Ultrastructural analysis of SARS-CoV-2 interactions with the host cell via high resolution scanning electron microscopy

Caldas, Lúcio Ayres; Carneiro, Fabiana A.; Higa, Luiza M.; Monteiro, Fábio Luiz; Silva, Gustavo Peixoto da; Costa, Luciana Jesus da; Durigon, Edison Luiz; Tanuri, Amílcar; Souza, Wanderley de

doi:10.1101/2020.06.10.144642

Cited by 23 publications

(43 citation statements)

References 37 publications

(24 reference statements)

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“…The SARS‐CoV‐2 SP‐induced LVCVs were further explored in our previous studies using SEM to inspect the infected cell's interior. Consistent with them, the electron tomography carried in the present work allowed the finding of virus particles of different sizes although presenting similar shapes (Figure 5 and Supplemental Material 1), previous corroborating data obtained by SEM and TEM (Caldas et al., 2020b; Zhao et al., 2020). Studies on cryo‐electron tomography showed that SARS‐CoV‐2 acquires its envelope, while budding through the LVCVs, which already display the S protein adhered to their internal face (Figure 3D) (Klein et al., 2020).…”

Section: Discussionsupporting

confidence: 91%

“…Since our previous study, we investigated the SARS‐CoV‐2 SP viroplasm by SEM (Caldas et al., 2020b); the membrane structures and compartments involved in the replication and morphogenesis of this virus were now studied by TEM herein. Therefore, this work contributes to understanding the interaction mechanisms between SARS‐CoV‐2 and the host cells, taking into account the general morphological alterations, the recruitment of organelles and cell components in the context of the intense membrane remodelling that takes place during infection.…”

Section: Discussionmentioning

confidence: 99%

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Intracellular host cell membrane remodelling induced by SARS‐CoV‐2 infection in vitro

Caldas

Carneiro

Monteiro

et al. 2021

Biology of the Cell

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Background Information Severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) infection induces an alteration in the endomembrane system of the mammalian cells. In this study, we used transmission electron microscopy and electron tomography to investigate the main structural alterations in the cytoplasm of Vero cells infected with a SARS‐CoV‐2 isolate from São Paulo state (Brazil). Results Different membranous structures derived from the zippered endoplasmic reticulum were observed along with virus assembly through membrane budding. Also, we demonstrated the occurrence of annulate lamellae in the cytoplasm of infected cells and the presence of virus particles in the perinuclear space. Conclusions and Significance This study contributes to a better understanding of the cell biology of SARS‐CoV‐2 and the mechanisms of the interaction of the virus with the host cell that promote morphological changes, recruitment of organelles and cell components, in a context of a virus‐induced membrane remodelling.

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Section: Discussionsupporting

confidence: 91%

Section: Discussionmentioning

confidence: 99%

Intracellular host cell membrane remodelling induced by SARS‐CoV‐2 infection in vitro

Caldas

Carneiro

Monteiro

et al. 2021

Biology of the Cell

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“…τ-STED microscopy demonstrates that fluorescent particles on the cell membrane were composed by single viruses, with ∼100-130 mm size, as well as very small clusters of about 2-3 virions (Figure 3, inset). These findings were in agreement with a previous electron microscopy study, which showed at 6h membrane-attached single and pair of wild-type SARS-CoV-2 viruses, with poor cell internalization 41,42 . The strong interaction of B.1.1.7 with cells at ~1h detected by microscopy was consistent with the short 1h exposure able to activate strong viral production after one day, at odds with the phenotype observed for B.1 (Figure 2b).…”

Section: Visualization Of Virus During Entry and Egresssupporting

confidence: 93%

A spatial multi-scale fluorescence microscopy toolbox discloses entry checkpoints of SARS-CoV-2 variants in Vero E6 cells

Storti

Quaranta

Primio

et al. 2021

Preprint

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We developed a multi-scale microscopy imaging toolbox to address some major issues related to SARS-CoV-2 interactions with host cells. Our approach harnesses both conventional and super-resolution fluorescence microscopy (Airyscan, STORM, and STED) and easily matches the spatial scale of single virus-cell checkpoints. We deployed this toolbox to characterize subtle issues related to the entry phase of SARS-CoV-2 variants in VeroE6 cells. Our results suggest that the variant of concern B.1.1.7, currently on the rise in several countries by a clear transmission advantage, in these cells outcompetes its ancestor B.1 in terms of a much faster kinetics of entry. Given the molecular scenario (entry by the late pathway and similar fraction of pre-cleaved S protein for B.1.1.7 and B.1), the faster entry of B.1.1.7 could be directly related to the N501Y mutation in the S protein, which is known to strengthen the binding of Spike RBD with ACE2. Remarkably, we also observed directly the significant role of clathrin as mediator of late entry endocytosis, which had been previously suggested in analogy with other CoVs and from experiments on pseudotyped virus models. On overall, we believe that our fluroescence microscopy-based approach is valuable for future studies addressing of how SARS-CoV-2 and its variants interact with cells.

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“…We suggest that this resembles the particle clustering by host defense protein BST-2 as it was observed for human coronavirus229E and quantified in HeLa cells by Wang et al [35]. However, the metal coating applied by Wang et al is clearly visible at high resolution in the SEM images as a rough layer on the cell membrane and hiding the true topography [24], [24], [36]. In contrast, the HIM images presented here not only allow for the quantification of particles and clusters, but also enable an unveiled view on the interaction of virus particles with the cell membrane.…”

Section: Resultssupporting

confidence: 63%

Imaging of SARS-CoV-2 infected Vero E6 Cells by Helium Ion Microscopy

Frese¹,

Schmerer²,

Wortmann³

et al. 2020

Preprint

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Helium ion microscopy (HIM) offers the opportunity to obtain direct views of biological samples such as cellular structures, virus particles, and microbial interactions. Imaging with the HIM combines sub-nanometer resolution, large depth of field, and high surface sensitivity. Due to its charge compensation capability, the HIM can image insulating biological samples without additional conductive coatings. Here, we present an exploratory HIM study of SARS-CoV-2 infected Vero E6 cells, in which several areas of interactions between cells and virus particles, as well as among virus particles, were imaged. The HIM pictures show the three-dimensional appearance of SARS-CoV-2 and the surface of Vero E6 cells at a multiplicity of infection of approximately 1 with great morphological detail. The absence of a conductive coating allows a distinction between virus particles bound to the cell membrane and virus particles lying on top of the membrane. After prolonged imaging, it was found that ion-induced deposition of hydrocarbons from the vacuum renders the sample sufficiently conductive to allow imaging even without charge compensation. The presented images demonstrate the potential of the HIM in bioimaging, especially for the imaging of interactions between viruses and their host organisms.

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Ultrastructural analysis of SARS-CoV-2 interactions with the host cell via high resolution scanning electron microscopy

Cited by 23 publications

References 37 publications

Intracellular host cell membrane remodelling induced by SARS‐CoV‐2 infection in vitro

Intracellular host cell membrane remodelling induced by SARS‐CoV‐2 infection in vitro

A spatial multi-scale fluorescence microscopy toolbox discloses entry checkpoints of SARS-CoV-2 variants in Vero E6 cells

Imaging of SARS-CoV-2 infected Vero E6 Cells by Helium Ion Microscopy

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