Phase T4 Head Assembly and Initiation of Infection

T4 阶段头部组装和感染启动

• 批准号: 6780511
• 负责人: LINDSAY W BLACK
• 金额: $ 18.56万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 1977
• 资助国家: 美国
• 起止时间: 1977-07-01 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6780511
• 关键词: DNA replication; Escherichia coli; bacteriophage T4; capsid; host organism interaction; protein biosynthesis; restriction endonucleases; virus DNA; virus assembly; virus genetics; virus infection mechanism; virus protein

DESCRIPTION (provided by applicant): This application proposes continuation of research toward two main objectives: 1) understanding the mechanism of terminase catalyzed viral DNA translocation into the procapsid, and 2) determining the host resistance mechanism leading to the evolution of the CTS- (capsid targeting sequence) initiated set of polymorphic internal proteins injected by the T-even phages. The objectives will be furthered by development of a new phage-derived technique for assessing protein interactions. As part of the first objective, the DNA packaging mechanism will be investigated by focusing on terminase and portal dodecamer structure and function. A multimeric activated ATPase form of the terminase large subunit can be produced by incubation with the terminase small subunit and with antibody against denatured terminase large subunit. Together with portal or functional portaI-GFP (green fluorescent protein) fusion, the reconstituted packasome motor (terminase + portal + DNA) will be characterized. Work incorporating portaI-GFP and HOC-portal fusions into functional proheads and phage suggests that the portal dodecamer of the prohead can be immobilized without blocking DNA packaging. Continuation of this work can determine whether a favored portal rotation model for DNA packaging is valid. Preliminary work reveals that NMR can solve the structure of the small terminase subunit. DNA binding and large subunit ATPase stimulating portions of the small subunit will be characterized biochemically and by NMR. T4 display has successfully identified a new terminase interactant, T4 late a factor gp55, required for replication-coupled T4 late transcription. Recent work has established a dependence of DNA packaging on this component; we believe the interaction serves to load the large terminase subunit onto DNA. This interaction could also serve to tightly couple packaging to DNA repair at the late replication-dependent transcription stage of T4 development. Reconstitution of active in vitro T4 DNA packaging will employ isolated gene 55 defective in vivo concatemers as well as T4 transcription assay plasmids. These DNAs will be activated for packaging by loading late transcription-replication components. In the second objective, a diverse family of CTS-injected proteins has evolved by gene expansion at the IPI locus of the T-even phages to challenge a corresponding set of resistance genes in natural hosts of these viruses. We have determined the host resistance mechanism by analysis of two resistance gene(s), IBEGs and IBEGd, cloned from one pathogenic strain of E. coli that are necessary and sufficient for phage T4 IPI- and other T-even phage resistance. These two cloned genes encode a novel two-subunit restriction endonuclease that targets glucosylated hydroxymethylcytosine (HmC)-containing T4 DNA. IPI shields the DNA from this nuclease. Evolutionary expansion and polymorphism of IPI locus genes among the T-even phages likely correspond to polymorphism of HmC adducts as specific targets for this type of previously undiscovered restriction endonuclease.

描述（由申请人提供）：本申请建议继续研究两个主要目标：1）了解终止酶催化病毒DNA易位到衣壳中的机制，以及2）确定导致由T-偶数噬菌体注射的CTS-（衣壳靶向序列）启动的一组多态性内部蛋白进化的宿主抗性机制。通过开发一种新的噬菌体衍生技术来评估蛋白质相互作用，将进一步推进这一目标。 作为第一个目标的一部分，将通过重点关注终止酶和门户十二聚体的结构和功能来研究 DNA 包装机制。多聚体活化的ATP酶形式的终止酶大亚基可以通过与终止酶小亚基和针对变性的终止酶大亚基的抗体一起温育来产生。与门户或功能性门户-GFP（绿色荧光蛋白）融合一起，将表征重建的包装体马达（终止酶+门户+DNA）。将 portaI-GFP 和 HOC-portal 融合体合并到功能性前头和噬菌体中的工作表明，前头的门十二聚体可以在不阻碍 DNA 包装的情况下固定。继续这项工作可以确定 DNA 包装所青睐的门户旋转模型是否有效。初步工作表明核磁共振可以解析小终止酶亚基的结构。 DNA 结合和小亚基的大亚基 ATP 酶刺激部分将通过生化和 NMR 进行表征。 T4 展示已成功鉴定出一种新的终止酶相互作用物，T4 晚期因子 gp55，这是复制偶联 T4 晚期转录所需的。最近的工作已经确定 DNA 包装对该成分的依赖性；我们相信这种相互作用有助于将大终止酶亚基加载到 DNA 上。这种相互作用还可以在 T4 发育的复制依赖性转录后期将包装与 DNA 修复紧密结合。活性体外 T4 DNA 包装的重建将采用分离的基因 55 缺陷体内串联体以及 T4 转录测定质粒。这些 DNA 将通过加载晚期转录复制组件来激活包装。 在第二个目标中，通过在 T-even 噬菌体的 IPI 基因座处进行基因扩展，进化出了不同的 CTS 注射蛋白家族，以挑战这些病毒自然宿主中相应的一组抗性基因。我们通过分析从大肠杆菌的一种致病菌株克隆的两个抗性基因 IBEG 和 IBEGd 确定了宿主抗性机制，这两个基因对于噬菌体 T4 IPI- 和其他 T-even 噬菌体抗性是必要和充分的。这两个克隆基因编码一种新型双亚基限制性内切核酸酶，该酶靶向含有葡萄糖基化羟甲基胞嘧啶 (HmC) 的 T4 DNA。 IPI 保护 DNA 免受这种核酸酶的影响。 T-偶数噬菌体中 IPI 位点基因的进化扩展和多态性可能与 HmC 加合物的多态性相对应，HmC 加合物是此类先前未发现的限制性内切核酸酶的特定靶标。