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

大分子的三维（3D）结构被组装以实现功能利用。然而，这种3D结构一旦符合，就能够根据需要进行转换和/或重新排列，以实现基本的功能交互。在宿主细胞感染期间，连续的大分子相互作用定义了病毒的生命周期，这是大分子结构功能利用高保真度的极好例子。然而，尽管对病毒生命周期进行了大量研究，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ssDNA细小病毒科成员小鼠微小病毒（MVM）的自然变异和新出现的毒力毒株作为病毒模型，来破译基因组相互作用引发的成功宿主细胞受体识别、细胞运输和衣壳成熟的结构要求。该模型系统也非常适合研究彼此相似但在宿主范围、组织趋向性和感染的致病结果方面存在显着差异的病毒株，这是由于其衣壳病毒蛋白的微小结构变化或氨基酸差异而改变所需的感染相互作用。这些变异通常会改变病毒与宿主细胞的相互作用，包括感染受体的利用，这在新兴病毒中很常见。这个项目包括一个多学科的方法，利用x射线晶体学、生物化学、生物物理学和病毒学，在四个不同机构的研究人员的合作努力下。研究结果将增加对病毒感染的功能域利用的理解，特别是对于非包膜病毒，它们必须进化出在往返复制位点的途中穿越复杂细胞环境的策略，并进一步了解病毒适应新宿主的机制，这一现象通常会导致新出现的病毒病原体。更广泛的影响：该项目的研究目标将整合到高中，本科生和研究生的教育培训中，使用多学科方法研究旨在破译大分子相互作用的结构-功能相关性。具体而言，将通过佛罗里达大学学生科学培训计划（SSTP）在夏季招募高中生参与该项目，三名本科生已经积极参与该项目，其中三个具体研究目标将构成研究生博士论文的研究组成部分。本科生和研究生也将参与在合作实验室进行的研究。此外，一个具体的推广目标将包括制作一个名为“结构生物学作为科学研究工具”的教育光盘，作为佛罗里达大学“科学之旅”计划的一部分，该计划基于网络，针对K-12学生的学习工具。这一目标还包括开发一个为期三天的研讨会，作为佛罗里达大学“教师学者”暑期项目的一部分，与光盘一起为跨学科的物理、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，这些知识可以融入他们的课堂教学策略。

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.