正链RNA病毒擅长重塑宿主细胞膜，以实现最佳病毒基因组复制和感染性后代的产生。这些超微结构的改变导致病毒膜细胞器的形成，并且可以通过不同的成像技术观察到，提供纳米分辨率。在共聚焦和电子显微镜的指导下，本研究描述了使用低温软 X 射线断层扫描 (cryo-SXT) 对 SARS-CoV-2 感染的人肺腺癌细胞生成宽视场体积。共聚焦显微镜显示，双链 RNA (dsRNA) 和核衣壳 (N) 蛋白在紧凑的核周结构中积累，在感染后期优先发现在中心体周围。透射电子显微镜（TEM）显示，受感染细胞核附近有膜结构的积累，形成病毒复制细胞器，其中含有特征性的双膜囊泡和较大囊泡结构内的病毒样颗粒。 Cryo-SXT 显示病毒复制细胞器与 TEM 观察到的病毒复制细胞器非常相似，但表明在 TEM 切片中观察到的囊泡细胞器确实是一个囊泡管状网络，在感染后期会扩大和拉长。总体而言，我们的数据为 SARS-CoV-2 复制细胞器的分子结构提供了更多见解。

Plus-strand RNA viruses are proficient at remodeling host cell membranes for optimal viral genome replication and the production of infectious progeny. These ultrastructural alterations result in the formation of viral membranous organelles and may be observed by different imaging techniques, providing nanometric resolution. Guided by confocal and electron microscopy, this study describes the generation of wide-field volumes using cryogenic soft-X-ray tomography (cryo-SXT) on SARS-CoV-2-infected human lung adenocarcinoma cells. Confocal microscopy showed accumulation of double-stranded RNA (dsRNA) and nucleocapsid (N) protein in compact perinuclear structures, preferentially found around centrosomes at late stages of the infection. Transmission electron microscopy (TEM) showed accumulation of membranous structures in the vicinity of the infected cell nucleus, forming a viral replication organelle containing characteristic double-membrane vesicles and virus-like particles within larger vesicular structures. Cryo-SXT revealed viral replication organelles very similar to those observed by TEM but indicated that the vesicular organelle observed in TEM sections is indeed a vesiculo-tubular network that is enlarged and elongated at late stages of the infection. Overall, our data provide additional insight into the molecular architecture of the SARS-CoV-2 replication organelle.