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bioRxiv · 10.1101/2021.03.31.437907

A spatial multi-scale fluorescence microscopy toolbox discloses entry checkpoints of SARS-CoV-2 variants in VeroE6 cells

Storti, B.·Quaranta, P.·Di Primio, C.·Clementi, N.·Spezia, P. G.·Carnicelli, V.·Lottini, G.·Paolini, E.·Freer, G.·Lai, M.·Costa, M.·Beltram, F.·Diaspro, A.·Pistello, M.·Zucchi, R.·Bianchini, P.·Signore, G.·Bizzarri, R.
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Abstract

We exploited 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 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 Vero E6 cells. Our results suggest that in these cells the variant of concern B.1.1.7, (aka Alpha variant), became the predominant circulating variant in several countries by a clear transmission advantage. In fact, in these cells B.1.1.7 outcompetes its ancestor B.1.177 in terms of a much faster kinetics of entry. Given the cell-entry scenario dominated by the endosomal "late pathway", the faster internalization 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 receptor binding domain with ACE2. Remarkably, we also directly observed the main role of clathrin as mediator of late-entry endocytosis, reconciling it with the membrane localization of the ACE2 receptor previously attributed to caveolin-enriched rafts. Overall, we believe that our fluorescence microscopy-based approach represents a fertile strategy to investigate the molecular features of SARS-CoV-2 interactions with cells.

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Storti, B., Quaranta, P., Di Primio, C., Clementi, N., Spezia, P. G., Carnicelli, V., Lottini, G., Paolini, E., Freer, G., Lai, M., Costa, M., Beltram, F., Diaspro, A., Pistello, M., Zucchi, R., Bianchini, P., Signore, G., Bizzarri, R.. 2021-03-31. A spatial multi-scale fluorescence microscopy toolbox discloses entry checkpoints of SARS-CoV-2 variants in VeroE6 cells. https://doi.org/10.1101/2021.03.31.437907

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