Institute of Virology, University Medical Center Ulm, Albert-Einstein-Allee, 89081 Ulm, Germany.
Cell Microbiol. 2013 Feb;15(2):305-14. doi: 10.1111/cmi.12077. Epub 2012 Dec 20.
Electron microscopy (EM) allows visualization of viruses in fixed cells with high resolution. High-pressure freezing for sample fixation in combination with freeze substitution and embedding in resin improves significantly the preservation of cellular structures and specifically of membranes. This advancement allows better visualization of human cytomegalovirus (HCMV) morphogenesis occurring at membranes. To obtain comprehensive information on viral phenotypes from ultrastructural images it is important to also quantify morphological phenotypes. This again can be much refined by three-dimensional visualization after serial sectioning. For elucidation of dynamic processes three-dimensional tomography is extremely helpful. We analysed interaction of HCMV particles with host cell membranes during final envelopment. Both wild-type virus and a viral mutant with impaired envelopment were analysed in fibroblasts, but also using in vivo relevant human endothelial cells and macrophages. The quantification of the EM data showed similar ultrastructural phenotypes regarding the envelopment efficiency in the different cell types indicating similar mechanisms in late stages of virus morphogenesis. Furthermore, thorough analysis of the viral assembly complex (AC) - a virus-induced cytosolic structure - by using three-dimensional visualization techniques combined with a quantitative analysis revealed that the events of final envelopment are equally distributed within the AC irrespective of different local membrane composition.
电子显微镜（EM）可用于高分辨率观察固定细胞中的病毒。高压冷冻固定与冷冻替代和树脂包埋相结合，可显著改善细胞结构的保存，特别是膜的保存。这一进步使人们能够更好地观察到发生在膜上的人巨细胞病毒（HCMV）形态发生。为了从超微结构图像中获得有关病毒表型的综合信息，对形态表型进行定量也很重要。通过对连续切片进行三维可视化，可以进一步细化这一定量。为了阐明动态过程，三维断层摄影术非常有帮助。我们分析了 HCMV 颗粒与宿主细胞膜在最终包膜过程中的相互作用。野生型病毒和包膜缺陷的病毒突变体均在成纤维细胞中进行了分析，但也在体内相关的人内皮细胞和巨噬细胞中进行了分析。对 EM 数据的定量分析显示，不同细胞类型的包膜效率具有相似的超微结构表型，表明在病毒形态发生的晚期存在相似的机制。此外，通过使用三维可视化技术结合定量分析，对病毒组装复合物（AC）（一种病毒诱导的细胞质结构）进行彻底分析表明，最终包膜的事件在 AC 内均匀分布，与不同的局部膜组成无关。
