NUCLEIC ACID INTERACTIONS IN FLOCK HOUSE VIRUS

本提案中概述的实验重点是鸡群屋病毒（FHV）（一种无包膜二十面体昆虫病毒）中的RNA-RNA相互作用。 FHV的基因组被分成两个不同的单链信使RNA分子，这两个分子被认为是单核苷酸化成单个病毒体。 选择每个基因组RNA的一个拷贝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，温和加热纯化的病毒粒子诱导形成一个稳定的异源二聚体RNA复合物的颗粒内，这表明，双核苷酸的核酸发生显着的结构重排。此外，FHV前病毒的轻微解离表明，这种复合物可能在加热之前就已经存在，尽管形式更不稳定。 异源二聚体的形成被假设为提供了一种手段，通过这种手段，两种RNA在病毒颗粒的组装过程中被共包装。 在本申请中，我们打算从纯化的病毒体中分离不稳定的异源二聚体和/或在体外重建它，并计划使用各种生物化学，物理和分子遗传学方法对其进行表征。 此外，在与约翰·E。约翰逊的实验室，一个新开发的低分辨率X射线晶体学技术将被用来可视化RNA结构的变化时，加热的FHV颗粒诱导。 从拟议的研究结果应该提供新的见解RNA包装在分段病毒和病毒粒子内的核酸组织的机制。 这些知识可以为制定如何干预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.