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

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.

SciScore for 10.1101/2021.03.31.437907: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Institutional Review Board Statement	not detected.
Randomization	not detected.
Blinding	not detected.
Power Analysis	In τ-STED mode, the 775 nm pulsed laser beam is superimposed at a typical power of 100 – 250 mW before the objective.
Sex as a biological variable	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
Primary antibodies for WB: mouse anti-S (1A9) 1:1000 (GTX632604, GeneTex)	anti-S suggested: None GTX632604 suggested: (GeneTex Cat# GTX632604, RRID:AB_2864418)
Secondary antibodies for Western blot analysis were HRP-conjugated anti-mouse purchased from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA. Cell infection (unsynchronized) for immunofluorescence …

SciScore for 10.1101/2021.03.31.437907: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Institutional Review Board Statement	not detected.
Randomization	not detected.
Blinding	not detected.
Power Analysis	In τ-STED mode, the 775 nm pulsed laser beam is superimposed at a typical power of 100 – 250 mW before the objective.
Sex as a biological variable	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
Primary antibodies for WB: mouse anti-S (1A9) 1:1000 (GTX632604, GeneTex)	anti-S suggested: None GTX632604 suggested: (GeneTex Cat# GTX632604, RRID:AB_2864418)
Secondary antibodies for Western blot analysis were HRP-conjugated anti-mouse purchased from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA. Cell infection (unsynchronized) for immunofluorescence study of virus entry: 105 VeroE6 cells were seeded in 35 mm glass bottom dishes (Willco, Amsterdam) with 2 ml of culture medium and cultured for 1 days at 37°C.	anti-mouse purchased from Santa Cruz Biotechnology , Inc . suggested: None
Primary antibodies for immunofluorescence studies: Secondary antibodies for immunofluorescence studies and combinations: Immunostaining of infected cells: Methanol-fixed infected cells and a methanol-fixed negative control were incubated overnight at 4 C° with 150 μl of a solution of anti-S IgG or anti-N IgG in PBS + 3% BSA (Sigma-Aldrich, Milan, Italy).	anti-S IgG suggested: None anti-N IgG suggested: None
Experimental Models: Cell Lines
Sentences	Resources
Kinetic study of virus growth in cells: VeroE6 cells were seeded in a 24 well plate at 105 cell/well in 1 ml of culture medium and cultured for 1 day at 37°C.	VeroE6 suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
The cDNA of Caco-2 and HepG2 cells was used as a positive control of TMPRSS2 amplification.	Caco-2 suggested: None HepG2 suggested: CLS Cat# 300198/p2277_Hep-G2, RRID:CVCL_0027)
Immunostaining of non-infected cells: 105 Vero-E6 cells were seeded in 35 mm glass bottom dishes (Willco, Amsterdam) with 2 ml of culture medium and cultured for 1 days at 37°C.	Vero-E6 suggested: None
Software and Algorithms
Sentences	Resources
The in-house one-step RT-qPCR reaction mixtures were run in a CFX Connect Real-Time PCR instrument (Bio-Rad Laboratories, Hercules, CA, USA) using a previously standardized thermal conditions (52.0 °C for 5 min, 95.0 °C for 10 s, 45 cycles of 10 s at 95.0 °C, and 62 °C for 30 s).	Bio-Rad Laboratories suggested: (Bio-Rad Laboratories, RRID:SCR_008426)
Lysates were quantified, loaded on 4-15% precast protein gels (BIORAD) and proteins were separated by SDS-PAGE and electro-blotted onto Hybond-C-Extra (Amersham Biosciences) nitrocellulose membranes.	BIORAD suggested: None
Colocalization of the green and far-red images was quantified by Pearson’s coefficient R according to the method by Costes et al. 65 by the colocalization threshold and colocalization test routines of Fiji.	Fiji suggested: (Fiji, RRID:SCR_002285)
Single molecule localization analysis: Acquired dSTORM stacks were processed by Thunderstorm, a Fiji plugin for PALM and STORM data analysis 66.	STORM suggested: (SToRM, RRID:SCR_006696)
Graphics and statistics: Graphs were prepared using Prism 7 (GraphPad) and IgorPro8 (Wavemetrics) software.	Prism suggested: (PRISM, RRID:SCR_005375) GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 20. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

Results from rtransparent:

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Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
No protocol registration statement was detected.

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

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