Protocol for live-cell fluorescence-guided cryoFIB-milling and electron cryo-tomography of virus-infected cells.

Here, we present a protocol for assessing virus-infected cells using electron cryo-tomography (cryoET). It includes the basic workflows of seeding cells, plunge-freezing, clipping, cryo-focused ion beam milling (cryoFIB-milling), and cryoET, as well as two optional modules: micropatterning and live-cell fluorescence microscopy. We use an A549 human cell line and the virus HAdV5-pIX-mcherry in this protocol, but the comprehensive workflow can be easily transferred to other cell types and different types of virus infection or treatment.

Cellular Electron Cryo-Tomography to Study Virus-Host Interactions.

Viruses are obligatory intracellular parasites that reprogram host cells upon infection to produce viral progeny. Here, we review recent structural insights into virus-host interactions in bacteria, archaea, and eukaryotes unveiled by cellular electron cryo-tomography (cryoET). This advanced three-dimensional imaging technique of vitreous samples in near-native state has matured over the past two decades and proven powerful in revealing molecular mechanisms underlying viral replication.