Molecular and cellular biology of the phage nucleus and spindle

噬菌体核和纺锤体的分子和细胞生物学

• 批准号: 10217195
• 负责人: JOSEPH A POGLIANO
• 金额: $ 49.09万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-06 至 2022-09-25
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10217195
• 关键词: Affect; Archaea; Bacteria; Bacterial Proteins; Bacteriophages; Biochemical; Biochemistry; Capsid; Cell Nucleus; Cells; Cellular biology; Coupled; Cryo-electron tomography; Cryoelectron Microscopy; Cytoplasm; DNA; DNA Packaging; Data; Development; Diffusion; Docking; Elements; Enzymes; Eukaryota; Genetic; Growth; Homologous Gene; Infection; Intracellular Transport; Ions; Lytic; Membrane; Messenger RNA; Metabolic; Modeling; Molecular and Cellular Biology; Movement; Mutation; Nuclear Pore Complex; Polymers; Proteins; Pseudomonas Infections; Pseudomonas aeruginosa; RNA Processing; Reproduction; Research; Resolution; Ribosomes; Role; Series; Structure; Subcellular structure; Surface; Tail; Techniques; Testing; Time; Transport Process; Tubulin; Viral; Work; alpha Tubulin; base; fascinate; image processing; insight; migration; organizational structure; particle; small molecule; structural biology; tool

We recently described the discovery of a nucleus like structure assembled by phage 201Φ2-1 after infection of Pseudomonas chlororaphis. The phage nucleus is composed of a protein shell (GP105) that segregates phage and host bacterial proteins according to function, with metabolic enzymes and ribosomes in the cytoplasm and proteins related to DNA and RNA processing inside the phage nucleus. This compartment is centered by a bipolar spindle composed of the phage-encoded tubulin PhuZ. Capsids assemble on the bacterial membrane and then migrate to and dock on the surface of the phage nucleus where phage DNA is packaged. Ultimately, capsids assemble with tails to create mature particles and the cell lyses. The GP105 shell appears in our preliminary cryoEM as an irregularly shaped 5 nm wide border that encloses phage DNA. Remarkably, this shell allows selective entry or retention of specific proteins. This work raises a number of questions such as: Is the GP105 shell essential for phage replication? Are other proteins required for shell assembly? What is the structural organization of GP105 within the shell and how does it assemble and incorporate new subunits as it grows? Does it contain pores that allow diffusion of mRNA, proteins and small molecules in and out of the structure? How is the PhuZ spindle organized over time as it pushes the growing GP105 to midcell? Does the spindle participate in other aspects of phage development such as capsid movement? Here, we propose to use a combination of genetics, biochemistry, structural biology, and cell biology to study the nucleus like structure assembled by GP105 and the spindle assembled by PhuZ and determine how these two structures participate in viral lytic growth.

我们最近描述了噬菌体组装的核样结构的发现 201Φ2-1感染绿针假单胞菌后组成噬菌体细胞核。 蛋白质壳（GP105）的分离噬菌体和宿主细菌蛋白质 通过细胞质中的代谢酶和核糖体以及蛋白质发挥作用 与噬菌体核内的 DNA 和 RNA 加工有关。 以由噬菌体编码的微管蛋白衣壳组成的双极纺锤体为中心。 在细菌膜上组装，然后迁移并停靠在细菌表面 噬菌体DNA最终在噬菌体细胞核中组装。 尾部产生成熟颗粒并且细胞裂解出现在我们的细胞中。 初步冷冻电镜为不规则形状的 5 nm 宽边界，包围噬菌体 值得注意的是，这种外壳可以选择性地进入或保留特定的蛋白质。 这项工作提出了许多问题，例如： GP105 外壳对于噬菌体来说是必需的吗？ 复制是否需要其他蛋白质？其结构是什么？ GP105在外壳内的组织以及它如何组装和合并新的 它生长时是否含有允许 mRNA、蛋白质和蛋白质扩散的孔？ 结构内外的小分子是如何组织的？ 当它将生长的 GP105 推向 midcell 时，主轴是否参与其他操作？ 噬菌体发育的各个方面，例如衣壳运动？在这里，我们建议使用 结合遗传学、生物化学、结构生物学和细胞生物学来研究 GP105组装的核状结构和PhuZ组装的主轴和 确定这两种结构如何参与病毒裂解生长。