Sf6 as a model system for understanding the mechanisms of virus: host recognition

Sf6作为理解病毒机制的模型系统：宿主识别

• 批准号: 9262961
• 负责人: Kristin N Parent
• 金额: $ 27.86万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-05-01 至 2020-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9262961
• 关键词: Adenoviruses; Affect; Affinity; Agriculture; Antiviral Therapy; Bacteriophage Genetics; Bacteriophages; Binding; Biochemical; Biochemical Genetics; Biochemistry; Biological; Biological Assay; Biological Models; Biophysics; Cell membrane; Cell physiology; Cells; Complex; Coupled; Cryoelectron Microscopy; DNA; Development; Ensure; Epidemic; Escape Mutant; Event; Fluorescence; Genetic; Genome; Genomics; Human; In Vitro; Infection; Injectable; Injection of therapeutic agent; Kinetics; Laws; Lipopolysaccharides; Location; Mediating; Membrane; Membrane Proteins; Molecular; Molecular Conformation; Monitor; Mutagenesis; Mutation; Outcome; Peptides; Play; Process; Production; Proteins; Receptor Cell; Research; Resistance; Resolution; Role; Route; Shigella; Shigella flexneri; Signal Transduction; Structure; Surface; System; Tail; Testing; Thick; Time; Tropism; Variant; Vesicle; Viral; Viral Genome; Viral Proteins; Virion; Virus; Virus Diseases; biophysical properties; biophysical techniques; cell type; electron tomography; genetic analysis; genetic manipulation; human disease; in vivo; insight; interest; mutant; particle; pathogen; permissiveness; protein S precursor; protein protein interaction; public health relevance; receptor; receptor binding; receptor function; tomography

 DESCRIPTION (provided by applicant): Viruses infect their respective hosts efficiently through a regulated process of recognizing specific receptors and subsequently transferring genomic material across cell membrane barriers. To understand the underlying mechanisms that control virus infection, it is important to characterize viral events in a biologically relevat manner. I have been able to study Sf6 infection in the context of its host, Shigella flexneri, through classic phage analyses and have identified two receptors important for Sf6 infection (S. flexneri outer membrane proteins A & C, "Omps A & C"). OmpA is the preferred receptor but Sf6 can utilize OmpC as an alternate route for infection. I plan to study the interactions between Sf6 and S. flexneri to determine regions in both the phage proteins and the Omps that are critical for proper infection. I also plan to extend our understanding of Sf6 infection dynamics since this phage mimics infection in vitro by binding and delivering its genome into host-derived Outer Membrane Vesicles (OMVs). This provides an ideal system for using cryo-electron tomography to visualize intermediates that arise during Sf6 infection, and variant receptors as the OMVs are considerably thinner (~100 nm thick) compared to whole Shigella cells (~2000 nm thick), which are currently outside the limits of effective cryo-tomography. In vitro genome ejections are also possible using purified receptors. This makes Sf6 one of a few model systems to correlate structural transitions that arise during host cell recognition and resulting genomic transfer, and s currently the only model system for Adenovirus with alternate receptors known. We developed a time-lapse fluorescence assay that monitors real time genome ejection in vivo at the single particle level. This, combined with decades of groundwork on bacteriophage genetic manipulation and biochemical/biophysical characterization, allows us to study the process of Sf6 infection from several different angles and will provide insight into the generalized mechanisms by which all viruses recognize, and infect hosts. Sf6, as well as many other phages and eukaryotic viruses, has several so-called "ejection proteins" that are passed from the virion into the host during infection. Ejection proteins of Sf6 are of particular interest since they interact intimately with the host, which is a human pathogen. The location of ejection proteins pre- or post-infection has yet to be determined for any phage, but it is likely that they play a role in protecting the genome as it is injected into the host. Therefore, we predict the ejection proteins to be released in a sequential manner, and to affect the rate and efficiency of genomic transfer. We will test this hypothesis through biophysical approaches.

 描述（由适用提供）：病毒通过识别特定受体并随后在细胞膜屏障中转移基因组材料的调节过程有效地感染了其各自的宿主。要了解控制病毒感染的潜在机制，重要的是以生物学相关的方式表征病毒事件。我能够通过经典的噬菌体分析研究SF6感染的宿主志贺氏菌Flexneri，并确定了对SF6感染很重要的两个受体（S. flexneri外膜蛋白A＆C，“ OMPS A＆C”）。 OMPA是首选的受体，但SF6可以利用OMPC作为感染的替代途径。我计划研究SF6和S. flexneri之间的相互作用，以确定噬菌体蛋白和对适当感染至关重要的OMP的区域。我还计划扩展我们对SF6感染动​​力学的理解，因为这种噬菌体模仿感染通过结合并将其基因组传递到宿主衍生的外膜囊泡（OMV）中。这提供了一个理想的系统，用于使用冷冻电子层析成像来可视化SF6感染期间出现的中间体，并且与整个志贺氏菌细胞（〜2000 nm厚）相比，OMV的变体接收器较薄（〜100 nm厚），这些（〜2000 nm厚）目前超出了有效的低温训练的极限。使用纯化的受体也可以驱逐体外基因组。这使得SF6成为将在宿主细胞识别和产生的基因组转移过程中产生的结构跃迁相关的少数模型系统之一，而S当前是腺病毒的唯一模型系统与已知的替代受体。我们开发了一种延时荧光测定法，该测定在单个颗粒水平的体内监测实时基因组弹出。结合了数十年的噬菌体遗传操纵和生化/生物物理特征的基础，使我们能够从几个不同角度研究SF6感染的过程，并将洞悉所有病毒识别和感染宿主的广义机制。 SF6以及许多其他噬菌体和真核病毒都具有几种所谓的“弹出蛋白”，这些“射血蛋白”在感染过程中从病毒体传递到宿主。 SF6的射血蛋白特别感兴趣，因为它们与宿主密切相互作用，这是人类病原体。感染前或感染后的射血蛋白的位置尚未确定任何噬菌体，但由于将其注入宿主时，它们很可能在保护基因组方面发挥作用。因此，我们预测射血蛋白会以顺序释放，并影响基因组转移的速率和效率。我们将通过生物物理方法检验这一假设。