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

 描述（由申请方提供）：病毒通过识别特异性受体并随后将基因组物质转移穿过细胞膜屏障的调节过程有效感染其各自的宿主。为了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。 通过经典的噬菌体分析，我已经能够在其宿主福氏志贺菌的背景下研究Sf6感染，并且已经鉴定出对Sf6感染重要的两种受体（S.弗氏外膜蛋白A & C，“Omps A & C”）。OmpA是优选的受体，但Sf6可以利用OmpC作为感染的替代途径。我计划研究Sf6和S之间的相互作用。以确定噬菌体蛋白和Omp中对适当感染至关重要的区域。我还计划扩展我们对Sf6感染动力学的理解，因为这种噬菌体通过结合并将其基因组递送到宿主衍生的外膜囊泡（OMV）中来模拟体外感染。这提供了一种理想的系统，用于使用冷冻电子断层扫描来可视化Sf6感染期间出现的中间体和变体受体，因为与目前在有效冷冻断层扫描的限制之外的整个志贺氏菌细胞（约2000 nm厚）相比，OMV相当薄（约100 nm厚）。使用纯化的受体也可以进行体外基因组排出。这使得Sf6成为将宿主细胞识别和所产生的基因组转移期间出现的结构转变相关联的少数模型系统之一，并且是目前唯一的具有已知替代受体的腺病毒模型系统。我们开发了一种延时荧光分析，监测真实的时间基因组喷射在体内的单粒子水平。这一点，结合几十年的噬菌体遗传操作和生物化学/生物物理特性的基础，使我们能够从几个不同的角度研究Sf6感染的过程，并将提供深入了解所有病毒识别和感染宿主的一般机制。 Sf6，以及许多其他的杆状病毒和真核病毒，有几个所谓的“排出蛋白”，在感染过程中从病毒体传递到宿主。Sf6的喷射蛋白是特别感兴趣的，因为它们与宿主（人类病原体）密切相互作用。对于任何噬菌体来说，感染前或感染后排出蛋白的位置还有待确定，但它们很可能在注射到宿主中时在保护基因组方面发挥作用。因此，我们预测排出蛋白以顺序的方式释放，并影响基因组转移的速率和效率。我们将通过生物物理学方法来检验这一假设。

 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.