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. For complete details on the use and execution of this protocol, please refer to Pfitzner et al. (2021).

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. Initial studies were restricted to cell peripheries and typically focused on early infection steps, analyzing surface proteins and viral entry. Recent developments including cryo-thinning techniques, phase-plate imaging, and correlative approaches have been instrumental in also targeting rare events inside infected cells. When combined with advances in dedicated image analyses and processing methods, details of virus assembly and egress at (sub)nanometer resolution were uncovered. Altogether, we provide a historical and technical perspective and discuss future directions and impacts of cryoET for integrative structural cell biology analyses of viruses.