Characterization of a bacteriophage tubulin involved in viral replication

描述（由申请人提供）：细胞骨架蛋白具有古老的起源，早于原核生物和真核生物的分化。虽然这些蛋白质在各种细胞过程中起着关键作用，但构成原核细胞骨架的蛋白质仍然定义不清。在细菌中，只有少数几个不同的微管蛋白家族被表征：FtsZ， 一种广泛分布的对细胞分裂至关重要的蛋白质，TubZ，涉及质粒分离和BtubA/BtubB，其功能仍然未知。我们最近发现了一个分歧的微管蛋白样细胞骨架蛋白，cytoskeleton Z，编码的非常大的（317 kb）绿针假单胞菌噬菌体，201 2-1。通过在假单胞菌中以低水平表达GFP标记的p53 Z，我们可以观察到裂解性噬菌体感染期间的丝状体形成。我们解决了1.67 A分辨率的结构的CITZ，并发现了一个保守的微管蛋白折叠与一个新的，延长的C-末端，我们表明是关键的聚合在体外和体内。令人惊讶的是，我们发现，在裂解生长过程中，噬菌体Z组装了一个动态纺锤体，将一个单一的噬菌体DNA大复合物定位在细胞的中心。此外，使用根据我们的结构设计的BZZ突变体，我们可以证明BZZ细丝的动态性质是噬菌体定中心所必需的。细菌病毒颗粒似乎聚集在这个中心DNA质量的周围，产生类似于疱疹病毒复制工厂的冠状结构，疱疹病毒与dsDNA噬菌体有很远的关系。这是第一个原核纺锤体的例子，它执行类似于真核生物微管纺锤体的作用的基因组中心功能。在这里，我们建议阐明的生物化学，结构和遗传基础的能力，cDNAZ中心的DNA和潜在的机制，该聚合物参与病毒裂解生长。聚合物和中心的合理作用包括：确定一个位点来协调复制和包装，促进噬菌体头部和/或尾部组装，以及促进细胞裂解。我们不仅将寻求回答这些问题，而且我们的工作还将为微管蛋白家族聚合物如何参与细胞分裂，质粒DNA分离和将DNA组织成复制工厂等不同功能提供新的见解。具体而言，我们提出以下研究目标：1.检查CXZ在病毒裂解生长中的作用。2.研究了在活体内，噬菌体组装和DNA复制和移动、噬菌体组装和细胞裂解之间可能存在的联系。3.从结构和功能上表征了体外组装的BZ丝的机制和性能。4.鉴定与BZ相互作用的噬菌体和宿主蛋白，并确定它们是否影响BZ聚合、定位或其他功能方面。5.在感染的各个阶段进行电子断层扫描和冷冻断层扫描，以获得高分辨率的洞察力，了解裂解生长过程中体内组装的病毒衣壳和病毒衣壳的结构组织。

DESCRIPTION (provided by applicant): Cytoskeletal proteins are of ancient origin, predating the divergence of prokaryotes and eukaryotes. Although these proteins play key roles in a variety of cellular processes, the proteins that make up the prokaryotic cytoskeleton are still poorly defined. In bacteria, only a few distinct families of tubulin have been characterized: FtsZ, a widely distributed protein critical for cell division, TubZ, involved in plasmid segregation and BtubA/BtubB, whose functions are still unknown. We recently discovered a divergent tubulin-like cytoskeletal protein, PhuZ, encoded by the very large (317 kb) Pseudomonas chlororaphis bacteriophage, 201ɸ2-1. By expressing a GFP-tagged PhuZ at low levels in Pseudomonas, we could observe filament formation during lytic phage infection. We solved the structure of PhuZ to 1.67A resolution, and found a conserved tubulin fold with a novel, extended C-terminus that we showed to be critical for polymerization both in vitro and in vivo. Surprisingly, we found that PhuZ assembles a dynamic spindle that positions a single large complex of phage DNA at the center of the cell during lytic growth. Moreover, using PhuZ mutants designed from our structure, we could show that the dynamic nature of PhuZ filaments is required for phage centering. Bacterial viral particles appear to assemble around the periphery of this central DNA mass, creating a corona-like structure similar to the replication factories of herpes viruses, whic are distantly related to dsDNA bacteriophage. This is the first example of a prokaryotic spindle that performs a genome centering function analogous to the role of microtubule-based spindles of eukaryotes. Here, we propose to elucidate the biochemical, structural, and genetic basis of the ability of PhuZ to center DNA and the underlying mechanisms by which the polymer participates in viral lytic growth. Plausible roles for the polymer and centering include: defininga site to coordinate replication and packaging, facilitating phage head and or tail assembly, and facilitating cell lysis. Not only will we seek to answer these questions, but our work will also provide new insights into how tubulin family polymers can participate in such divergent functions as cell division, separation of plasmid DNA and organizing DNA into replication factories. Specifically, we propose the following research aims: 1. Examine the role of PhuZ in viral lytic growth. 2. Examine the possible connections between PhuZ assembly and DNA replication and movement, phage assembly and cell lysis in vivo. 3. Structurally and functionally characterize the mechanism and properties of PhuZ filaments assembled in vitro. 4. Identify phage and host proteins that interact with PhuZ and determine if they affect PhuZ polymerization, localization or other aspects of function. 5. Perform electron tomography and cryoTomography at various stages of infection to gain high resolution insights into the structural organization of PhuZ and viral capsids assembled in vivo during lytic growth.