Characterization of a bacteriophage tubulin involved in viral replication

描述(申请人提供)：细胞骨架蛋白起源古老，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发挥着关键作用，但组成原核细胞骨架的蛋白质仍然缺乏明确的定义。在细菌中，只有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，参与了质粒分离和BtubA/BtubB，其功能尚不清楚。我们最近发现了一个发散的微管蛋白样细胞骨架蛋白PhuZ，它由非常大(317kb)的绿假单胞菌噬菌体201？2-1编码。通过在假单胞菌中低水平表达GFP标记的PhuZ，我们可以观察到裂解噬菌体感染过程中细丝的形成。我们将PhuZ的结构解析到1.67A分辨率，发现了一个保守的微管蛋白折叠，它具有一个新的、延长的C末端，我们证明这对体外和体内的聚合都是关键的。令人惊讶的是，我们发现PhuZ组装了一个动态的纺锤体，在裂解生长过程中将单个大型的噬菌体DNA复合体定位在细胞的中心。此外，使用根据我们的结构设计的PhuZ突变体，我们可以证明PhuZ细丝的动态性质是噬菌体中心所必需的。细菌病毒颗粒似乎聚集在这个中心DNA团的外围，形成一种类似于疱疹病毒复制工厂的日冕状结构，疱疹病毒与dsDNA噬菌体有遥远的亲缘关系。这是原核生物纺锤体执行基因组中心功能的第一个例子，类似于真核生物基于微管的纺锤体的作用。在这里，我们建议阐明PhuZ以DNA为中心的能力的生化、结构和遗传基础，以及该聚合物参与病毒裂解生长的潜在机制。聚合物和中心的合理作用包括：定义一个协调复制和包装的位置，促进噬菌体头部和/或尾部的组装，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、分离质粒DNA和将DNA组织到复制工厂等不同功能提供新的见解。具体地说，我们提出了以下研究目标：1.检测PhuZ在病毒裂解生长中的作用。2.在体内检测PhuZ组装与DNA复制和移动、噬菌体组装和细胞裂解之间的可能联系。3.对体外组装的PhuZ纤维的结构和性能进行了表征。4.鉴定与PhuZ相互作用的噬菌体和宿主蛋白，并确定它们是否影响PhuZ聚合、定位或其他方面的功能。5.在感染的不同阶段进行电子断层扫描和冷冻断层扫描，以高分辨率了解PhuZ和病毒衣壳在体内裂解生长过程中组装的结构组织。

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, 201?2-1. 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.