key: cord-1024679-sy37gtn2
authors: Storti, Barbara; Quaranta, Paola; Di Primio, Cristina; Clementi, Nicola; Spezia, Pietro Giorgio; Carnicelli, Vittoria; Lottini, Giulia; Paolini, Emanuele; Freer, Giulia; Lai, Michele; Costa, Mario; Beltram, Fabio; Diaspro, Alberto; Pistello, Mauro; Zucchi, Riccardo; Bianchini, Paolo; Signore, Giovanni; Bizzarri, Ranieri
title: A spatial multi-scale fluorescence microscopy toolbox discloses entry checkpoints of SARS-CoV-2 variants in VeroE6 cells
date: 2021-04-14
journal: bioRxiv
DOI: 10.1101/2021.03.31.437907
sha: 62e285d87ea74096431b7c2423a26caa29651ea0
doc_id: 1024679
cord_uid: sy37gtn2

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 only 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, as already suggested for other CoVs and from pseudovirus-based infection models. Overall, we believe that our fluroescence microscopy-based approach represents a valuable tool for evaluating the entry kinetic of SARS-CoV-2 and its variants.

The mechanistic knowledge of virus entry in cells is relevant for developing drugs tailored to prevent infection 16 17 . For instance, therapeutic strategies aimed at inhibiting TMPRSS2 protease activity are currently under evaluation 18, 19 . The proposed, yet unclear and non-exclusive involvement of clathrin and caveolin-1 as mediators of endocytosis in the late pathway may afford further molecular targets to stop pathogenesis 20 . Nonetheless, mutations in the S protein may be crucial for the first step of viral transmission of novel SARS-CoV-2 variants, with significant epidemiological consequences. In particular, D614G early became the dominant mutation in SARS-CoV-2 lineages that have been circulating worldwide since spring 2020. D614G is associated with a selective infectivity advantage 21 , possibly due a more favorable entry phase 22 .

Recently, the B.1.1.7 SARS-CoV-2 variant 21 has become the dominant lineage in UK 23 and is now taking over in many other countries 24 , due to Its higher transmissibility (40-70% compared to other lineages), possibly associated to ~30% higher mortality rates 25 . Beside D614G, two additional mutations in the S protein seem significant for the enhanced transmissibility of B.1.1.7 (Scheme 1b): 1) N501Y, which is thought to increase the affinity for ACE2 receptor 26, 27 ; 2) P681H, which is next to the furin cleavage S1/S2 site. Actually, both D614G 28, 29, 30 and P681H allegedly modulate the amount of cleaved S protein harbored by infecting viruses influencing their entry mechanism 15, 31 . image single viruses of different families at<100 nm, also in the cellular context 33, 34, 35 . To our knowledge, however, no super-resolution imaging of full (or pseudotyped) SARS-CoV-2 interacting with cells has been described yet in literature.

In this study, we deploy for the first time a multi-scale fluorescence microscopy toolbox to investigate entry checkpoints of SARS-CoV-2 with two general goals: 1) demonstrate that imaging SARS-CoV-2 at single virus level help answering biological questions that are only partially addressed by in vitro techniques, and 2) highlight the ability of super-resolution techniques to afford morphology details of virus structure and its molecular interactions with the cell. Our multiscale toolbox was organized according to the resolution capability of each technique: confocal and TIRF microscopy (200-300 nm) were applied to visualize interactions at cell level; superresolution microscopy techniques (structured illumination in airyscan mode: 120-180 nm, STED: 70-100 nm, SMLM: 25-40 nm) were applied to reveal single virus morphology and interactions with cell substructures. By our approach we shed light on the different entry kinetics of variant B.1.1.7 compared to its ancestor B.1 lineage, and on the role of clathrin and caveolin in mediating the first endocytic step in the late pathway in VeroE6 cells. Beside their own relevance, we believe that our results are representative of a new and fertile approach in the study of SARS-CoV-2 interactions with cells. For safety reasons, SARS-CoV-2 was manipulated in a biohazard safety level 3 (BSL3) and

imaging of virus cell interactions was performed on fixed cells by immunocytochemistry and following indirect labeling. Adherent VeroE6 cells infected by B.1 or B.1.1.7 were methanol-fixed and immunostained by anti-S or anti-N rabbit antibodies followed by fluorescently-labeled antirabbit secondary antibodies. The use of Alexa488 and Alexa647 dyes was suitable for both confocal/structured illumination (airyscan) and SMLM by the direct STORM approach (dSTORM). Indeed, dSTORM exploits the intrinsic cycling of these fluorophores between bright (on) and dark (off) states to image and localize sparse single molecules in different times across a large field of view and reconstruct a pointillist super-resolved map of the labeled specimen 37 .

Conversely, STED nanoscopy requires stable and non-blinking fluorophores because the resolution improvement is performed by the targeted detection of non-depleted fluorophores and high photon flux is necessary 38 . Thus, we selected Atto594 and Atto647 for two-colour STED imaging. The STED has always been performed in the separation of photons by lifetime tuning (SPLIT) modality 39 easily enabled by the Leica Stellaris 8 (Leica Microsystems, Mannheim, Germany) and commercially called τ-STED.

To our knowledge, a detailed comparison of B.1 and B.1.1.7 replication kinetics in cell culture is still unreported. Therefore, we infected VeroE6 cells at low multiplicity of infection (m.o.i = 0.001) to enable multicycle replication and the amount of virus in the external medium was Since the entry mechanism of SARS-CoV-2 in VeroE6 seems crucially related to the presence of S1/S2 cleaved virus by furin or other proteases during egress, we set out to determine the cleavage ratio of S protein by Western Blot at 48 hpi. In both cases we found out that uncleaved viral particles constitute the larger fraction of released viruses ( Figure 1d ). More specifically, the cleaved protein accounted for the 14% and 21% of S pool of B.1 and B.1.1.7, respectively. The slight difference between lineages excludes a strong effect of the P681H on the adjacent furin cleavage site (Scheme 1b). 

Given the nanoscale resolution of dSTORM in the xy plane (average localization precision: 30 nm) and the strong z-sectioning of TIRF imaging mode (100-120 nm), we set out to investigate Figure S1 ). Given the cylindrical symmetry of the illumination system, we calculated the localization density as a function of the distance (ρ) from the center of a viral particle located at different distances from the basal plane (Supplementary Information, Figure S1 ), in order to mimic different experimental conditions. Our simulation showed that the localization density grows up from ρ=0 nm to ρ =48-58 nm, depending on the labeling site on the S protein, to decrease slowly farther off (Supplementary Information, Figure S2 ). This implies that the maximum fluorescent intensity of a S-labeled virus must be expected slightly above its envelope radius, in good agreement with our cluster analysis results.

In agreement with literature data 46 , we found out that VeroE6 cells express no membrane protease TMPRSS2, whereas control CaCo-2 cells do (Figure 6a ). In absence of TMPRSS2, SARS-CoV-2 is thought to enter cells by the "late pathway", i.e. by the endosomal route 13 . Accordingly, the early events of the "late pathway" were investigated for B.1 by adopting an infection scheme that enabled synchronization of virus entry 47 . Cells were pre-incubated with B.1 for 3h at 4 °C, allowing membrane attachment of the virus but preventing its endocytosis. After the chilling step, the nonattached virions were removed, and cells were incubated at 37 °C to promote viral entry. Viral particles were clearly visible near the cell membrane at 2-3 hpi (Figure 6b ). Clathrin-mediated and caveolar endocytosis represent the most common initial step of virus endocytosis 48 . Remarkably, dual-color airyscan images alleged a significant colocalization between viral particles and clathrin, but not caveolin-1 (Figure 7 ). This pattern was quantitatively confirmed by Pearson's coefficient R, which measures the stoichiometric correlation between the two fluorescent partners as a proxy of their functional association (Table 1) .

Perfect stoichiometric correlation (R=1) can never be achieved, owing to incomplete labeling, fluorescence background, and slight spatial mismatch of colors due to residual chromatic aberration. Accordingly, a positive control made of green/far-red doubly immunostained ACE2 receptor set the maximum achievable R to 0.69±0.01. With this reference, we found a medium/strong functional association of S with clathrin, but a poor or negligible association with caveolin-1 ( Table 1 ). Table 1 ). Also, we found a significant degree of colocalization of ACE2 with CD71, the transferrin receptor ( Figure 10b , Table 1 ). CD71 is known as a marker of the non-raft regions of the cell membrane 51 and its clathrin-mediated endocytosis upon stimulation is well documented 52 .

Conversely, Airyscan images highlighted that ACE2 colocalizes with caveolin-1 to a negligible extent ( Figure 10c , Table 1 ). 

The ongoing COVID-19 pandemic makes imperative the full understanding of virus-host interactions. In this context, it has been early recognized the pivotal role of the surface Spike (S) protein, which mediates both the docking with the host cell receptor (ACE2) and the fusion process. The subtle interplay of S with the ACE2 receptor, its ability to hijack the cell endocytic machinery, and its intrinsic tunable fusogenic properties, are directly related to the viral tropism.

The SARS-CoV-2 spike is the antigen encoded by available vaccines 1 . Also, the S glycoprotein represents the main target of therapeutic approaches aimed at neutralizing virus infectivity. S appears also key to viral adaptation to humans under selective pressure, and its sequence variability has already enabled the emergence of dominant viral variants such as the D614G clade B.1 and, more recently, the variant of concern B.1.1.7.

In spite of the accumulated knowledge insofar, the elucidation of unclear checkpoints of S- Michele Oneto (IIT Nanophysics) are gratefully acknowledged for technical assistance and support.

African green monkey kidney cells (VeroE6) were obtained from ATCC (CRL-1586). VeroE6

were cultured in DMEM high glucose medium supplemented with heat-inactivated 10% fetal bovine serum (FBS) (Sigma-Aldrich, Milan, Italy), 2 mM L-glutamine, 10 U/ml penicillin and 10 mg/ml streptomycin (Sigma-Aldrich, Milan, Italy), at 37°C in the presence of 5% CO2. in DMEM culture medium supplemented with 5% FBS was added. Cells were incubated at 37 °C and 5% CO2. On day 3 the plaques became detectable, the cells were fixed overnight with 500 µl/well of 4% buffered formalin solution (Sigma-Aldrich), and then stained with 1% crystal violet (Sigma-Aldrich, Milan, Italy).

VeroE6 cells were seeded in a 24 well plate at 10 5 cell/well in 1 ml of culture medium and cultured for 1 day at 37°C. Subsequently the medium was removed and the cells were inoculated for 1h 

VeroE6 cells were seeded in 6 well plate at 2•10 5 cell/well and cultured for 1 day at 37°C. Three wells were washed once with PBS, after that 1ml TRIZOL (Thermo Fisher) was added. Samples were then transferred in 4 ml tubes and processed for total RNA extraction with RNA micro kit 

Acquired dSTORM stacks were processed by Thunderstorm, a Fiji plugin for PALM and STORM data analysis 66 . At first, we set the properties of acquisition by the "Camera setup" menu: pixel size = 158.7 nm, Photoelectrons per A/D count: 2.5, Base level: 100 counts, EM gain: 300. Then, we carried out the localization algorithm ("Run analysis"), setting the following parameters: a) pre-filter: difference of averaging filters with 3 and 6 pixels as first and second kernel size, respectively; b) approximate localization of molecules by local maximum method with threshold 200 and 8-neighbourhood connectivity; c) sub-pixel localization by the Integrated Gaussian method, performing least squares multi-fitting (threshold p=1E-6) with initial sigma 1.6 pixels and fitting radius 3 pixels, maximum 5 molecule for fitting region with limit intensity range 1-1000

photons. Eventually, we cleaned the obtained results from drift and those localizations not strictly lying on the focal plane by the following post-filtering algorithm: a) removal of first 500 frames; b) drift correction by correlation; c) merging reactivated molecules (max distance: 20 nm, max off frames: 1, limited frames per molecule); d) removal of localizations with: (intensity = 1000 AND sigma >180 nm AND uncertainty > 130 nm). 

Lifetime-tuning STED (τ-STED) measurements were performed by means of a Leica STELLARIS 8 Falcon τ-STED (Leica Microsystems, Mannheim, Germany) inverted confocal/STED microscope. Excitation was provided by a White Light Laser and selecting the following wavelengths by the acousto-optical tunable filter (AOTF): 488 nm, 560 nm, and 638 nm. Detection has been performed by the embedded tunable spectrometer in the 500 -550 nm, 570 -630 nm, 660-750 nm ranges respectively, and three Power HyD detectors. Pinhole was set to 0.6-1 Airy size. Line scanning speed ranged from 10 to 1400 Hz in standard acquisition mode. In τ-STED mode, the 775 nm pulsed laser beam is superimposed at a typical power of 100 -250 mW before the objective. Two-colors τ-STED has been performed sequentially by line for the red and far-red fluorophores. Green fluorophores are not affected by the depletion beam at 775nm.

Graphs were prepared using Prism 7 (GraphPad) and IgorPro8 (Wavemetrics) software. Data are shown as the mean +/-SEM. Statistical analysis was performed by Prism 7 (GraphPad).

Secondary antibodies for immunofluorescence studies and combinations • donkey anti-rabbit IgG Alexa488-labeled monoclonal antibody (a21206, ThermoFisher), dilution: 1:500 (confocal, airyscan, and dSTORM-TIRF experiments

• donkey anti-rabbit IgG Alexa647-labeled monoclonal antibody (a31573, ThermoFisher), dilution: 1:500 (confocal, airyscan, and dSTORM-TIRF experiments

• donkey anti-mouse IgG Alexa488-labeled monoclonal antibody (a21202, ThermoFisher), dilution: 1:500 (confocal, airyscan, and dSTORM-TIRF experiments

• donkey anti-mouse IgG Alexa647-labeled monoclonal antibody (a31571, ThermoFisher), dilution: 1:500 (confocal, airyscan, and dSTORM-TIRF experiments

• goat anti-rabbit IgG Atto647N-labeled monoclonal antibody (40839, Merck)

• goat anti-mouse IgG Atto594-labeled monoclonal antibody (76085, Merck)

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

After rinsing four times with PBS + 0.5% BSA (PBB), infected cells and negative controls were incubated for 1h with a a solution of 1-2 fluorescentlylabeled secondary antibody/ies in PBB (see: secondary antibody section in Materials and Methods) and then rinsed four times with PBB

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. Then, cells were fixed with PFA 2% in PBS for 15 min, rinsed three times with PBS, permeabilized for 15 min with

Fixed cells were incubated overnight at 4 C° with 200 μl of a solution of anti-ACE2 IgG, and either Spike RBD-mFC BIBLIOGRAPHY

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