Structural studies of bacteriophage T4 and their potential medical applications

项目摘要 大多数细菌病毒（“噬菌体”或只是“噬菌体”）在它们的能力是非常有效的， 感染它们通常非常特殊的细菌宿主。一般来说，只有一个或几个病毒颗粒， 成功感染一个细菌所必需的。因此，长期以来， 噬菌体可用作抗生素的希望尚未实现。对替代抗生素的需求是 由于常见细菌病原体正在产生耐药性突变， 使得迫切需要开发诸如噬菌体疗法的替代方案。 我们在噬菌体T4的结构和功能研究方面都有丰富的经验。我们 现在希望扩展该知识并使其可用于医学应用。计划是 分析了DNA包装机衣壳（Specific Aim 1）的结构和组装 （具体目标2）、尾部组装（具体目标3）、尾部与头部组装（具体目标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、晶体学和电子显微学。分子生物学 研究将产生足够数量的纯样品用于结构和功能研究。 晶体学研究将是蛋白质成分，提供三维信息， 近原子分辨率冷冻电子显微镜重建将提供三维 关于病毒内蛋白质成分的组织的数据。结合这些技术 将在病毒生命周期的不同阶段生成病毒的“伪原子”解析结构。 我们的研究将促进噬菌体在医学上的应用。为 例如，在临床试验中遇到的问题之一是任何病毒的宿主范围都很窄， 一种特殊的噬菌体和快速降解的抗生素时，在哺乳动物中使用。 宿主范围在一定程度上是由我们将要研究的病毒酶控制的，这些酶需要 穿透宿主的细胞壁和其他防御。由于噬菌体头部的重复性 衣壳，它们可用于产生展示其他病毒抗原表位的高抗原性颗粒。 从而创造了新疫苗的潜力。其他医疗应用包括 强大的DNA包装马达，例如，可以用于基因的传递。 基因治疗的发展。

PROJECT SUMMARY Most bacterial viruses ("bacteriophages" or just "phage") 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. 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 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. Cryoelectron 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. Our investigations will advance the use of bacteriophages for medical applications. For instance, among the problems encountered in clinical trials were the narrow host range of any one particular phage and the rapid degradation of phages when used as antibiotics in mammals. The host range is, in part, controlled by the viral enzymes that we will study, which are required to penetrate the host's cell wall and other defenses. Because of the repetitive nature of phage head capsids, they can be used to produce highly antigenic particles displaying epitopes of other viral pathogens thereby creating the potential for new vaccines. Other medical applications involve the powerful DNA packaging motor that might be used, for instance, in the delivery of genes in the development of gene therapy.