Molecular and cellular biology of the phage nucleus and spindle

我们最近描述了一种由噬菌体组装的类核结构的发现 201 Φ 2 - 1感染绿针假单胞菌。噬菌体核由 分离噬菌体和宿主细菌蛋白质的蛋白质外壳（GP105）的 与细胞质和蛋白质中的代谢酶和核糖体一起发挥作用 与噬菌体核内的DNA和RNA加工有关。该隔室 由噬菌体编码的微管蛋白pZ组成的双极纺锤体为中心。衣壳 在细菌膜上组装，然后迁移到 噬菌体DNA被包装的噬菌体核。最终，衣壳与 形成成熟的颗粒，然后细胞裂解。GP105外壳出现在我们的 初步cryoEM作为包围噬菌体的不规则形状的5 nm宽的边界 DNA.值得注意的是，这种外壳允许特定蛋白质的选择性进入或保留。这 这项工作提出了一些问题，如：GP105外壳是噬菌体必需的吗？ 复制？壳组装需要其他蛋白质吗？什么是结构 GP105在外壳内的组织，以及它如何组装和整合新的 亚单位，因为它的增长？它是否含有允许mRNA、蛋白质和 小分子在结构中的进出如何组织的Z-Z主轴在 时间，因为它推动不断增长的GP105到中间细胞？主轴是否参与其他 噬菌体的发展方面，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 由GP105组装的类核结构和由GSTZ组装的纺锤体， 确定这两种结构如何参与病毒裂解生长。

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.