A novel immunogold cryoelectron microscopic approach to investigate the structure of the intracellular and extracellular forms of vaccinia virus
A novel immunogold cryoelectron microscopic approach to investigate the structure of the intracellular and extracellular forms of vaccinia virus
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DOI：
10.1002/j.1460-2075.1996.tb00590.x
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发表时间：
1996-05-15
期刊：
影响因子：
11.4
通讯作者：
Griffiths, G
中科院分区：
文献类型：
--
作者：
Roos, N;Cyrklaff, M;Griffiths, G
我们介绍了一种将免疫金标记与薄玻璃化标本的冷冻电子显微镜相结合的新方法，该方法利用了悬浮液中的颗粒在空气-水界面处浓缩并在随后的免疫金标记过程中保持在那里的观察，随后，可以形成薄水膜，该膜被玻璃化并通过冷冻电子显微镜观察，在我们看来，牛痘病毒装配的关键早期步骤，即球形未成熟病毒的形成，涉及内质网和高尔基体之间的中间室的特化池状结构域的形成。使用这种新的冷冻电子显微镜的方法，我们表明，在细胞内成熟病毒（IMV）的核心仍然包围着一个膜池，脱落的病毒核心治疗后，与二硫苏糖醇，暴露抗原的病毒表面：核心。互补蛋白酶的研究表明，IMV可能是密封的膜融合，但由蛋白质结构，中断外膜，我们还描述了结构和膜拓扑结构的第二种感染形式的牛痘，细胞外包膜病毒，并确认这种形式具有额外的膜覆盖IMV。
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.