multimerization- and glycosylation-dependent receptor binding of sars-cov-2 spike proteins ·...

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Multimerization- and glycosylation-dependent receptor binding of SARS-1

CoV-2 spike proteins 2

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Kim M. Bouwman1*, Ilhan Tomris1*, Hannah L. Turner2, Roosmarijn van der 4

Woude1, Gerlof P. Bosman1, Barry Rockx3, Sander Herfst3, Bart L. Haagmans3, 5

Andrew B. Ward2, Geert-Jan Boons1,3,4,5, and Robert P. de Vries1# 6

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1 Department of Chemical Biology & Drug Discovery, Utrecht Institute for 8

Pharmaceutical Sciences, Utrecht University, Universiteitsweg 99, 3584CG 9

Utrecht, The Netherlands 10

2 Department of Integrative Structural and Computational Biology, The 11

Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 12

92037, USA 13

3 Department of Viroscience, Erasmus University Medical Center, 14

Rotterdam, The Netherlands. 15

4 Bijvoet Center for Biomolecular Research, Utrecht University, Utrecht, 16

The Netherlands. 17

5 Complex Carbohydrate Research Center, University of Georgia, 315 18

Riverbend Rd, Athens, GA 30602, USA. 19

6 Department of Chemistry, University of Georgia, Athens, GA 30602, 20

USA. 21

* Contributed equally 22

# for correspondence: [email protected] 23

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preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted September 4, 2020. ; https://doi.org/10.1101/2020.09.04.282558doi: bioRxiv preprint

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Receptor binding studies using recombinant SARS-CoV proteins have 26

been hampered due to challenges in approaches creating spike protein 27

or domains thereof, that recapitulate receptor binding properties of native 28

viruses. We hypothesized that trimeric RBD proteins would be suitable 29

candidates to study receptor binding properties of SARS-CoV-1 and -2. 30

Here we created monomeric and trimeric fluorescent RBD proteins, 31

derived from adherent HEK293T, as well as in GnTI mutant cells, to 32

analyze the effect of complex vs high mannose glycosylation on receptor 33

binding. The results demonstrate that trimeric fully glycosylated proteins 34

are superior in receptor binding compared to monomeric and immaturely 35

glycosylated variants. Although differences in binding to commonly used 36

cell lines were minimal between the different RBD preparations, 37

substantial differences were observed when respiratory tissues of 38

experimental animals were stained. The RBD trimers demonstrated 39

distinct ACE2 expression profiles in bronchiolar ducts and confirmed the 40

higher binding affinity of SARS-CoV-2 over SARS-CoV-1. Our results 41

show that fully glycosylated trimeric RBD proteins are attractive to 42

analyze receptor binding and explore ACE2 expression profiles in tissues. 43

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KEYWORDS 51

Coronavirus spike protein, fluorescent reporter protein, multimerization domain, 52

glycosylation, ACE2, SARS 53

54

INTRODUCTION 55

SARS-CoV-2 has sparked a society changing pandemic, and additional means 56

to understand this virus will facilitate counter-measures. SARS coronaviruses 57

carry a single protruding envelope protein, called spike, that is essential for 58

binding to and subsequent infection of the host cell. The trimeric spike protein 59

consists of 3 protomers of 140kD each, containing 60 N-linked glycosylation 60

sites. The spike is composed of an S1 and S2 domain, in which S2 contains 61

membrane fusion activity. S1 of coronaviruses can be further divided into N-62

terminal and C-terminal domains (NTD & CTD), both can contain the receptor-63

binding domain. In SARS-CoV-1 and -2 this domain is located in the CTD and 64

referred to as the receptor-binding domain (RBD). The RBD binds to 65

angiotensin-converting enzyme 2 (ACE2) [1-3], which functions as an entry 66

receptor for SARS-CoV. After binding and internalization, several proteinases 67

induce the spike protein into its fusogenic form allowing the fusion of the viral 68

and target membrane. Although this pathway is known, the details that are of 69

importance for receptor binding and what differentiates SARS-CoV-2 from its 70

predecessor SARS-CoV-1, are incompletely understood. It has been shown 71

that the affinity of the SARS-CoV-2 spike to ACE2 is significantly higher 72

compared to SARS-CoV-1 [2, 4]. However, how this relates to tissue and cell 73

tropism remains to be determined. 74

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Several recombinant protein approaches to create coronavirus spike proteins 76

have been utilized with success for the development of serological assays [5], 77

elucidating of spike structures, and isolation of neutralizing antibodies [6, 7]. 78

However, the conformation of the receptor-binding domain of the spike, which 79

can be in the “up” and “down” configuration [8-12], is highly variable. This 80

variability is important in receptor binding as only in the “up” conformation can 81

RBD bind ACE2. Approaches to control the conformation for vaccine purposes 82

using stabilization mutations appear to keep the RBD conformation in their 83

down-state [11, 12], making them non preferred proteins to analyze receptor-84

binding properties. Also, the multimerization status of the RBD is critical to allow 85

the analysis of ACE2 interactions. While monomeric RBD proteins can be 86

efficiently made in large quantities [5], and bind ACE2, they are hardly used in 87

receptor binding assays to cells and tissues. The majority of studies analyzing 88

RBD protein binding to cells and tissues, utilize Fc-tagged proteins [2]. Fc-tags 89

on recombinant proteins are extremely convenient for mammalian cell 90

expression and purification systems, which result in dimeric recombinant spikes 91

that are biologically functional. However, the coronavirus spike is trimeric and 92

thus Fc tagged spikes do not fully recapitulate native properties. 93

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We hypothesized that fluorescent trimeric RBD proteins would provide 95

complementary means to study RBD-receptor interactions. In this study, we 96

compared monomeric and trimeric SARS-CoV RBD with full-length trimeric 97

spikes expressed in cells producing proteins with either complex or high 98

mannose glycosylation. The fusion of sfGFP and mOrange2 at the C-terminus 99

has previously been shown to increase expression yields, protein stability, and 100


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afford additional means for fluorescent-based experiments [13], and thus are 101

attractive to be fused to RBD proteins. The resulting proteins were analyzed for 102

binding to cell culture cells and paraffin-embedded tissues of various hosts 103

including susceptible and non-susceptible animals. The results demonstrate 104

that fully glycosylated trimeric SARS-CoV-2 RBD proteins reveal the 105

differences in ACE2 expression between cell cultures and tissue sections. 106

These trimeric RBD proteins bind ACE2 efficiently in a species-dependent 107

manner and can be used to profile ACE2 tissue expression. Finally, we 108

observed distinct expression of ACE2 in bronchioles of experimental animal 109

models. 110

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RESULTS 112

Generation of fluorescent coronavirus receptor-binding domain spike proteins 113

To create recombinant fluorescent soluble full-length ectodomains, NTDs and 114

RBDs, we cloned these open reading frames (ORF) in plasmids with and 115

without the GCN4 trimerization domain, fused to either sfGFP or mOrange (Fig 116

1A). The monomeric and trimeric RBDs were efficiently expressed in both HEK 117

293T as well as GnTI cells (data not shown), with an increased yield up to 2- to 118

5-fold when fused to a C-terminal sfGFP (Fig 1B). Expression yields of the 119

mOrange2 fusions were comparable to that of sfGFP fusions (data not shown). 120

To illustrate the expression yields of SARS spike proteins or domains thereof 121

we measured the fluorescence in the cell culture supernatant (Fig 1C). The 122

wild-type full-length ectodomains were difficult to express even with the addition 123

of sfGFP or mOrange2 fusion (Fig 1B). To increase yields for the full-length 124

ectodomain we introduced the 2P and additional hexapro mutations [14], and 125


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analyzed the fluorescence in cell culture supernatants five days post-126

transfection after incubation at 33 or 37°C. Although we did not observe a large 127

increase in yields, we were able to purify sufficient protein to compare full-length 128

ectodomain trimers vs monomeric and trimeric RBD and NTD proteins. 129

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Spike RBD domains in frame with a C-terminal GCN4 and fluorescent reporter 131

protein display multimeric features on gel and maintain antigenicity 132

After purification, all RBD proteins were analyzed on gel under non- and 133

reducing conditions (Fig 2A). Without reducing agent, monomeric RBD proteins 134

revealed dimeric fractions which could be reduced to a single monomeric form. 135

The NTD trimers were reduced under non-reducing conditions, thus solely by 136

SDS. The trimeric RBD variants, on the other hand, revealed dimers and trimers 137

that could be reduced. Besides, the NTD of prototypical γ-coronavirus IBV-M41 138

and influenza A virus HA PR8 as control proteins were included. Finally, we 139

determined the extent of N-glycosylation maturation on purified proteins 140

expressed in either GnTI or 293T by subjecting the monomeric and trimeric 141

proteins to PNGaseF and EndoH treatment (Fig S1). Upon PNGaseF 142

treatment, all N-glycans were trimmed whereas 293T derived proteins were 143

insensitive to Endo H, as expected. Surprisingly trimeric RBDs derived from 144

GnTI cells were partially resistant to Endo H treatment, whereas the monomers 145

derived from GnTI cells where fully deglycosylated using Endo H. 146

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Fluorescent multimeric Spike RBD proteins maintain antigenicity 148

Next, we examined the antigenicity of the SARS-CoV-1 and -2 proteins using 149

serum collected from macaques 21 days post-infection with SARS-CoV-2 [15]. 150


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Both SARS-CoV-2 RBD monomers and trimers derived from 293T cells were 151

efficiently recognized, indicating proper folding (Fig 2B). As expected, SARS-152

CoV-1 RBD proteins were poorly recognized, and the negative controls M41 153

NTD and PR8 HA displayed baseline binding identical to pre-infection serum. 154

The NTD trimers were likewise not recognized by the serum (not shown), 155

indicating that the majority of antibodies in naïve animals after infection are 156

directed against the SARS-CoV RBD [16]. Similar results were obtained using 157

GnTI-derived proteins, with the RBD trimer being less efficiently recognized by 158

the macaque serum than its monomeric counterpart. This is in line with recent 159

observations that insect cell-derived proteins are less well bound by serum 160

antibodies [5], indicating the importance of mature N-glycans. 161

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RBD fused to GCN4 and sfGFP fold as trimers with three RBD and sfGFP 163

molecules divided by the trimerization coiled-coil. 164

To determine whether the fluorescent RBD trimers are indeed structured in a 165

trimeric manner we subjected these proteins to negative stain single-particle 166

EM. The EM data revealed that the RBD proteins form stable trimers that 167

resemble known spike structures (Fig. 2C). Initially, 58,018 individual particles 168

were picked, placed into a stack, and submitted to reference-free two-169

dimensional (2C) classification. From the initial 2D classes, particles that did 170

not resemble RBD were removed, resulting are final particle stacks of 32,152 171

particles, which were then subject to Relion 2D classification. All resultant 172

classes demonstrated evident and distinct trimeric RBD, GCN4, and three 173

sfGFP protein structures that could be identified in the EM images. From the 174

EM images, we generated a model in which we took the crystal structures of 175


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sfGFP, the GCN4 trimerization domain (PDB:2O7H), and the SARS-CoV-2 176

RBD (PDB: 6XM4) to demonstrate the likely structure of our RBD trimer (Fig 177

2D). 178

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Fluorescent multimeric Spike RBD proteins bind cell lines in an ACE2 180

dependent manner and similar to the full-length ectodomain. 181

To determine the biological activity of our RBD proteins we stained A549 and 182

VERO cells that are reported to support SARS-CoV replication, with the latter 183

being more susceptible [17]. However, A549 cells were bound by all our RBD 184

proteins with a slight increase in intensity from monomeric GnTI derived RBD 185

proteins to trimeric 293T derived RBDs (Fig 3). Trimeric 293T RBD binding was 186

efficiently blocked using 4μM recombinant ACE2 whereas 400nM ACE2 pre-187

incubation was not sufficient to prevent binding of fully glycosylated trimeric 188

RBD proteins to cells completely. SARS-CoV-2 RBD proteins bound slightly 189

more intensely to A549 cells compared to the same SARS-CoV-1 RBD 190

proteins. A similar pattern was observed for VERO-E6 cells, however, the fully 191

glycosylated SARS-CoV-2 RBD trimer bound markedly stronger compared to 192

the other RBD preparations (Fig S2A). Importantly, the full-length ectodomain 193

also bound efficiently to A549 cells (Fig S2B). We did not observe any binding 194

of the trimeric NTD domains to A549 cells (Fig S2B). MDCK cells, derived from 195

canine kidney, served as negative controls, to which we indeed did not observe 196

any binding with any of the indicated proteins (Fig S2C). 197

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Fluorescent RBD proteins reveal strong binding to bronchioles in both ferret 199

and Syrian golden hamster lungs. 200


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Ferrets are a susceptible animal model for SARS-CoV-2 [18, 19] and closely 201

related minks are easily infected on farms [20]. Formalin-fixed, paraffin-202

embedded tissue slides, will most closely resemble the complex membrane 203

structures to which spike proteins need to bind. First, ACE2 expression was 204

assessed using an ACE2 antibody which allowed for comparisons with SARS-205

CoV-RBD protein binding localization. Here, ACE2 expression was observed in 206

bronchiolar epithelium cells of ferret lungs (Fig 4A), while the antibody failed to 207

bind mouse lungs. Comparably, the SARS-CoV RBD protein preparations did 208

not stain mouse lungs. In ferret lung tissues, we observed only minimal binding 209

of monomeric RBD proteins derived from GnTI cells (Fig 4B). The fully 210

glycosylated counterparts showed a slight increase in fluorescent intensity, 211

which was further increased when fully glycosylated trimeric RBDs were 212

applied. In all cases, SARS-CoV-2 displayed a higher avidity compared to 213

SARS-CoV-1. A similar trend of binding intensities was observed for 214

monomeric, trimeric, and different N-glycosylated SARS-CoV-RBD proteins 215

fused to mOrange2 (Fig S3A). Again specific binding was seen to the epithelium 216

of terminal bronchioles and, to a much lower extent, to alveoli and endothelium. 217

The results were confirmed using horseradish peroxidase readout with a 218

hematoxylin counterstain (Fig S3B), which output is enzyme driven and purely 219

qualitative, however, we did observe similar differences in staining intensities. 220

Here, very minimal staining using the SARS-CoV NTD domains was observed 221

(Fig S3B), which we did not detect using a fluorescent readout (Fig S3C). To 222

determine if the binding was ACE2 dependent we pre-incubated trimeric RBD 223

proteins with recombinant ACE2. While 4 μM was sufficient to block binding to 224


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cell culture cells (Fig 4C), 8μM was needed to prevent all detectable binding to 225

ferret lung tissue. 226

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To confirm our observations of different binding on tissues, we quantified the 228

intensities of the ACE2 antibody and SARS-CoV-1 and -2 RBD proteins, except 229

for the monomeric GnTI derived proteins as these were almost at the 230

background (Fig 4D). As expected a noteworthy trend was observed of 231

increasing binding strength from SARS-CoV1 GnTI derived monomers to 232

SARS-CoV-2 fully glycosylated RBD trimers. Interestingly multimerization 233

appears to be more important for strong ACE2 interaction to tissue compared 234

to the glycosylation status. 235

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Finally, we applied our RBD proteins on the lung tissue of Syrian hamsters, 237

which are currently the preferred small animal model and known to be highly 238

susceptible to SARS-CoV-1 and -2 [21-23]. Here again, trimeric fully 239

glycosylated RBD proteins were superior in binding compared to their 240

monomeric counterparts. Interestingly, SARS-CoV-2 bound with an even 241

higher intensity to terminal bronchioles compared to SARS-CoV-1. Importantly, 242

all binding could be inhibited by pre-incubation of the RBD proteins with 243

recombinant ACE2, indicating that binding intensity differences are likely ACE2 244

related. 245

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DISCUSSION 247

In this report, we describe the generation of fluorescent trimeric RBD proteins 248

derived from SARS-CoV-1 and 2. These proteins can be efficiently expressed 249


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in adherent cells and purified directly from the cell culture supernatant using the 250

increased protein yields provided by the introduction of a C-terminal sfGFP or 251

mOrange fusion. The fluorescent RBD trimers were superior in receptor binding 252

on tissues compared to their monomeric RBD counterparts. 253

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Mature N-glycosylation on the spike protein appears to be important for the avid 255

binding of trimeric RBD proteins to ACE2 in different assays. There is a wealth 256

of information on site-specific N- and O- glycan conformations on spike proteins 257

[24-26], with some disagreement on the nature of the RBD N-glycans. In our 258

protein preparations, they appear to be complex as the majority could not be 259

cleaved by EndoH (Fig S1). Furthermore, the N-glycosylation of the NTD 260

domain has been shown to affect the conformation of the RBD in full-length 261

spikes [27], however similar analyses on the RBD N-glycans are lacking. 262

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Currently, most knowledge of ACE2 expression is based on genomic and 264

transcriptomic data [28]. However, these analyses are limited as it does not 265

determine expression biochemically on epithelial cells that make contact to the 266

outside world [29]. Several groups have transfected variants of ACE2 in cells 267

to analyze transduction by SARS-CoV-pseudo viruses, although informative 268

these studies do not provide information on the natural expression pattern of 269

ACE2 in susceptible hosts [30, 31]. A detailed understanding of the difference 270

between animal species and cell-specific expression of ACE2 at the molecular 271

level is essential, as this can provide valuable knowledge on potential hosts 272

that can be susceptible to SARS-like coronaviruses. Here we demonstrate that 273


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our fluorescent trimeric RBD proteins are complementary to study ACE2 274

expression and SARS-CoV receptor binding dynamics. 275

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An intriguing observation was the abundant expression of ACE2 in terminal 277

bronchioles both in the ferret and Syrian hamster lungs, which are used as 278

experimental animal models. SARS-CoV-RBD protein binding correlated 279

perfectly with the staining of the ACE2 antibody and a ferret infection study [18]. 280

In another ferret infection study, however, the infection was observed in nasal 281

epithelium and many type I pneumocytes and fewer type II pneumocytes in the 282

lungs, with few bronchial epithelial cells expressing viral antigen [19]. Some 283

studies have been performed with anti-ACE2 antibodies, but these stainings 284

appear not to correlate with infection patterns [32, 33]. We observed a similar 285

trend indicating that several other factors may be important to infect cells and 286

that infection could, in turn, regulate ACE2 expression. 287

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MATERIAL AND METHODS 289

Expression and purification of coronavirus spike proteins for binding studies 290

Recombinant SARS-CoV-1 and -2 envelope proteins and their subunits were 291

cloned using Gibson assembly from cDNAs encoding codon-optimized open 292

reading frames of full-length SARS-CoV-1 and -2 spikes (A kind gift of Rogier 293

Sanders, Amsterdam Medical Centre, The Netherlands). The pCD5 expression 294

vector as described previously [34], was adapted to clone the SARS-1 295

(GenBank: MN908947.3) and 2 (GenBank: MN908947.3), ectodomains 296

(SARS-2 15-1213, SARS-1 15-1195), N-terminal S1 (SARS-2 15-318, SARS-1 297

15-305, M41 19 to 272 ) and RBDs (SARS-2 319-541, SARS-1 306-527) 298


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sequences coding for a secretion signal sequence, a GCN4 trimerization 299

domain (RMKQIEDKIEEIESKQKKIENEIARIKK) followed by a seven amino 300

acid cleavage recognition sequence (ENLYFQG) of tobacco etch virus (TEV), 301

a super folder GFP [13], or mOrange2 [35] and the Twin-Strep 302

(WSHPQFEKGGGSGGGSWSHPQFEK); IBA, Germany). Alongside we 303

expressed infectious bronchitis virus M41 spike RBD (GenBank: AY851295.1) 304

[36], ) and influenza A virus PR8 HA (GenBank: NP_040980) [13]. The viral 305

envelope proteins were purified from cell culture supernatants after expression 306

in HEK293T or HEK293GnTI(-) cells as described previously [34]. In short, 307

transfection was performed using the pCD5 expression vectors and 308

polyethyleneimine I. The transfection mixtures were replaced at 6 h post-309

transfection by 293 SFM II expression medium (Gibco), supplemented with 310

sodium bicarbonate (3.7 g/L), Primatone RL-UF (3.0 g/L), glucose (2.0 g/L), 311

glutaMAX (Gibco), valproic acid (0,4 g/L) and DMSO (1,5%). At 5 to 6 days after 312

transfection, tissue culture supernatants were collected. 313

314

For ACE2 inhibition studies, ACE2 was expressed in a highly identical fashion, 315

ACE2 (Addgene Plasmid #145171) was cloned into a pCD5 expression vector 316

with SacI and BamHI restriction enzymes. The adapted pCD5 expression 317

vector with an N-terminal HA leader (MKTIIALSYIFCLVFA) peptide, ACE2, and 318

Twin-Strep (WSHPQFEKGGGSGGGSWSHPQFEK); IBA, Germany) was 319

purified from HEK293T cell culture supernatant. 320

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Determining expression yield 322


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We measure fluorescence in the cell culture supernatant when applicable using 323

a polarstar fluorescent reader with excitation and emission wavelengths of 480 324

nm and 520 nm for sfGFP and 520nm and 550nm for mOrange2, respectively. 325

Spike protein expression was confirmed by western blotting using a StrepMAB-326

HRP classic antibody. Proteins are purified using a single-step with strepTactin 327

sepharose beads in batch format. Purified proteins were pre-treated (were 328

indicated) with PNGase F or Endo H (New England Biolabs, USA) according to 329

the manufacturer’s protocol before analysis by Western blotting using Strep-330

MAb-HRP classic antibody. 331

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Antigen ELISA 333

Plates were coated with 2 μg/mL spike protein in PBS for 16 hours at 4°C, 334

followed by blocking by 3% bovine serum albumin (BSA, VWR, 421501J) in 335

phosphate-buffered saline-Tween 0,1% (PBS-T 0,1%). After the block, the 336

proteins were detected using a 21-days post-infection macaque serum and 337

serum of the same monkey before infection. Serum starting dilution was 1:100 338

and diluted 1:1 for 5 times and incubated at RT for 2 hrs. Next, wells were 339

treated with goat-α-human HRP secondary antibody for 1 hour at room 340

temperature. Serum spike binding antibodies were detected using ODP and 341

measured in a plate reader (Polarstar Omega, BMG Labtech) at 490 nm. 342

343

Negative stain electron microscopy structural analysis 344

SARS-CoV2-RBD-GCN4-sfGFP in 10mM Tris, 150mM NaCl at 4°C was 345

deposited on 400 mesh copper negative stain grids and stained with 2% uranyl 346

formate. The grid was imaged on a 200KeV Tecnai F20 electron microscope 347


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and a 4k x 4k TemCam F416 camera. Micrographs were collected using 348

Leginon [37] and then uploaded to Appion [38]. Particles were picked using 349

DoGPicker [39], stacked. 2D processing was undertaken using Relion. Images 350

showing Trimeric RBD, a GCN4 connector, and three sfGFPs. 351

352

Immunofluorescent cell staining 353

Vero-E6, A549, and MDCK cells grown on coverslips were analyzed by 354

immunofluorescent staining. Cells were fixed with 4% paraformaldehyde in 355

PBS for 25 min at RT after which permeabilization was performed using 0,1% 356

Triton in PBS. Subsequently, the coronavirus spike proteins were applied at 357

50μg/ml for 1 h at RT. Primary Strep-MAb classic chromeo-488 (IBA) and 358

secondary Alexa-fluor 488 goat anti-mouse (Invitrogen) were applied 359

sequentially with PBS washes in between. DAPI (Invitrogen) was used as 360

nuclear staining. Samples were imaged on a Leica DMi8 confocal microscope 361

equipped with a 10x HC PL Apo CS2 objective (NA. 0.40). Excitation was 362

achieved with a Diode 405 or white light for excitation of Alexa488 and 363

Alexa555, a pulsed white laser (80MHz) was used at 488 nm and 549 nm, and 364

emissions were obtained in the range of 498-531nm and 594-627 nm 365

respectively. Laser powers were 10 – 20% with a gain of a maximum of 200. 366

LAS Application Suite X was used as well as ImageJ for the addition of the 367

scale bars. 368

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Tissue staining 370

Sections of formalin-fixed, paraffin-embedded ferret, Syrian hamster, and 371

mouse lungs were obtained from the Department of Veterinary Pathobiology, 372


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Faculty of Veterinary Medicine, Utrecht University and the department of 373

Viroscience, Erasmus University, The Netherlands, respectively. Tissue 374

sections were rehydrated in a series of alcohol from 100%, 96% to 70%, and 375

lastly in distilled water. Tissues slides were boiled in citrate buffer pH 6.0 for 10 376

min at 900 kW in a microwave for antigen retrieval and washed in PBS-T three 377

times. Endogenous peroxidase activity was blocked with 1% hydrogen peroxide 378

for 30 min. Tissues were subsequently incubated with 3% BSA in PBS-T 379

overnight at 4 °C. The next day, the purified viral spike proteins (50μg/ml) were 380

added to the tissues for 1 h at RT. With rigorous washing steps in between the 381

proteins were detected with mouse anti-strep-tag- antibodies (IBA) and goat 382

anti-mouse IgG antibodies (Life Biosciences). Where indicated recombinant 383

RBD and ACE2 proteins were pre-incubated O/N at 4 °C before application on 384

lung tissues or cells. 385

386

Quantifying and plotting fluorescent image data 387

Image quantification was performed by measuring the intensity of gray pixels 388

of the stained ferret and Syrian hamster lung tissue slides with ImageJ version 389

1.52p. For stained tissues, background correction was performed by 390

subtracting the average signal intensity of the antibody control from the images 391

stained with ACE2 antibody, SARS-CoV1, and SARS-CoV2 recombinant 392

proteins. Regions of interest were set by highlighting the area using the Image 393

-> Adjust -> Threshold setting. The 8-bit images were checked for saturation by 394

plotting the distribution of gray values with Analyze -> Histogram. Subsequent 395

analysis was performed by quantifying the mean ± standard deviation of 396

alveoli/cells stained with distinct proteins in various images (Analyze -> 397


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Measure). Plotting means ± standard deviation values were performed in 398

GraphPad Prism v8.0.1. A 3D Surface plot was generated for several staining 399

conditions with Analyze -> 3D Surface Plot. 400

401

ACKNOWLEDGEMENTS 402

R.P.dV is a recipient of an ERC Starting Grant from the European Commission 403

(802780) and a Beijerinck Premium of the Royal Dutch Academy of Sciences. 404

Electron microscopic imaging work in the Ward lab was supported by Bill and 405

Melinda Gates Foundation OPP1170236. SH was funded by NIH/NIAID 406

(contract number HHSN272201400008C). The authors would like to thank 407

Rogier Sanders and Phillip Brouwer of the Amsterdam Medical Centre for 408

sharing the SARS-CoV 1 & 2 open reading frames, Andrea Gröne, Darryl 409

Leydekkers, and Hélène Verheije from the Division of Pathology, Department 410

Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht 411

University, for sharing tissues, IBV M41 plasmids and editing of the manuscript. 412

413

REFERENCES 414

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573

FIGURE LEGENDS 574


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Figure 1. Expression of coronavirus spike proteins using sfGFP and 575

mOrange2 fusions 576

(A) Spike expression plasmids with and without GCN4 trimerization 577

motif fused with either sfGFP or mOrange2 578

Schematic representation of the used spike expression cassette. The spike 579

open reading frame is under the control of a CMV-promotor and was cloned in 580

frame with DNA sequences coding for the CD5 signal peptide. At the C-581

terminus a GCN4 trimerization domain followed by sfGFP or mOrange2, and a 582

TEV cleavable Strep-tag II. 583

(B) Expression analyses of non and sfGFP fused monomeric and 584

trimeric RBD proteins and the full-length spike 585

Denatured samples of the cell culture supernatant were subjected to SDS-586

PAGE and western blot analyzes stained with an anti-Streptag-HRP antibody. 587

(C) Quantification 588

sfGFP emission was directly measured in the supernatants. 589

(D) Full-length spike adaptation to the hexapro variant. 590

Full-length spike expression vectors containing the wildtype, 2P or Hexapro 591

were expressed at 33 or 37C and fluorescence was measured in cell culture 592

supernatant 4 days post transfection. 593

594

595

Figure 2. Molecular analyses 596

(A) SDS-PAGE analyses of purified RBD proteins 597

500 ng of purified proteins were loaded on a gel with or without preheating for 598

30 minutes at 98C in the presence of a reducing agent. 599


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(B) Antigenic analyses using 21-day post-infection macaque serum 600

2μg/ml SARS-CoV-RBD proteins were coated on 96-well plates. Proteins were 601

detected with macaque serum for 2hrs at RT. An anti-human IgG-HRP was 602

used to detect RBD specific antibodies. Both 293T and GnTI derived proteins 603

were analyzed. As negative controls, IBV-M41-NTD and HA-PR8D were 604

included. 605

(C) Negative stain EM of trimeric RBD fused to sfGFP 606

Negative-stain 2D class averages of soluble RBD proteins demonstrate that 607

they are well-folded trimers. The C-terminal helices and fusion proteins are 608

visible in some class averages. 609

(D) Structural model 610

Based on the crystal structure of a SARS-CoV-2 RBD in the up conformation, 611

with a GNC4 trimerization domain with 3 sfGFP domains added. 612

613

Figure 3. Binding of RBD proteins to A549 cells 614

SARS-CoV-RBD proteins were applied at 50μg/ml onto A549 cells and where 615

indicated pre-incubated with recombinant ACE2 protein. SARS-CoV proteins 616

were detected using an anti-Streptag and a goat-anti-mouse antibody 617

sequentially. sfGFP fused SARS-CoV-RBD proteins were applied from GnTI 618

monomers to 293T trimers, left to right. Scalebar is 50μm. 619

620

Figure 4. Binding of SARS-COV-RBD proteins and ACE2 antibody to 621

mouse and ferret lung tissue slides 622

(A) ACE2 antibody, antibody control, and SARS-CoV-1 and -2 on 623

mouse lung. Scalebar is 100μm. 624


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(B) SARS-CoV-RBD fluorescent protein localization in ferret lung tissue. 625

SARS-CoV-RBD proteins were applied at 50μg/ml and detected using an anti-626

Streptag and goat-anti-mouse antibodies sequentially. DAPI was used as a 627

nucleic stain. Scalebar is 100μm. 628

(C) SARS-CoV-RBD trimer produced in HEK 293T cells pre-incubated with 629

recombinant ACE2 before application on ferret lung tissue slides. 630

(D) Quantification 631

The intensity of gray pixels of stained ferret lung tissue slides with ImageJ 632

version 1.52p. 633

634

Figure 5. Binding of SARS-CoV-RBD proteins to Syrian hamster tissue 635

slides 636

SARS-COV-RBD proteins (50μg/ml) were applied to Syrian hamster lung tissue 637

slides and detected using additional anti-Streptag and goat-anti-mouse 638

antibodies sequentially. Where indicated SARS-CoV-RBD proteins were pre-639

incubated with recombinant ACE2 protein. 640

Scalebar is 100μM. 641

642

643

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646

647

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Figure 1. 650

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Figure 2. 675

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Figure 3 677

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Figure 4. 685

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Figure 5 690

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706

Supplemental Figure 1. 707

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Supplemental figure 1. Glycosylation analyses of the SARS-CoV protein 724

preparations. 725

0.5μg of protein was subjected without or with PNGase F or EndoH for 1hr and 726

subjected to SDS-PAGE and western blot analyzes. 727

728

729

730


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732

Supplemental figure 2. Binding of RBD proteins to cell lines 733

(A) Protein binding of RBD proteins observed on VERO E6 cells. 734

Proteins were applied 50μg/ml and where indicated pre-incubated with 735

recombinant ACE2 protein. Spike proteins were detected using anti-strep and 736

goat-anti-mouse antibodies. Scalebar is 5μm. 737

(B) Binding of full-length SARS-CoV-2 ectodomain, IBV-M41, antibodies 738

only, and NTD spike proteins to A549 cells. Proteins were applied 50μg/ml 739

and detected using anti-strep and goat-anti-mouse antibodies. Scalebar is 740

50μm. 741

(C) Non-binding of RBD trimers to MDCK cells. 742


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743

744


746

Supplemental figure 3. Binding of RBD proteins to tissues 747

(A) Binding of RBD proteins fused to mOrange2 to ferret lung tissues 748


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Proteins were applied 50μg/ml and detected using anti-strep and goat-anti-749

mouse antibodies. Scalebar is 100μm. 750

(B) Binding of RBD proteins fused to sfGFP proteins to ferret lung tissues, 751

using HRP as a readout. 752

Identical experiment to (A) but using an HRP readout using anti-strep and goat-753

anti-mouse antibodies. Scalebar is 100μm. 754

(C) Control staining on ferret lung tissues using HRP as readout. 755

M41, NTD trimers of SARS-CoV-1 and -2 and antibodies only. Proteins were 756

applied 50μg/ml and detected using anti-strep and goat-anti-mouse antibodies. 757

Scalebar is 100μm. 758

(D) Lack of NTD binding to ferret lung tissue using fluorescence 759

Proteins were applied 50μg/ml and detected using anti-strep and goat-anti-760

mouse antibodies. Scalebar is 100μm. 761

(E) Control stainings to Syrian hamster tissues, antibodies only and M41 762

Proteins were applied 50μg/ml and where indicated pre-incubated with 763

recombinant ACE2 protein. Scalebar is 100μm. 764

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https://doi.org/10.1101/2020.09.04.282558

multimerization- and glycosylation-dependent receptor binding of sars-cov-2 spike proteins ·...

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