Structural studies of bacteriophage T4 and their potential medical applications

描述(由申请人提供)：大多数细菌病毒(“噬菌体”或简称“噬菌体”)在感染其通常非常特定的细菌宿主的能力方面是高效的。一般来说，只需要一个或几个病毒颗粒就能成功感染一种细菌。因此，噬菌体可以用作抗生素的希望由来已久，但一直没有实现。随着常见的细菌病原体发生耐药突变，对替代抗生素的需求变得越来越迫切，因此迫切需要开发替代抗生素，如噬菌体疗法。此外，重组噬菌体被认为是一种基因治疗手段，也是疫苗开发的潜在抗原。噬菌体T4是研究病毒结构和功能的模型系统，具有先进的生物复合体组装和蛋白质-蛋白质相互作用的生化原理。我们在噬菌体T4的结构和功能研究方面拥有丰富的经验。我们现在希望扩展这一知识，使其可用于潜在的医疗应用。该计划将分析衣壳的结构和组装(特异性目的1)、DNA包装机(特异性目的2)、尾部组装(特异性目的3)、尾部带头组装(特异性目的4)以及噬菌体纤维对宿主的识别(特异性目的5)。我们的主要工具将是分子生物学、蛋白质化学、结晶学和电子显微镜。分子生物学研究将产生足够数量的纯样本，用于结构和功能研究。结晶学研究将是对蛋白质成分的研究，提供接近原子分辨率的三维信息。冷冻电子显微镜重建将提供有关病毒内蛋白质成分组织的三维数据。将这些技术结合起来，将在病毒生命周期的不同阶段产生病毒的“伪原子”解析结构。

DESCRIPTION (provided by applicant): Most bacterial viruses ("bacteriophages" or just "phages") are highly efficient in their ability to infect their often very specific bacterial hosts. In general, only one or a few viral particles are necessary to successfully infect one bacterium. As a consequence there has been a long, unfulfilled hope that phages could be used as antibiotics. The need for alternative antibiotics is becoming ever more acute as common bacterial pathogens are developing resistant mutations, making it urgent to develop alternatives such as phage therapy. In addition, recombinant phages have been suggested as a means for gene therapy and as potential antigens for development of vaccines. Bacteriophage T4 has been a model system to study virus structure and function, having advanced biochemical principles of the assembly of biological complexes and protein-protein interactions. We have extensive experience in both structural and functional studies of phage T4. We now wish to expand this knowledge and make it available for potential medical applications. The plan is to analyze the structure and assembly of the capsid (Specific Aim 1), the DNA packaging machine (Specific Aim 2), the tail assembly (Specific Aim 3), assembly of the tail with head (Specific Aim 4) and the recognition of the host by the phage fibers (Specific Aim 5). Our primary tools will be molecular biology, protein chemistry, crystallography and electron microscopy. Molecular biology studies will produce pure samples in sufficient quantity for structural and functional studies. Crystallographic studies will be of protein components, providing three-dimensional information at near atomic resolution. Cryo-electron microscopic reconstructions will provide three-dimensional data on the organization of the protein components within the virus. Combining these techniques will generate "pseudo-atomic" resolution structures of the virus at different stages of its life cycle.