Roos N, Cyrklaff M, Cudmore S, Blasco R, Krijnse-Locker J, Griffiths G
Cell Biology Programme, European Molecular Biology Laboratory, Heidelberg, Germany.
EMBO J. 1996 May 15;15(10):2343-55.
We introduce a novel approach for combining immunogold labelling with cryoelectron microscopy of thin vitrified specimens. The method takes advantage of the observation that particles in suspension are concentrated at the air-water interface and remain there during the subsequent immunogold labelling procedure. Subsequently, a thin aqueous film can be formed that is vitrified and observed by cryoelectron microscopy. In our view, a key early step in the assembly of vaccinia virus, the formation of the spherical immature virus, involves the formation of a specialized cisternal domain of the intermediate compartment between the endoplasmic reticulum and the Golgi. Using this novel cryoelectron microscopy approach, we show that in the intracellular mature virus (IMV) the core remains surrounded by a membrane cisterna that comes off the viral core upon treatment with dithiothreitol, exposing an antigen on the surface of the viral core. Complementary protease studies suggest that the IMV may be sealed not by membrane fusion but by a proteinaceous structure that interrupts the outer membrane. We also describe the structure and membrane topology of the second infectious form of vaccinia, the extracellular enveloped virus, and confirm that this form possesses an extra membrane overlying the IMV.
我们介绍了一种将免疫金标记与超薄玻璃化标本的冷冻电子显微镜相结合的新方法。该方法利用了悬浮颗粒会在空气-水界面聚集，并在随后的免疫金标记过程中保留在那里的这一观察结果。随后，可以形成一层薄水膜，将其玻璃化并通过冷冻电子显微镜进行观察。我们认为，痘苗病毒组装过程中的一个关键早期步骤，即球形未成熟病毒的形成，涉及在内质网和高尔基体之间的中间隔室中形成一个特殊的池状结构域。使用这种新型冷冻电子显微镜方法，我们发现，在细胞内成熟病毒（IMV）中，核心仍然被一个膜池包围，在用二硫苏糖醇处理后，该膜池会从病毒核心脱离，暴露出病毒核心表面的一种抗原。互补的蛋白酶研究表明，IMV可能不是通过膜融合来封闭，而是通过一种中断外膜的蛋白质结构来封闭。我们还描述了痘苗病毒的第二种感染形式，即细胞外被膜病毒的结构和膜拓扑结构，并证实这种形式在IMV上有一层额外的膜。
