NUCLEIC ACID INTERACTIONS IN FLOCK HOUSE VIRUS

这项建议中概述的实验重点是鸡舍病毒(FHV)中的RNA-RNA相互作用，FHV是一种无包膜的二十面体昆虫病毒。FHV的基因组分裂成两个截然不同的单链信使RNA分子，这两个分子被认为被封装成一个单一的病毒粒子。选择每个基因组RNA的一个拷贝以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，对纯化的病毒粒子进行温和加热可以在粒子内形成稳定的异二聚体RNA复合体，这表明被包裹的核酸经历了重大的结构重排。此外，FHV前体的温和解离表明，这种复合体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认为是为了提供一种在病毒颗粒组装过程中共同包裹两个RNA的方法。在这一应用中，我们打算从纯化的病毒粒子中分离出不稳定的异源二聚体和/或在体外重建它，并计划用各种生化、物理和分子遗传学方法对其进行表征。此外，与约翰·E·约翰逊博士的实验室合作，将利用一种新开发的低分辨率X射线结晶学技术来可视化FHV颗粒加热时引起的RNA结构的变化。拟议的研究结果将为RNA包装在分段病毒中的机制以及病毒粒子内核酸的组织提供新的见解。这些知识可以为制定如何干扰具有重要医学意义的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.