究分节病毒的RNA包装机制和病毒粒子内核酸的组织结构提供新的见解。这些知识可以为制定如何干扰医学上重要病毒的生命周期的策略提供基础。

The experiments outlined in this proposal focus on RNA-RNA interactions in Flock House virus (FHV), a nonenveloped, icosahedral insect virus. The genome of FHV is split between two distinct, single-stranded, messenger-sense RNA molecules which are thought to be encapsidated into a single virion. The mechanism by which one copy of each genomic RNA is selected for packaging into a single particle is unknown. Preliminary studies from our laboratory have demonstrated that mild heating of purified virions induces formation of a stable heterodimeric RNA complex within the particle suggesting that the encapsidated nucleic acids undergo significant structural rearrangements. Furthermore, mild dissociation of FHV provirions has revealed that this complex may already exist prior to heating, albeit in a more labile form. Heterodimer formation is hypothesized to provide a means by which the two RNAs are copackaged during assembly of virus particles. In this application, we intend to isolate the labile heterodimer from purified virions and/or reconstitute it in vitro and plan to characterize it using various biochemical, physical and molecular genetic methods. In addition, in collaboration with Dr. John E. Johnson's laboratory, a newly developed low resolution X-ray crystallography technique will be utilized to visualize changes in RNA structure induced upon heating of FHV particles. The results from the proposed studies should provide new insights into the mechanisms of RNA packaging in segmented viruses and the organization of the nucleic acid inside the virion. This knowledge could provide a basis for developing strategies of how to interfere with the life cycle of medically important viruses.