Localization of the Houdinisome (Ejection Proteins) inside the Bacteriophage P22 Virion by Bubblegram Imaging.
Localization of the Houdinisome (Ejection Proteins) inside the Bacteriophage P22 Virion by Bubblegram Imaging.
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DOI：
10.1128/mbio.01152-16
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发表时间：
2016-08-09
期刊：
影响因子：
6.4
通讯作者：
Steven AC
中科院分区：
文献类型：
--
作者：
Wu W;Leavitt JC;Cheng N;Gilcrease EB;Motwani T;Teschke CM;Casjens SR;Steven AC
P22衣壳是T=7的二十面体对称蛋白质壳，在一个5重顶点处具有门户蛋白十二聚体。从该顶点向外延伸的是一个短尾，向内延伸的是一个15 nm长的α-螺旋桶，由门蛋白亚基的C-末端结构域形成。除了密集包装的基因组外，衣壳还含有三种“排出蛋白”（E-蛋白[gp 7，gp 16和gp 20]），这些蛋白在DNA递送到靶细胞的过程中从紧密密封的衣壳中排出。我们通过定量SDS-PAGE估计它们的拷贝数为gp 16和gp 7的每个病毒粒子约12个分子和gp 20的30个拷贝。为了定位它们，我们使用了气泡图成像，这是一种低温电子显微镜的适应，其中通过长时间照射在蛋白质中诱导的气泡用于绘制蛋白质的位置。我们将这种技术应用于野生型P22，一个三重突变体缺乏所有三个E-蛋白，和三个突变体，每个缺乏一个E-蛋白。我们的结论是，所有三个E-蛋白松散地聚集在门户轴周围，在该地区径向向内位移的门户冠。气泡图数据表明，在门静脉晶体结构中看到的α螺旋桶的大约一半在成熟病毒体中是无序的，并且部分无序区域存在E蛋白的结合位点。如此定位，E-蛋白被策略性地放置以在感染期间从衣壳离开时向下通过缩短的桶并通过门环和尾部。虽然人们长期以来一直认为衣壳是病毒基因组的传递载体，但现在越来越多的人意识到病毒也将蛋白质传递到其宿主细胞中。P22有三种这样的蛋白质（排出蛋白[E蛋白]），尽管它们的量很大（总共2.5 MDa），但它们在病毒体中的初始位置仍然未知。这项研究成功地定位他们的新技术的气泡成像。P22 E蛋白分布于门管开口周围。有趣的是，这个晶体结构中15 nm长的桶只有原位的一半长：剩下的无序部分似乎是E蛋白的结合位点。这些观察记录了一个壮观的例子，在一个超分子系统中的监管秩序-无序过渡，并证明了气泡图成像的潜力，以映射其他病毒的组件，以及细胞复合物。
The P22 capsid is a T=7 icosahedrally symmetric protein shell with a portal protein dodecamer at one 5-fold vertex. Extending outwards from that vertex is a short tail, and putatively extending inwards is a 15-nm-long α-helical barrel formed by the C-terminal domains of portal protein subunits. In addition to the densely packed genome, the capsid contains three “ejection proteins” (E-proteins [gp7, gp16, and gp20]) destined to exit from the tightly sealed capsid during the process of DNA delivery into target cells. We estimated their copy numbers by quantitative SDS-PAGE as approximately 12 molecules per virion of gp16 and gp7 and 30 copies of gp20. To localize them, we used bubblegram imaging, an adaptation of cryo-electron microscopy in which gaseous bubbles induced in proteins by prolonged irradiation are used to map the proteins’ locations. We applied this technique to wild-type P22, a triple mutant lacking all three E-proteins, and three mutants each lacking one E-protein. We conclude that all three E-proteins are loosely clustered around the portal axis, in the region displaced radially inwards from the portal crown. The bubblegram data imply that approximately half of the α-helical barrel seen in the portal crystal structure is disordered in the mature virion, and parts of the disordered region present binding sites for E-proteins. Thus positioned, the E-proteins are strategically placed to pass down the shortened barrel and through the portal ring and the tail, as they exit from the capsid during an infection. While it has long been appreciated that capsids serve as delivery vehicles for viral genomes, there is now growing awareness that viruses also deliver proteins into their host cells. P22 has three such proteins (ejection proteins [E-proteins]), whose initial locations in the virion have remained unknown despite their copious amounts (total of 2.5 MDa). This study succeeded in localizing them by the novel technique of bubblegram imaging. The P22 E-proteins are seen to be distributed around the orifice of the portal barrel. Interestingly, this barrel, 15 nm long in a crystal structure, is only about half as long in situ: the remaining, disordered, portion appears to present binding sites for E-proteins. These observations document a spectacular example of a regulatory order-disorder transition in a supramolecular system and demonstrate the potential of bubblegram imaging to map the components of other viruses as well as cellular complexes.