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

 描述(由申请人提供)：病毒通过识别特定受体并随后跨越细胞膜屏障转移基因组材料的调控过程，有效地感染各自的宿主。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。我已经能够通过经典的噬菌体分析在其宿主福氏志贺氏菌的背景下研究SF6感染，并确定了两个对SF6感染重要的受体(福氏志贺氏菌外膜蛋白A和C，即OMPS A和C)。OmpA是首选受体，但SF6可以利用OMPC作为感染的替代途径。我计划研究SF6和福氏志贺菌之间的相互作用，以确定噬菌体蛋白和OMP中对正确感染至关重要的区域。我还计划扩大我们对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传递到宿主来源的外膜小泡(OMV)来模拟体外感染。这为使用冷冻电子断层扫描显示SF6感染过程中出现的中间产物和不同的受体提供了理想的系统，因为与整个志贺氏菌细胞(约2000 nm厚)相比，OMV要薄得多(约100 nm厚)，后者目前超出了有效的冷冻断层扫描的限制。在体外，使用纯化的受体也可以进行基因组弹射。这使得SF6成为少数几个将宿主细胞识别和由此产生的基因组转移过程中出现的结构转变联系起来的模型系统之一，S也是目前已知的唯一具有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方法，可以在单个粒子水平上实时监测体内基因组的喷射。结合几十年来在噬菌体遗传操作和生化/生物物理特性方面的基础工作，我们可以从几个不同的角度研究SF6感染的过程，并将深入了解所有病毒识别和感染宿主的一般机制。与许多其他噬菌体和真核病毒一样，SF6有几种所谓的“喷射蛋白”，在感染期间从病毒粒子进入宿主。SF6的喷射物蛋白特别令人感兴趣，因为它们与宿主密切相互作用，宿主是人类的病原体。对于任何噬菌体，感染前或感染后喷出蛋白的位置尚未确定，但它们很可能在基因组被注入宿主时发挥保护作用。因此，我们预测喷射蛋白将以顺序的方式释放，并影响基因组转移的速度和效率。我们将通过生物物理学的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.