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

• 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
• 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

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，只有几个不同的微管蛋白家族被表征：FtsZ，