Characterization of a bacteriophage tubulin involved in viral replication

参与病毒复制的噬菌体微管蛋白的表征

基本信息

批准号：
8420103
负责人：
DAVID A. AGARD
金额：
$ 38.89万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-08-01 至 2017-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8420103
关键词：
Affect Algorithms Bacteria Bacteriophages Biochemical Biological Assay Capsid Cell division Cell physiology Cells Complex Cryoelectron Microscopy Cytolysis Cytoskeletal Proteins Cytoskeleton DNA DNA biosynthesis Deltastab Diffusion Equipment Eukaryota Event Family Filament GTP Binding Genetic Genome Growth Head Herpesviridae Hydrolysis Image In Vitro Infection Institution Kinetics Lead Life Lighting Lytic Lytic Phase Methods Microscope Microscopy Microtubules Modeling Molecular Morphology Movement Mutagenesis Nature Photobleaching Plasmids Play Polymers Positioning Attribute Process Prokaryotic Cells Property Proteins Proteomics Pseudomonas Regulation Reproduction Research Resolution Role Series Signal Transduction Site Staging Structure Tail Testing Time Tubulin Viral Viral Physiology Width Work base cell assembly design electron tomography in vivo insight mutant novel particle plasmid DNA polymerization public health relevance reconstruction segregation

项目摘要

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.

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。尽管这些蛋白质在多种细胞过程中发挥着关键作用，但构成原核细胞骨架的蛋白质仍然知之甚少。在细菌中，仅对几个不同的微管蛋白家族进行了表征：FtsZ、 TubZ 是一种广泛分布的对细胞分裂至关重要的蛋白质，参与质粒分离，而 BtubA/BtubB 的功能仍不清楚。我们最近发现了一种不同的微管蛋白样细胞骨架蛋白 PhuZ，由非常大 (317 kb) 绿针假单胞菌噬菌体 201?2-1 编码。通过在假单胞菌中低水平表达 GFP 标记的 PhuZ，我们可以观察裂解噬菌体感染期间的丝状形成。我们将 PhuZ 的结构解析至 1.67A 分辨率，并发现了一个保守的微管蛋白折叠，具有一个新颖的、延伸的 C 末端，我们证明它对于体外和体内的聚合至关重要。令人惊讶的是，我们发现 PhuZ 组装了一个动态纺锤体，在裂解生长过程中将单个大型噬菌体 DNA 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 PhuZ 突变体，我们可以证明 PhuZ 丝的动态性质是噬菌体居中所必需的。细菌病毒颗粒似乎聚集在这个中心 DNA 团块的周围，形成类似于疱疹病毒复制工厂的冠状结构，而疱疹病毒与 dsDNA 噬菌体有远亲关系。这是原核纺锤体的第一个例子，它执行类似于真核生物基于微管的纺锤体的基因组定心功能。在这里，我们建议阐明 PhuZ 使 DNA 居中的能力的生化、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。聚合物和定心的可能作用包括：定义协调复制和包装的位点、促进噬菌体头和/或尾部组装以及促进细胞裂解。我们不仅要寻求回答这些问题，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。具体来说，我们提出以下研究目标： 1.检查PhuZ在病毒裂解生长中的作用。 2. 检查PhuZ组装与体内DNA复制和运动、噬菌体组装和细胞裂解之间可能的联系。 3. 从结构和功能上表征体外组装的 PhuZ 丝的机制和特性。 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、定位或功能的其他方面。 5. 在感染的各个阶段进行电子断层扫描和冷冻断层扫描，以获得对裂解生长过程中体内组装的 PhuZ 和病毒衣壳的结构组织的高分辨率见解。