Zhang Peijun, Mendonca Luiza, Howe Andrew, Gilchrist James, Sun Dapeng, Knight Michael, Zanetti-Domingues Laura, Bateman Benji, Krebs Anna-Sophia, Chen Long, Radecke Julika, Sheng Yuewen, Li Vivian, Ni Tao, Kounatidis Ilias, Koronfel Mohamed, Szynkiewicz Marta, Harkiolaki Maria, Martin-Fernandez Marisa, James William
University of Oxford.
Diamond Light Source.
Res Sq. 2021 Jan 19:rs.3.rs-134794. doi: 10.21203/rs.3.rs-134794/v1.
Since the outbreak of the SARS-CoV-2 pandemic, there have been intense structural studies on purified recombinant viral components and inactivated viruses. However, structural and ultrastructural evidence on how the SARS-CoV-2 infection progresses in the frozen-hydrated native cellular context is scarce, and there is a lack of comprehensive knowledge on the SARS-CoV-2 replicative cycle. To correlate the cytopathic events induced by SARS-CoV-2 with virus replication process under the frozen-hydrated condition, here we established a unique multi-modal, multi-scale cryo-correlative platform to image SARS-CoV-2 infection in Vero cells. This platform combines serial cryoFIB/SEM volume imaging and soft X-ray cryo-tomography with cell lamellae-based cryo-electron tomography (cryoET) and subtomogram averaging. The results place critical SARS-CoV-2 structural events â€" e.g. viral RNA transport portals on double membrane vesicles, virus assembly and budding intermediates, virus egress pathways, and native virus spike structures from intracellular assembled and extracellular released virus - in the context of whole-cell images. The latter revealed numerous heterogeneous cytoplasmic vesicles, the formation of membrane tunnels through which viruses exit, and the drastic cytoplasm invasion into the nucleus. This integrated approach allows a holistic view of SARS-CoV-2 infection, from the whole cell to individual molecules.
自新冠疫情爆发以来，人们对纯化的重组病毒成分和灭活病毒进行了深入的结构研究。然而，关于严重急性呼吸综合征冠状病毒2（SARS-CoV-2）在冷冻水合的天然细胞环境中感染如何进展的结构和超微结构证据却很稀少，并且对于SARS-CoV-2复制周期缺乏全面了解。为了将SARS-CoV-2诱导的细胞病变事件与冷冻水合条件下的病毒复制过程相关联，我们在此建立了一个独特的多模态、多尺度冷冻关联平台，用于对Vero细胞中的SARS-CoV-2感染进行成像。该平台将连续冷冻聚焦离子束/扫描电子显微镜（cryoFIB/SEM）体积成像和软X射线冷冻断层扫描与基于细胞薄片的冷冻电子断层扫描（cryoET）及亚断层平均相结合。结果将关键的SARS-CoV-2结构事件——例如双膜囊泡上的病毒RNA转运通道、病毒组装和出芽中间体、病毒释放途径以及细胞内组装和细胞外释放病毒的天然病毒刺突结构——置于全细胞图像的背景中。后者揭示了众多异质性的细胞质囊泡、病毒通过其排出的膜隧道的形成以及细胞质对细胞核的剧烈侵袭。这种综合方法能够从全细胞到单个分子对SARS-CoV-2感染进行全面观察。
