Structural Analysis of Virulence Determinants and Genomic DNA Packaging Constraints in a ssDNA Virus

组装大分子的三维(3D)结构以实现功能利用。然而，这种3D结构一旦形成，就能够根据需要进行转换和/或重新排列，以实现基本的功能相互作用。在宿主细胞感染期间，定义病毒生命周期的连续的大分子相互作用是大分子结构功能利用的高保真的极好例子。然而，尽管对病毒生命周期的研究很多，但关于促进衣壳-宿主相互作用、结构衣壳转变和衣壳-基因组相互作用的3D结构信息有限。该项目将利用小鼠微小病毒(MVM)的自然发生变体和新出现的毒株作为病毒模型，以破译由基因组相互作用引发的成功的宿主细胞受体识别、细胞运输和衣壳成熟的结构要求。该模型系统也是研究病毒株的理想选择，这些病毒株彼此相似，但由于衣壳病毒蛋白上微小的结构变化或氨基酸差异，改变了所需的感染相互作用，因此在宿主范围、组织嗜性和感染的致病结果方面存在显著差异。这些变异通常会改变病毒宿主与细胞的相互作用，包括在新出现的病毒中常见的感染性受体利用。该项目包括一种多学科方法，利用X射线结晶学、生物化学、生物物理学和病毒学，来自四个不同机构的研究人员共同努力。研究成果将增加对病毒感染功能域利用的理解，特别是对于非包膜病毒，这些病毒必须进化出在往返其复制位点的途中穿越复杂细胞环境的策略，并提供更深入的见解(S)，以适应新宿主的机制，这一现象通常会导致新的病毒病原体出现。广泛的影响：该项目的研究目标将被整合到高中、本科生和研究生的教育培训中，使用多学科方法进行研究，旨在破译大分子相互作用的结构与功能之间的关系。具体来说，高中生将通过佛罗里达大学学生科学培训计划(SSTP)在夏季几个月内被招募参与这个项目，三名本科生已经积极参与到这个项目中，其中三个具体的研究目标将构成一名研究生博士论文的研究组成部分。本科生和研究生也将参加在合作实验室进行的研究。此外，一个具体的外联目标将包括制作题为“作为科学研究工具的结构生物学”的教育光盘，作为佛罗里达大学“科学之旅”方案的一部分，以K-12学生为对象的基于网络的学习工具。这一目标还将包括开发一个为期三天的研讨会，作为佛罗里达大学“教师作为学者”暑期计划的一部分，并伴随着光盘，为跨学科的物理、数学和生物高中教师提供实践经验和结构生物学的理论知识，这些知识可以整合到他们的课堂教学策略中。

The three-dimensional (3D) structure of a macromolecule is assembled to enable functional utilization. This 3D structure, however, once conformed, is capable of transitions and/or re-arrangements, as required, to enable essential functional interactions. The consecutive macromolecular interactions that define the life-cycle of viruses during host-cell infection represent excellent examples of the high fidelity of functional utilization of macromolecular structure. However, despite much research on viral life cycles, there is limited 3D structural information on the facilitating capsid-host interactions, structural capsid transitions, and capsid-genome interactions. This project will utilize naturally-occurring variants and emerging virulent strains of the Minute Virus of Mice (MVM), a member of the ssDNA Parvoviridae, as a viral model to decipher the structural requirements for successful host-cell receptor recognition, cellular trafficking and capsid maturation triggered by genomic interaction. This model system is also ideal for studies of virus strains that are similar to one another yet have pronounced differences in their host-range, tissue tropism, and pathogenic outcomes of infection due to minor structural variations or amino-acid differences on their capsid viral proteins which modify required infectious interactions. These variations often confer altered viral host-cell interactions, including infectious receptor utilization, which is common in emerging viruses. This project includes a multi-disciplinary approach, utilizing X-ray crystallography, biochemistry, biophysics, and virology, in a collaborative effort among investigators from four different institutions. Research outcomes will augment the understanding of functional domain utilization for viral infection in general, particularly for non-enveloped viruses that must evolve strategies for traversing the complex cellular milieu en route to and from their sites of replication, and provide further insight into the mechanism(s) of viral adaptations to new hosts, a phenomenon that often gives rise to emerging viral pathogens.Broader Impacts: The research objectives of this project will be integrated into the educational training of high school, undergraduate and graduate students in the use of a multidisciplinary approach for studies aimed at deciphering structure-function correlations for macromolecular interactions. Specifically, high school students will be recruited to work on this project during the summer months through the University of Florida Student Science Training Program (SSTP), three undergraduate students are already actively involved in this project, and three of the specific research aims will form the research component of the Ph.D thesis of a graduate student. Undergraduate and graduate students will also participate in the research studies to be conducted in the collaborating laboratories. In addition, a specific outreach aim will involve the production of an educational CD-ROM titled "Structural biology as a tool for scientific research" as part of the University of Florida "Excursion in Science" Program web based learning tools aimed at K-12 students. This aim will also include the development of a three day workshop as part of the University of Florida "Teachers as Scholars" summer program to accompany the CD-ROM that will provide interdisciplinary physics, math, and biology high school teachers with hands-on experience and theoretical knowledge of structural biology that can be integrated into their classroom teaching strategies.