Structural studies of bacteriophage T4 and their potential medical applications

 描述(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方面是高效的。通常，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。计划分析衣壳的结构和组装(特异性目标1)，噬菌体纤维识别宿主(特异性目标2)，DNA包装机(特异性目标3)，包装机与头部的连接(特异性目标4)，以及基因传递的机制(特异性目标5)。这些研究是针对针对特定原核细胞(作为抗生素)和真核细胞(如癌细胞)的特定医学应用而设计的噬菌体T4，以输送毒素。此外，我们正在对T4的表面进行工程改造，使其成为CARR的多个表位副本，用于疫苗开发。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。结合这些技术将产生病毒在其生命周期不同阶段的“伪原子”解析结构，用于功能解释和验证。

 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), recognition of the host by the phage fibers (Specific Aim 2), the DNA packaging machine (Specific Aim 3), attachment of the packaging machine to the head (Specific Aim 4), and the mechanism of gene delivery (Specific Aim 5). These studies are directed towards engineering phage T4 for specific medical applications to target specific prokaryotic cells (as an antibiotic) and eukaryotic cells (such as cancer cells) for the delivery of toxins. In addition, we are engineering the surface of T4 to carr multiple copies of an epitope for vaccine development. 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 for functional interpretation and verification.