Structural studies of bacteriophage T4 and their potential medical applications

项目总结 大多数细菌病毒(“噬菌体”或简称“噬菌体”)的能力都很强。 来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是 成功感染一种细菌所必需的。因此，已经有很长一段时间， 噬菌体可以用作抗生素的希望没有实现。对替代抗生素的需求是 随着常见细菌病原体产生抗药性突变，情况变得越来越严重， 因此迫切需要开发替代疗法，如噬菌体疗法。 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，使其可用于医疗应用。我们的计划是 分析DNA包装机衣壳(特指1)的结构和组装 (特定目标2)、尾部组装(特定目标3)、尾部与头部组装(特定目标4) 以及噬菌体纤维对宿主的识别(特定目标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品，用于结构和功能研究。 将对蛋白质成分进行结晶学研究，提供三维信息，请访问 近原子分辨率。冷冻电子显微镜重建将提供三维 关于病毒内蛋白质组分组织的数据。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。 我们的研究将推动噬菌体在医疗应用中的应用。为 例如，在临床试验中遇到的问题之一是任何 一种特殊的噬菌体，以及在哺乳动物中用作抗生素时噬菌体的快速降解。 宿主范围在一定程度上由我们将研究的病毒酶控制，这些酶需要 穿透宿主的细胞壁和其他防御系统。因为噬菌体头部的重复性质 衣壳，它们可以用来产生展示其他病毒表位的高抗原性颗粒 病原体，从而创造了新疫苗的可能性。其他医疗应用包括 强大的DNA包装马达，例如，可以用于基因在 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.