Correlative Multi-scale Cryo-imaging Unveils SARS-CoV-2 Assembly and Egress

Abstract

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 – 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.

Secretory Vesicles Are the Principal Means of SARS-CoV-2 Egress

“…The cryoFIB/SEM study cited above also reported the occurrence of plasma membrane discontinuities next to virus particles outside the cell, suggesting possible viral release through membrane lesions [13]. We observed no such discontinuities, with our electron micrographs instead suggesting viral release without plasma membrane rupture via the possible exocytosis of small vesicles, most of which contained a single viral particle.…”

“…A recent cryoFIB/SEM imaging study of Vero cells infected with SARS-CoV-2 has suggested the presence of "exit tunnels" connecting large intracellular virus-containing vesicles to the plasma membrane [13]. The authors suggested that these tunnels resulted from the fusion of these large virus-containing vesicles with the cell membrane, allowing the release of numerous SARS-CoV-2 viral particles through an exocytosis-like mechanism.…”

“…On section 10, another viral release event involving a small se-cretory vesicle containing a single viral particle can be visualised (thin white box in the upper part of the image, also shown in the inset). The numbers on the panels (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15) indicate the successive serial TEM sections.…”

“…However, it is unknown whether these intracellular compartments containing viral particles can fuse with the plasma membrane for virion release. A recent cryoFIB/SEM study (cryotechniques with a focused ion-beam scanning electron microscope) of SARS-CoV-2-infected cells suggested the presence of "exit tunnels", linking virion-rich intracellular vacuoles to the extracellular space [13]. The authors suggested that SARS-CoV-2 could egress through these tunnels by a mechanism of exocytosis from these large intracellular cisterns.…”

Computational methods for in situ structural studies with cryogenic electron tomography

“…The 3D structures of SARS-CoV-2 ( Turoňová et al., 2020 ; Calder et al., 2022 ) by cryo-ET have helped researchers to explore the functional mechanism and promote the development of vaccines and drugs. Critical SARS-CoV-2 structures are resolved in Zhang et al. (2021) , such as the virus egress pathway and native virus spike structures.…”