key: cord-0866444-pch2twgl
authors: Storti, Barbara; Quaranta, Paola; Di Primio, Cristina; Clementi, Nicola; Mancini, Nicasio; Criscuolo, Elena; Giorgio Speziax, Pietro; 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 Vero E6 cells
date: 2021-11-02
journal: Comput Struct Biotechnol J
DOI: 10.1016/j.csbj.2021.10.038
sha: ba4e5383375777c1cb16c7cc78c2cd007555103b
doc_id: 866444
cord_uid: pch2twgl

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. After its validation through the characterization of infected cells and virus morphology, we leveraged this toolbox to reveal subtle issues related to the entry phase of SARS-CoV-2 variants in Vero E6 cells. Our results show that in Vero E6 cells the B.1.1.7 strain (aka Alpha Variant of Concern) is associated with much faster kinetics of endocytic uptake compared to its ancestor B.1.177. 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 central role of clathrin as a mediator of endocytosis in the late pathway of entry. In keeping with the clathrin-mediated endocytosis, we highlighted the non-raft membrane localization of ACE2. 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.

Since late 2019, SARS-CoV-2 has rapidly spread worldwide generating a pandemic with devastating social consequences. The development of a handful of novel and effective vaccines 1 represented a brilliant scientific achievement and it holds promise for a rapid end of the pandemic.

Nonetheless, the way out of pandemic could be slowed by the emergence of novel SARS-CoV-2 lineages endowed with better ability to spread and infect humans while featuring lower in vitro susceptibility to neutralizing monoclonal and serum antibodies 2 . In this context, elucidation of structure-property relationships that modulate virus-cell host checkpoints, such as entry, replication, and egress, is crucial to assess the role of genome mutation on virus infectivity. SARS-CoV-2 contains four structural proteins, namely spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins (Scheme 1a). S is a ~180 kDa glycoprotein anchored in the viral membrane and protruding as homotrimers from the viral surface (the "corona") 3 . S plays the most important roles in viral attachment, fusion and entry 4, 5 . The N-terminal S1 subunit contains the receptor binding domain (RBD) that mediates SARS-CoV-2 binding to the cell membrane receptor ACE2 6 . Yet, this interaction could not be unique in accounting for virus interaction at cell membrane, as the host role of heparan sulfates 7, 8, 9 and neuropilin-1 10 were recently highlighted.

The C-terminal S2 subunit (Scheme 1b) is responsible for the fusion of the viral envelope with cellular membranes to deliver the viral RNA 11 . The S-mediated membrane fusion follows two proteolytic events: i) the "priming" cleavage that occurs at the S1/S2 interface, which yields S1 and S2 non-covalently bound in a pre-fusion conformation, and ii) the "activation" cleavage that occurs within the S2 subunit (S2') to trigger the fusion process 12 . For several CoVs, including SARS-CoV-2, fusion can occur either at the plasma or endosomal membrane, according to the "early" and the "late" pathways of entry 13 . Availability of the transmembrane-bound protease TMPRSS2 favors virus entry through the early pathway 14 . Conversely, in TMPRSS2-negative cell lines, CoVs internalize by the late pathway and fusion is triggered by the Cathepsin B/L proteases 13 .

Quite remarkably, SARS-CoV-2 bears a polybasic amino acid insert (PRRA) at the S1/S2 junction (Scheme 1b). This site is potentially cleavable by furin, a protease commonly found in the secretory pathway of most cell lines 15 . Accordingly, a few studies suggest that SARS-CoV-2 may be primed at S1/S2 by furin (or related proteases) during maturation, thereby harboring a cleaved The mechanistic knowledge of virus entry in cells is relevant for developing drugs tailored to prevent infection 20 21 . For instance, therapeutic strategies aimed at inhibiting TMPRSS2 protease activity are currently under evaluation 22, 23 . 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 24 . 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 became a dominant mutation in SARS-CoV-2 lineages that have been circulating worldwide since spring 2020. Indeed, mutation D614G seems associated with a selective infectivity advantage 25 , possibly due to a more favorable entry phase 26 compared to wild-type SARS-CoV-2. A further evolutionary step of the virus occurred in late 2020 in the UK, where the B.1.1.7 SARS-CoV-2 variant (recently denominated as "Alpha" Variant of Concern by WHO) was firstly detected 25 . B.1.1.7 rapidly outcompeted older D614G strains in many countries 27, 28 , due to its 40-70% higher transmissibility that could be associated with ~30% higher mortality rates 29 . Although B.1.1.7 retains D614G, the enhanced transmissibility of this lineage appears to be related to two additional mutations in the S protein: 1) N501Y, which resides in the Receptor Binding Motif of S, and 2) P681H, which is next to the furin cleavage S1/S2 site (Scheme 1b). Yet, the exact mechanism that confers dominance to B.1.1.7 over the older D614G strains is still unclear, although it has been proposed that N501Y may increase the affinity for ACE2 receptor 30, 31 and/or P681H may modulate the amount of cleaved S protein harbored by infecting viruses thereby influencing their entry mechanism 32, 33 .

Recent advances in fluorescence microscopy opened the way to individual virus imaging as a tool to understand viral life cycle. The dynamic and heterogeneous nature of virus-cell interactions is the perfect framework for highly sensitive imaging systems such as confocal fluorescence microscopy and Total Internal Reflection Fluorescence (TIRF) microscopy. Of note, TIRF enables imaging of a 100-150 nm layer above the coverslip where 2D cell cultures are adhered, it is thus ideally tailored to follow dynamic processes occurring at the cell membrane like viral entry. Yet, viruses such as CoVs have a size around 100 nm, i.e. well below the optical resolution of confocal and TIRF microscope on the focal plane (200-300 nm), and details of single viral particles interacting with subcellular structures may be only partially revealed with these techniques.

Optical super-resolution methods that break the light-diffraction barrier either by leveraging on the photophysical properties of the fluorescent probe or by structuring the excitation light, may easily reach the 20-150 nm spatial scale 34 . Indeed, STimulated Emission Depletion (STED) and

Single Molecule Localization Microscopy (SMLM) have been recently applied to image single viruses of different families at <100 nm, also in the cellular context 35, 36, 37 . To our knowledge, however, no super-resolution imaging of full (or pseudotyped) SARS-CoV-2 interacting with cells was yet described in the literature, albeit some studies relying on conventional diffraction-limited microscopy have appeared 38 .

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 does help answering biological questions that can only be partially addressed by in vitro techniques, and 2) highlight the ability of super-resolution techniques to afford morphology details of virus structure and molecular interactions with the cell.

Our multi-scale 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;

super-resolution microscopy techniques (image scanning microscopy, ISM 39 , in airyscan mode 40, 41 : 120-180 nm, STED: 70-100 nm, SMLM: 25-40 nm) were applied (and validated) to reveal single-virus morphology and interactions with cell substructures. By our approach we shed light on the different endocytic uptake kinetics of variant B.1.1.7 compared to B.1.177, an older D614G lineage with large diffusion in Europe in late 2020, 42 as well as on the role of clathrin and caveolin in mediating the endocytic uptake of the virus in the late pathway in Vero E6 cells. Beside their own relevance, we believe that our results are representative of a new and fertile approach for the study of SARS-CoV-2 interactions with cells. Figure S1 ) B.1 originated from a B.1 strain (hCoV-19/Italy/LOM-UniSR1/2020, GISAID Accession ID: EPI_ISL_413489) whose passaging in VeroE6 yielded a CS devoid of 685 RS 686 sequence (Scheme 1). For this reason, B.1 cannot be proteolytically cleaved by trypsin/furin-like proteases during virus maturation. In our study, B.1 has two benchmark roles: 1) it retains a mutation pattern of S protein like B.1.177, whose B.1 is direct ancestor, 2) it is a good structural benchmark of S protein of our B.1.1.7 strain, whose S protein in not S1/S2 cleaved as witnessed by Western Blot analysis (Supplementary Information, Figure S1 ). Adherent Vero E6 cells infected by B.1.177, B.1.1.7 or B.1 were methanol-fixed and immunostained by orthogonal anti-S or anti-N antibodies followed by fluorescently labeled secondary antibodies. The concomitant use of Alexa488 and Alexa647 dyes was suitable for both confocal/ISM (airyscan) and direct STORM (dSTORM). dSTORM exploits the intrinsic cycling of these fluorophores between bright (on) and dark (off) states to image and localize sparse single molecules at different times across a large field of view and reconstruct a pointillist super-resolved map of the labeled specimen 43 . Albeit Alexa488 is less efficient than Alexa647 in providing fluorescent photocycling, these two common dyes may be used together when two-color dSTORM is carried out by adopting tailored buffer solutions 44 . 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 45 . Thus, we selected Atto594 and Atto647 for two-color STED imaging. Two-color STED images were acquired according to the separation of photons by lifetime tuning modality 46 (hereafter abbreviated as τ-STED). In some cases, we acquired two-color STED/Confocal images on samples prepared for dSTORM, by using Alexa488 as reporter for STED and Alexa647 as reporter for confocal imaging.

At first, we assessed the relative infection capabilities of Vero E6 by our three strains. Cells were The N protein was found to accumulate in perinuclear regions (Figure 1b , red) and in small clusters near the plasma membrane (Figure 1c , red). These regions may be identified with sites of Nproduction or of N/RNA complexes in liquid-phase separated assemblies 48, 49 . Very interestingly, S and N protein colocalized only in a subset of egressing virions along the membrane (Figure 1d ). In these spots, a smaller N-particle was embedded into a larger S-particle (Figure 1g ,h), in agreement with the core-shell structure of SARS-CoV-2. Dual-color -STED clearly highlighted in the cytoplasm several N-particles surrounded by a banana-shaped S-enriched region ( Figure   1h ). These systems likely correspond to ERGIC regions where the envelope coating of the Npacked virus genome is taking place 47 . Of note, both S-coated and bare N-particles were characterized by FWHM 110 nm (Figure 1h ), yielding d = 85 nm, which is again in good ∼ agreement with the actual diameter of the virus core (vide infra).

Given the higher resolution of dSTORM (average localization precision: 30 nm) and the strong ∼ z-sectioning of TIRF imaging mode (100-120 nm), we set out to investigate the size of single viral particles detected by dSTORM-TIRF on the basal plasma membrane during the entry (vide infra) and egress phases ( Figure 2 ).

To retrieve the particle size, single-molecule localization data were analyzed by density-based spatial clustering of applications with noise (DBSCAN), a clustering algorithm based on localization maps that can discover clusters of arbitrary shapes 50 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 S2 ), 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 an S-labeled virus must be expected slightly above its envelope radius, in good agreement with our cluster analysis results.

Next, we set out to investigate the biological mechanism of virus access to the Vero E6 cells by our spatial multiscale imaging platform. Recent data pointed out how the entry pathway of SARS-CoV-2 is critically dependent on the presence of TMPRSS2 protease on the cell membrane 51 .

Accordingly, we first checked for the presence of TMPRSS2 on our Vero E6 cell line. In full agreement with literature data 52 , we found out that Vero E6 express almost no TMPRSS2 ( Figure   3a ). In this experiment, HepG2 and Caco-2 cells were used as positive control of low and high TMPRSS2 expression. In absence of TMPRSS2, SARS-CoV-2 is thought to enter cells by the late pathway, i.e. by the endosomal route 17, 51 , whose milestone is the cleavage of S protein by endosomal cathepsins, The early endocytic events of the "late pathway" were investigated by our multiscale imaging platform adopting an infection scheme that enabled synchronization of virus entry 53 . Cells were pre-incubated with our strains for 1-3h at 4 °C, allowing membrane attachment of the virus but preventing its endocytosis. After the chilling step, the non-attached virions were removed, and cells were incubated at 37 °C to promote viral entry until fixation at 1-3 hpi. This time span ensured the absence of almost any virus production and egress 51, 54 . Clathrin-mediated and caveolar endocytosis represent the most common initial step of virus endocytosis 56 . Accordingly, we investigated the endocytosis pathway of B.1.177 by quantitative colocalization imaging 57 , by concomitantly labeling the S protein and clathrin or caveolin-1.

Remarkably, dual-color ISM images alleged a significant colocalization between viral particles and clathrin, but not caveolin-1 ( Figure 6 ). 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 ). A further support to clathrin-mediated late entry of SARS-CoV-2 was provided by τ-STED measurements, which addressed the apical submembrane level where most colocalized signal was visible ( Figure 8 ). τ-STED images clearly showed single virions embedded into larger clathrin vesicles (170±90 nm) that, albeit not resolved into the structural triskelion, can safely be attributed to clathrin pits. The clathrin-mediated endocytosis of SARS-CoV-2 is at odds with the controversial hypothesis that ACE2 resides in caveolin-enriched raft regions of the cell membrane in several cell lines,

including Vero E6 58, 59 . Accordingly, we set out to investigate the localization and functionality of ACE2 in the Vero E6 membrane by our microscopy toolbox. Confocal and TIRF imaging confirmed that ACE2 shows a prevalent membrane localization (Figure 9a) , with some minor cytoplasmic staining. The functional receptor activity of membrane ACE2 towards SARS-CoV-2 was corroborated by the large colocalization with recombinant RBD of the S protein ( Figure 9b , Table 1 ). Also, we found a significant degree of colocalization of ACE2 with CD71, the transferrin receptor ( Figure   9b , Table 1 ). CD71 is known as a marker of the non-raft regions of the cell membrane 60 and its clathrin-mediated endocytosis upon stimulation is well documented 61 . Conversely, ISM images highlighted negligible ACE2 colocalization with caveolin-1 (Figure 9c , Table 1 ).

We can conclude that ACE2 localizes poorly in caveolin-1-enriched membrane regions in Vero In the second part of the work, we applied our multiscale imaging toolbox to investigate the early phase of virus entry in cells. The "late pathway" of SARS-CoV-2 entry can be split in two different phases: first the activation of endocytic internalization of the viral particle (the "penetration" phase 67 ), then the endosome maturation which ends in the cathepsin-mediated activation of the Spike protein and the release of viral genome into the cytoplasm (the "uncoating" phase 67 ). While recent studies shed light on many mechanistic details of the second phase, including its crucial dependence on structural characteristics of S such as the S1/S2 cleavage ratio, the first phase is still rather obscure. By direct imaging, we highlighted that B.1.1.7 is much faster in activating the endocytic internalization than B.1.177 and B.1. This property does not seem to depend on the S1/S2 cleavage ratio, as B. Figure S1 ). We are tempted to attribute the faster phenotype of In conclusion, we believe that our fluorescence microscopy imaging toolbox represents a fertile strategy to address urgent questions on virus-cell checkpoints at the single virus level, while avoiding the conflicting results sometimes obtained using models unable to recapitulate the desired viral phenotypes. Michele Oneto (IIT Nanophysics) are gratefully acknowledged for technical assistance and support.

For safety reasons, all the procedures of infection were performed in a Biohazard Safety Level 3 facility.

African green monkey kidney cells (Vero E6) were obtained from ATCC (CRL-1586). Vero E6

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% CO 2 .

TMPRSS2-expressing Vero E6 cells (Vero E6 TMRPSS2(+) ) were kindly provided by the National Institute for Biological Standards and Control (NIBSC) and were supplemented with 10% FBS and 1 mg/mL geneticin (G418). 

Vero E6 cells (3•10 5 cells/mL) were seeded into 96-well plates and cultured for 1 day at 37°C.

Then, the cells were washed three times with PBS after 1 h of virus adsorption (0.001 MOI) at 37°C, and cells were incubated for 1, 3, 6, 12, 18, 24, 48, and 72 hpi. The cell supernatants were collected at the different time points as well. Viral genome from the supernatants of both experimental settings was extracted and analyzed by real-time RT-PCR.

Vero E6 cells (3•10 5 cells/mL) or Vero E6 TMPRSS2(+) were seeded into 96-well plates and cultured for 1 day at 37°C. Subsequently, cells were incubated 1h before infection with 10 µM E64d, a All values were normalized by the housekeeping gene RPL13A. All samples were run in duplicate. and then rinsed four times with PBB and three times with PBS. When required, cell nuclei were stained by exposure for 5 min to 1 mg/100 ml Hoechst 33342 (ThermoFisher) in water. 

Acquired dSTORM stacks were processed by Thunderstorm, a Fiji plugin for PALM and STORM data analysis 77 . 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 molecules 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).

Single molecule localization maps and cluster analysis were collected by the LocAlization Microscopy Analyzer software (LAMA), available for download at http://share.smb.unifrankfurt.de/index.php/software-menue/lama. Before LAMA analysis, the localization list 

Lifetime-tuning STED (τ-STED) measurements were performed using 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 acoustic-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. The 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). For comparisons amongst Cycle thresholds (Ct) the 2way ANOVA multiple comparisons with corrections were performed by using Prism 9 (GraphPad).

Cells were incubated at 37°C and 5% CO 2 for 1,3,6, and 48 h. Next, the medium was removed, cells were washed 3 times with 500 µl of PBS, and finally fixed and permeabilized with ice-cold 100% methanol for 15 minutes. After methanol was removed, cells were rinsed again three times in PBS

Sino Biological), dilution: 1:200 • anti-N IgG mouse monoclonal antibody (#33717, Cell Signaling), dilution: 1:6400 • anti-ACE2 IgG rabbit monoclonal antibody (ab15348, AbCam)

• anti-clathrin IgG mouse monoclonal antibody

dilution: 1:200 • anti-CD71 IgG mouse monoclonal antibody (sc-65882, SantaCruz), dilution: 1:100 • anti-α-tubulin IgG mouse monoclonal antibody

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

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

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

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

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

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

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