Abstract
Since the outbreak of the SARS-CoV-2 pandemic, there have been intense structural studies on purified viral components and inactivated viruses. However, structural and ultrastructural evidence on how the SARS-CoV-2 infection progresses in the native cellular context is scarce, and there is a lack of comprehensive knowledge on the SARS-CoV-2 replicative cycle. To correlate cytopathic events induced by SARS-CoV-2 with virus replication processes in frozen-hydrated cells, 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. Here we report critical SARS-CoV-2 structural events – e.g. viral RNA transport portals, virus assembly intermediates, virus egress pathway, and native virus spike structures, in the context of whole-cell volumes revealing drastic cytppathic changes. This integrated approach allows a holistic view of SARS-CoV-2 infection, from the whole cell to individual molecules.
Highlights
Since the outbreak of the SARS-CoV-2 pandemic, there have been intense structural studies on purified viral components and inactivated viruses
A molecular pore has been described in murine hepatitis coronavirus (MHV) and SARS-CoV-2 that can serve as export portal for the mRNA and positive-strand viral genome copies[27]
To image and investigate SARS-CoV-2 replication in the context of the intracellular universe under nearphysiological conditions, we infected Vero cells grown on indexed electron microscopy (EM) grids with SARS-CoV-2 at 0.1 and 0.5 multiplicity of infection (MOI)
Summary
Since the outbreak of the SARS-CoV-2 pandemic, there have been intense structural studies on purified viral components and inactivated viruses. We exploited a unique correlative multi-modal multi-scale cryo-imaging approach to investigate SARS-CoV-2 replication in Vero cells under near-native conditions This approach empowers a holistic view of SARS-CoV-2 infection, from the whole cell to individual virus spike molecules, revealing pathways of SARS-CoV-2 assembly and egress and cytopathic effects of SARS-CoV-2 infection. These results substantiate previous findings obtained by thin-sectioning electron microscopy (EM) and serial focused ion beam scanning electron microscopy (FIB/SEM) of stained plastic-embedded samples[36] and further expand our knowledge of SARS-CoV-2 assembly and egress[4], as well as its presence in other membrane compartments It validates serial cryoFIB/SEM and soft X-ray tomography as techniques to investigate whole-cell morphology correlated with highresolution cryo-electron tomography (cryoET), with the advantages of frozen-hydrated conditions and fast and straightforward sample preparation
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