ABSTRACT
Pathogenesis induced by SARS-CoV-2 is thought to result from both an inflammation-dominated cytokine response and virus-induced cell perturbation causing cell death. Here, we employ an integrative imaging analysis to determine morphological organelle alterations induced in SARS-CoV-2-infected human lung epithelial cells. We report 3D electron microscopy reconstructions of whole cells and subcellular compartments, revealing extensive fragmentation of the Golgi apparatus, alteration of the mitochondrial network and recruitment of peroxisomes to viral replication organelles formed by clusters of double-membrane vesicles (DMVs). These are tethered to the endoplasmic reticulum, providing insights into DMV biogenesis and spatial coordination of SARS-CoV-2 replication. Live cell imaging combined with an infection sensor reveals profound remodeling of cytoskeleton elements. Pharmacological inhibition of their dynamics suppresses SARS-CoV-2 replication. We thus report insights into virus-induced cytopathic effects and provide alongside a comprehensive publicly available repository of 3D datasets of SARS-CoV-2-infected cells for download and smooth online visualization.
Subject(s)
COVID-19/genetics , Endoplasmic Reticulum/ultrastructure , SARS-CoV-2/ultrastructure , Viral Replication Compartments/ultrastructure , COVID-19/diagnostic imaging , COVID-19/pathology , COVID-19/virology , Cell Death/genetics , Endoplasmic Reticulum/genetics , Endoplasmic Reticulum/virology , Humans , Microscopy, Electron , Pandemics , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity , Viral Replication Compartments/metabolism , Virus Replication/geneticsABSTRACT
The visualization of cellular ultrastructure over a wide range of volumes is becoming possible by increasingly powerful techniques grouped under the rubric "volume electron microscopy" or volume EM (vEM). Focused ion beam scanning electron microscopy (FIB-SEM) occupies a "Goldilocks zone" in vEM: iterative and automated cycles of milling and imaging allow the interrogation of microns-thick specimens in 3-D at resolutions of tens of nanometers or less. This bestows on FIB-SEM the unique ability to aid the accurate and precise study of architectures of virus-cell interactions. Here we give the virologist or cell biologist a primer on FIB-SEM imaging in the context of vEM and discuss practical aspects of a room temperature FIB-SEM experiment. In an in vitro study of SARS-CoV-2 infection, we show that accurate quantitation of viral densities and surface curvatures enabled by FIB-SEM imaging reveals SARS-CoV-2 viruses preferentially located at areas of plasma membrane that have positive mean curvatures.