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HCoV-HKU1 Spike protein uses O-acetylated sialic acid as an attachment 1

receptor determinant and employs HE protein as a receptor-destroying enzyme 2 3

Short title: HCoV-HKU1 virus receptor and RDE function of HE 4 5 Xingchuan Huang1, Wenjuan Dong1,2, Aleksandra Milewska3, Anna Golda3, Yonghe Qi1,4, 6 Quan K. Zhu5, Wayne A. Marasco5, Ralph S. Baric6,7, Amy C. Sims7*, Krzysztof Pyrc3,8*, 7 Wenhui Li1*# and Jianhua Sui1*# 8 9 1 National Institute of Biological Sciences, No. 7 Science Park Road, Zhongguancun Life 10

Science Park, Changping, Beijing, 102206, China. 11 12 2 China Agricultural University Graduate Program, National Institute of Biological Sciences, 13

Beijing, 102206, China. 14 15 3 Microbiology Department, Faculty of Biochemistry, Biophysics and Biotechnology, 16

Jagiellonian University, Gronostajowa 7, 30-387 Krakow, Poland 17 18 4 Beijing Normal University Graduate Program, National Institute of Biological Sciences, 19

Beijing, 102206, China 20 21 5 Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute; Department of 22

Medicine, Harvard Medical School, Boston, Massachusetts, 02115, USA 23 24 6 Department of Microbiology and Immunology, University of North Carolina, Chapel Hill, 25

North Carolina, 27599, USA 26 27 7 Department of Epidemiology, University of North Carolina, Chapel Hill, North Carolina, 28

27599, USA 29 30 8 Malopolska Centre of Biotechnology, Jagiellonian University, Gronostajowa 7, 30–387 31

Krakow, Poland 32

* A.C.S., K.P., W.L. and J.S. are co-senior authors of the paper 33

# Address correspondence to W.L. (liwenhui@nibs.ac.cn) or J.S. (suijianhua@nibs.ac.cn) 34 35

Word count for the abstract: 241 36

Word count for the text: 6370 37

Number of figures: 4 figures 38

39 Key words: Coronavirus, hCoV-HKU1, receptor, hemagglutinin-esterase, HE, Spike protein, 40 neuraminidase, sialic acid 41

42

JVI Accepted Manuscript Posted Online 29 April 2015J. Virol. doi:10.1128/JVI.00854-15Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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Abstract 43

44

Human coronavirus (hCoV) HKU1 is one of six hCoVs identified to date and the only one 45

with an unidentified cellular receptor. HCoV-HKU1 encodes a hemagglutinin-esterase (HE) 46

protein that is unique to the betacoronaviruses group a (group 2a). The function of HKU1-HE 47

remains largely undetermined. In this study, we examined binding of the S1 domain of 48

hCoV-HKU1 spike to a panel of cells and found that the S1 could specifically bind on the cell 49

surface of a human rhabdomyosarcoma cell line, RD. Pretreatment of RD cells with 50

neuramidase (NA) and trypsin greatly reduced the binding, suggesting that the binding was 51

mediated by sialic acids on glycoproteins. However, unlike other group 2a CoVs, e.g. 52

hCoV-OC43 for which 9-O-acytylated sialic acid (9-O-Ac-Sia) servers as a receptor 53

determinant, HKU1-S1 neither bound with 9-O-Ac-Sia containing glycoprotein(s), nor rat and 54

mouse erythrocytes. Nonetheless, the HKU1-HE was similar to OC43-HE, also possessed 55

sialate-O- acetylesterase activity and acted as a receptor-destroying enzyme (RDE) capable 56

of eliminating the binding of HKU1-S1 to RD cells, whereas the O-acetylesterase inactive 57

HKU1-HE mutant lost this capacity. Using primary human ciliated airway epithelial cell 58

cultures (HAE), the only in vitro replication model for hCoV-HKU1 infection, we confirmed that 59

pretreatment of HAE cells with HE but not the enzymatically inactive mutant blocked 60

hCoV-HKU1 viral infection. These results demonstrate that hCoV-HKU1 exploits O-AC-Sia as 61

a cellular attachment receptor determinant to initiate the infection of host cells, and its HE 62

protein possesses the corresponding sialate-O-acetylesterase RDE activity. 63

64

65

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Importance statement 66

67

Human coronaviruses (hCoV) are important human respiratory pathogens. Among the six 68

hCoVs identified to date, only hCoV-HKU1 has no defined cellular receptor. It is also unclear 69

whether hemagglutinin-esterase (HE) protein plays a role in viral entry. In this study, we found 70

that, similar to other members of the group 2a CoVs, sialic acid moieties on glycoproteins are 71

critical receptor determinants for the hCoV-HKU1 infection. Interestingly, the virus seems to 72

employ different type of sialic acid than other group 2a CoVs. In addition, we determined that 73

the HKU1-HE protein is an O-acetylesterase and acts as a receptor-destroying enzyme (RDE) 74

for hCoV-HKU1. This is the first study to demonstrate that hCoV-HKU1 uses certain types of 75

O-acytylated sialic acid residues on glycoproteins to initiate the infection of host cells, and 76

HKU1-HE protein possesses sialate-O-acetylesterase RDE activity. 77

78

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Introduction 79

80

Human coronaviruses (hCoV) are enveloped RNA viruses. They are usually associated 81

with mild to moderate respiratory tract illnesses, but can also cause severe and highly lethal 82

disease depending on the virus strain (1). Six hCoV strains have been identified to date and 83

belong to four different groups, including hCoV-229E and hCoV-NL63 in the 84

alphacoronaviruses (group 1); hCoV-OC43 and hCoV-HKU1 in the betacoronaviruses group a 85

(group 2a); severe acute respiratory syndrome CoV (SARS-CoV) in the betacoronaviruses 86

group b (group 2b); and Middle East Respiratory Syndrome CoV (MERS-CoV) in the 87

betacoronaviruses group c (group 2c). Infections by viruses in groups 1 and 2a are common 88

worldwide, and can also cause severe disease in young children or immunocompromised 89

adults. SARS-CoV (2-4) and MERS-CoV (5, 6) are two highly virulent hCoVs causing severe 90

respiratory diseases with high morbidity and mortality (7); the latter strain is still circulating in 91

human populations. 92

93

Cellular receptor specificity plays an important role in viral cell and tissue tropism, 94

pathogenesis, interspecies transmission and adaptation. The CoV Spike (S) glycoprotein is 95

generally responsible for binding to cellular receptors and mediating viral entry. S protein is a 96

large type-I transmembrane glycoprotein that exists as a trimer protruding from the surface of 97

virions (8). S proteins have an amino-terminal (NT) S1 domain that mediates binding with 98

cellular receptors and a carboxy-terminal (CT) S2 domain that mediates subsequent virus-cell 99

membrane fusions. A wide-range of diverse cellular receptors specifically recognized by the 100

S1 domains has been identified for all the aforementioned hCoVs except hCoV-HKU1. 101

Human aminopeptidase N (CD13) is the cellular receptor for hCoV-229E (9). 9-O-acytylated 102

sialic acid (9-O-Ac-Sia) is the cellular receptor determinant for hCoV-OC43 (10). hCoV-NL63 103

and SARS-CoV both employ human angiotensin-converting enzyme 2 (ACE2) to mediate 104

cellular entry (11, 12), while hCoV-NL63 utilizes heparan sulfate proteoglycans for attachment 105

to target cells (13). MERS-CoV utilizes dipeptidyl peptidase 4 (DPP4 or CD26) receptor to 106

enter host cells (14). 107

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HCoV-HKU1 was initially identified in 2005 from a pneumonia patient in Hong Kong (15). 108

It was subsequently found to be as common and widespread as previously known hCoVs, 109

hCoV-229E, hCoV-OC43 and hCoV-NL63 (16-19). Characterization of hCoV-HKU1 has been 110

challenging due to the lack of a convenient cell line-based culture system. It was recently 111

demonstrated that hCoV-HKU1 replicates to high titer in an in vitro culture system that uses 112

primary human ciliated airway epithelial cells (HAE) or type II alveolar epithelial cells (20-22), 113

however the functional receptor(s) of hCoV-HKU1 and other important aspects of virus-host 114

interaction remain unknown. Being a member of group 2a CoVs, HKU1-CoV also carry 115

another viral surface protein hemagglutinin-esterase (HE) encoding gene that is present 116

exclusively in this group of CoVs genomes (23). The HE protein is also a type-I 117

transmembrane glycoprotein comprised of two functional domains: an O-acetylated sialic acid 118

binding domain and a corresponding sialate O-acetylesterase domain (24). HE protein 119

functions primarily as a receptor-destroying enzyme (RDE) for CoVs, e.g. hCoV-OC43 and its 120

proposed zoonotic ancestor bovine coronavirus (BCoV) (25). Both viruses bind to receptor 121

9-O-Ac-sia via their S proteins, and their HE proteins mediate RDE activity late in the infection 122

cycle via the sialate-9-O-acetylesterases domain to facilitate the release of viral progeny and 123

escape from attachment on non-permissive host cells (23, 26). In contrast, mouse hepatitis 124

virus (MHV), another member of group 2a CoVs infects cells via the interaction of S protein 125

with its principle receptor carcinoembryonic antigen-related cell adhesion molecule 126

(CEACAM1a), while the MHV HE protein functions at very early viral attachment steps 127

through a concerted action of its O-acetylated sialic acid binding and RDE activities (27, 28). 128

To date the function and role of the hCoV-HKU1 HE protein remains undefined. 129

130

In this study, we found that hCoV-HKU1 S protein mediated viral attachment by utilizing 131

O-acytylated sialic acids on glycoprotein(s) as a receptor determinant or initial attachment 132

factors. The HE protein of hCoV-HKU1 did not exhibit sialic acid binding activity but instead 133

mediated sialate-O-acetylesterase RDE activity specific to the O-acytylated sialic acids 134

recognized by the S protein. Interestingly, HKU1-HE protein displayed similar 135

sialate-9-O-acetylesterase RDE activity as OC43-HE and BCoV-HE. In the hCoV-HKU1 in 136

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vitro replication model, we further demonstrated that the HE protein but not an enzymatically 137

inactive HE mutant acted as a RDE and completely blocked or greatly reduced infection 138

depending on the dose of inoculating hCoV-HKU1. These findings revealed that early viral 139

entry steps for hCoV-HKU1 are similar to but also distinct from other members of group 2a 140

CoVs. Like hCoV-OC43 and BCoV, hCoV-HKU1 employs O-acytylated sialic acids as a 141

primary receptor determinant or attachment factor and its HE protein as corresponding RDE; 142

however, hCoV-HKU1 also uniquely requires additional receptor determinants than 143

hCoV-OC43 and BCoV. 144

145

Materials and Methods 146

147

Construction of expression plasmids. A synthetic codon-optimized sequence for the 148

HKU1-S1 gene (Genebank accession number NC_006577.2) encoding aa15-600 was cloned 149

into a mammalian expression vector containing a CD5 signal peptide and a C-terminal Fc tag 150

from mouse IgG2a (mFc). The expression cassette was under a CAG (CMV early 151

enhancer/chicken β actin) promoter. The resulting construct, pCAGGS-HKU1-S1(600)-mFc, 152

encodes a chimeric S1 protein with an N-terminal CD5 signal peptide and mFc at its C 153

terminus. Similarly, plasmids encoding other proteins were constructed, including the NT 154

domain of HKU1-S1 (aa15-268), the NT domain of hCoV-OC43-S1 (aa15-268) (ATCC 155

VR-759 strain, AAT84354), NT domain of S1 of CoV-HKU3 (aa16-323) (DQ022305). The 156

extracellular domain of HE proteins from different CoVs were also similarly constructed, 157

including HKU1-HE (aa14-358, NC_006577.2), hCoV-OC43-HE protein (aa19-376, 158

AAX85668.1), BCoV-HE (aa19-377, AAA92991.1), and MHV-S-HE (aa 25-393, AAX08110.1). 159

Plasmids encoding mutants of HKU1-HE were generated by site-directed QuikChange 160

mutagenesis method (Stratagene). All mutations were confirmed by DNA sequencing, in 161

which the codon for the esterase catalytic residue Ser40 was substituted by Ala (S40A mutant) 162

or the catalytic triad S40, H329, and D326 were all substituted by Ala (S40A/H329A/D326A). 163

164

Expression and purification of recombinant proteins. HEK293T cells were transiently 165

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transfected with the expression plasmids using polyethyleneimine (Polysciences). At 12 h 166

after transfection, the medium was replaced by 293 SFM II expression medium (Life 167

Technology). Tissue culture supernatants were harvested 3 days after transfection, and the 168

recombinant proteins were purified by protein A-affinity chromatography. 169

170

Flow cytometry (FACS) analysis. HKU1-S1(600)-mFc or other proteins at different 171

concentrations were diluted in FACS buffer (PBS containing 0.5% BSA and 0.1% NaN3) and 172

then incubated with 0.5-1x106 of RD cells or red blood cells (RBCs) from mouse or rat blood 173

samples at 4ºC for 0.5-1 hr. Cells were then washed three times with FACS buffer and 174

followed by incubation with FITC-labeled anti-mouse Fc antibody at a dilution following the 175

manufacturer’s instruction (Sigma or Pierce) at 4 ºC for 30 mins. Cells were washed as above, 176

and the binding of proteins to cells was analyzed by a BD FACS LSRII (Beckon,Dickinson) 177

flow cytometer and FCS Express software (De NoVo Software). For FACS analysis to 178

examine the inhibition of HKU1-S1 binding to RD cells, the cells were pretreated with 179

indicated HE proteins or enzymes at different concentrations, then incubated with HKU1-S1 180

and analyzed binding as described above. The NA was from clostridium perfringens (Sigma), 181

trypsin was from bovine pancreas and TPCK-treated (Sigma). For both NA and trypsin, the 182

pretreatment was carried out at 37 ºC for 1 hr ; for HE proteins the pretreatment was carried 183

out at 4 ºC for 1 hr. 184

185

Indirect immunofluorescence. RD cells were seeded on glass cover slips one day before 186

staining. Cells were washed three times with PBS, blocked with 0.5%BSA/PBS at 37 ºC for 30 187

mins, incubated with HKU1-600-mFc or HKU3-323-mFc at 20 μg/mL in PBS at 4 ºC for 1 hr, 188

followed by washing three times with PBS, then incubated with FITC-Goat anti-Mouse Fc 189

antibody (Sigma) at 4 ºC for 1 hr. Cells were washed three times, then incubated with 5 μg/mL 190

Hoechst 33258 at 37 ºC for 10 mins, followed by three additional washes and finally incubated 191

with 5 μg/mL FM-4-64 on ice for 1 min. Cells were analyzed and imaged with a 63x oil 192

objective using an Zeiss LSM510 Meta Confocal Microscope. Representative images are 193

shown. 194

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195

Hemagglutination assay. About 0.25-0.5% suspension of RBCs prepared from mouse 196

(BALB/c) or rat (Sprague Dawley) blood were added to a round-bottom 96-well plate at 50 197

μL/well. S1 proteins were two-fold serial diluted with 0.5%BSA/PBS and added at 50 μL/well 198

to the wells containing RBCs. For HE protein inhibition assay, RBCs were preteated with 199

two-fold serial diluted HE first, washed by PBS and followed by adding 50 μL/well of 10μg/mL 200

of hCoV-OC43-S1 to the wells containing the HE pretreated and washed RBCs. The plates 201

were left at room temperature for 60 minutes or longer until hemagglutination developed or 202

RBCs gradually settled. Positive hemagglutination results formed a uniform reddish color 203

across the well, whereas negative results appeared as dots in the center of round-bottomed 204

plates due to RBCs sedimentation. 205

206

ELISA assay. The binding of S1 proteins to BSM was determined by an ELISA assay as 207

previously described with modification (28). MaxisorpTM 96-well plates (NUNC) were coated 208

overnight at 4ºC with BSM (Sigma) at 10 μg/mL at 100 μL/well. The wells were washed with 209

washing buffer (PBST, 0.05% Tween-20 in PBS) and treated with blocking buffer (PBS, 0.05% 210

Tween-20, 2% nonfat milk) for 1 hr at RT. Serial diluted S1 proteins were prepared in blocking 211

buffer (starting concentration 20 μg/mL) and then added to the BSM-coated wells at 100 212

μl/well. Incubation was continued for 1 hr followed by washing with PBST for six times. 213

Binding was detected using an HRP-conjugated goat anti-mouse IgG (1:10,000 in blocking 214

buffer; Pierce) followed by washing again. The optical density at 450 nm was measured after 215

incubation of the peroxidase tetramethylbenzidine (TMB) substrate and stop solution. 216

217

Acetylesterase activity assay. Chromogenic p-nitrophenyl acetate (pNPA, Sigma) 218

substrate was serially diluted in two-fold, and then incubated with 1 or 2 μg/mL HE-mFc 219

protein or its mutants in 100 μL volume in PBS (pH7.4) at 37 ºC for different time period as 220

indicated. The acetylesterase activity was determined by measuring the release of 221

para-nitrophenol (OD at 405 nm) at the end of each reaction in microtiter plates with a 222

microplate spectrophotometer (Bio-RAD). An unrelated protein was used as a control in the 223

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enzymatic assay, the OD405 for HE protein or mutants was subtracted with that of this control. 224

The Km value of HE protein was calculated from the Michaelis-Menten Enzyme Kinetics using 225

Graphpad Prism 5 software. 226

227

Neuraminidase activity assay. An Amplex red neuraminidase assay kit (Molecular 228

Probes, Invitrogen) was used to measure NA activity. Briefly, 25 μg/mL HKU1-HE protein 229

was serially diluted in 50 μL of 1x reaction buffer followed by addition of 50 μL of 2x working 230

solution containing of 100 μM Ample Red reagent containing 0.2 U/mL of HRP, 4 U/mL of 231

galactose oxidase and the fetuin substrate serially diluted 100-fold from 2.5 mg/mL to 2.5 232

pg/mL. The mixture was incubated at 37 °C for 10 mins under dark, the fluorescence signal 233

was then measured by at 595 nm wavelength and the values were used to indicate relative 234

NA activity. 235

236

HKU1 infection of HAE. HAE culture system has been described previously (20). Briefly, 237

the apical surface of HAE were washed three times in situ with phosphate-buffered saline 238

(PBS) and then treated with testing reagents or controls by incubation at 32°C for 1 hr 239

followed by washing with PBS to remove testing reagents. The treatment and washing were 240

repeated two more times. Then HAE was inoculated with 100 µl of viral stock. Following 241

incubation for 2 hrs at 32°C, the unbound virus was removed by washing with 500 μL for 10 242

min at 32°C for three times, and the HAE were maintained at an air-liquid interface for the 243

remainder of the experiment at 32°C. HKU1 replication kinetics were determined at specific 244

time points post-inoculation as indicated, 120 μL of PBS was applied to the apical surface of 245

HAE, and after 10 mins of incubation at 32°C the apical sample was harvested for RNA 246

isolation. The RNA was then analyzed by real-time reverse transcriptase (RT)-PCR to 247

determine viral genomic mRNA copies (20). 248

249

Results 250

251

S1 domain of hCoV-HKU1 binds to RD cells. 252

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As CoV S1 domains generally mediate the interactions with cellular receptor(s) to trigger 253

subsequent viral-host cell membrane fusion to initiate viral entry, we first expressed codon 254

optimized soluble HKU1 S1 domain (amino acids (aa) 15 to 600) and fused it to the Fc domain 255

from murine IgG2a (HKU1-S1(600)-mFc) (Fig. 1A ) to identify the cellular receptor/attachment 256

factor for hCoV-HKU1. As a control, we also expressed the NT of the bat coronavirus HKU3 257

(29) S1 domain (aa16-323) fused to mFc, HKU3-S1(323)-mFc. To determine, which if any, 258

immortalized cell lines expressed the cellular receptor for hCoV-HKU1, we probed cell lines 259

that were isolated from several different species and tissues with our HKU1-S1 protein using 260

flow cytometry. These cell lines included 293T (human embryonic kidney cells), HeLa (human 261

cervical adenocarcinoma), CHO (Chinese hamster ovary cells), A549 (human lung epithelial 262

adenocarcinoma cells), Caco2 (human epithelial colorectal adenocarcinoma cells), HepG2 263

(human liver hepatocellular carcinoma cell line), Huh-7 (human hepatoma cells), RD (human 264

rhabdomyosarcoma/muscle tumor cells), HRT-18 (human colon adenocarcinoma cells), Lovo 265

(human colon adenocarcinoma cells), MDCK (Madin-Darby canine kidney cells), Vero 266

(African green monkey kidney cells). Interestingly, only RD cells showed specific strong 267

binding with 5 μg/mL of HKU1-S1(600)-mFc as compared with the control protein (Fig. 1B - 268

left), no specific binding was found for all other cell lines tested, a representative negative 269

staining result on HeLa cells was shown in Fig.1B - right. To independently confirm that 270

HKU1-S1 binds to the surface of RD cells, we incubated cells with either HKU1-S1(600) or 271

HKU3-S1(323) followed by fluorescent-labeled secondary antibody and then used FM-4-64, a 272

lipophilic probe that fluoresces intensely upon binding to the outer leaf of the plasma 273

membrane. As shown in Fig. 1C - left, HKU1-S1(600) and FM-4-64 had similar staining 274

patterns on the cell membrane of RD cells, suggesting that both were labeling the surface of 275

the cells. In contrast, no HKU3-S1(323) could be detected on the surface of the RD cells (Fig. 276

1C - right). FACS analysis also showed that the binding of HKU1-S1 with RD cells was in a 277

dose-dependent manner (Fig. 1D), the binding can be detected at a low concentration of 278

HKU1-S1(600)-mFc protein at 0.61 μg/mL. These results indicate that a cellular attachment 279

factor or receptor(s) for hCoV-HKU1 is present on the surface of the RD cells. We further 280

tested whether the HKU1-S1’s NT can bind with RD cells. The NT aa15-268 of HKU1-S1 was 281

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expressed as a mFc-fusion protein (HKU1-S1(268)-mFc) (Fig. 1A), and analyzed for binding 282

with RD cells by FACS analysis. As shown in Fig. 1E - left, the NT domain did not bind to RD 283

cells even at a high concentration of 10 μg/mL, suggesting that the binding of HKU1-S1 to RD 284

cells requires regions beyond the NT of S1, the NT domain alone is not sufficient to support 285

the binding. In contrast, the NT of hCoV-OC43-S1(268) protein could bind to RD cells in a 286

dose dependent manner (Fig. 1E - right), but the binding activity was relatively weaker than 287

that of HKU1-S1(600) to RD cells. 288

289

Binding of HKU1-S1 to RD cells is sialic acid dependent. 290

Sialic acids serve as attachment factors or receptors for a number of viruses (28, 30, 31). 291

9-O-Ac-sia was found to be essential for viral entry of BCoV and hCoV-OC43 (10, 30). To 292

investigate whether sialic acids are also involved in the binding of HKU1-S1 to RD cells, we 293

first pretreated cells with neuraminidase (NA), a sialidase that removes terminal free or 294

modified sialic acids which are α2,3-, α2,6- or α2,8-linked to the subterminal residue of a 295

sugar chain. Treated cells were then incubated with 5 μg/mL of HKU1-S1(600)-mFc. As 296

shown in Fig. 2A - left, pretreatment with NA ranging from 20- 500 mU/mL markedly reduced 297

the binding of HKU1-S1(600) to RD cells in a dose-dependent manner; 500 mU/mL of NA 298

reduced the binding to about 3-5% of the levels observed in the mock treated cells. Similarly, 299

NA treatment of RD cells resulted in the reduction of OC43-S1(268)-mFc binding (Fig. 2A 300

–middle). Whereas a control protein SIRPα binding to RD cells via its protein receptor CD47 301

(32) expressed on RD cell surface was not affected by NA treatment (Fig. 2A –right). This 302

result indicated that cell surface sialic acids participated in the binding of HKU1-S1(600) 303

protein to RD cells. To further test whether sialic acids involved in the binding are attached to 304

glycoprotein(s), RD cells were pretreated with TPCK-trypsin protease and then inoculated 305

with 5 μg/mL of HKU1-S1(600)-mFc. As shown in Fig. 2B, pretreatment with trypsin 306

dose-dependently reduced the binding of HKU1-S1 to RD cells, the maximum dose tested at 307

20 μg/mL trypsin reduced the binding to about 20% of the levels in the mock treated cells. 308

Trypsin treatment also similarly reduced binding of OC43-S1 to RD cells. Taken together, 309

these findings suggest that HKU1-S1 protein, similar to OC43-S1, can bind to sialic acids that 310

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are attached to glycoprotein(s). 311

312

Unlike S1 of hCoV-OC43, HKU1-S1 cannot bind to 9-O-acetylated sialic acid containing 313

glycoprotein or RBCs. 314

To understand the sialic acid specificity and preference of HKU1-S1, bovine submaxillary 315

mucin (BSM), which mainly contains 9-O-Ac-sia and 8,9-di-O-Ac-sia (33), was first tested for 316

binding with HKU-S1 by ELISA. As shown in Fig. 2C, no binding was observed for BSM 317

coated on ELISA plate, whereas the positive control OC43-S1(268)-mFc bound to BSM under 318

the same condition tested. 319

Engagement of sialic acid by viruses usually correlates with the capacity to agglutinate 320

red blood cells (RBCs) from different animal species. hCoV-OC43 and influenza C viruses use 321

9-O-Ac-sia for attachment, and consequently both viruses have hemagglutination (HA) 322

activity specific for rat and mouse RBCs, which have a high concentration of 9-O-Ac-sia on 323

their surface (33, 34), but not for human, sheep, or horse RBC as they have little to no 324

9-O-Ac-sia expressed (10, 35, 36). Using OC43-S1(268)-mFc protein as a positive control, 325

both rat and mouse RBCs could be agglutinated by the OC43-S1 protein dose-dependently 326

as expected, but HKU1-S1(600) protein showed no HA activity on RBCs from both species 327

(Fig. 2D). Consistently, HKU1-S1 showed no binding whereas OC43-S1 strongly bound to 328

both RBCs dose-dependently in a FACS analysis (Fig. 2E). These results suggest that 329

hCoV-HKU1 is different from influenza C, hCoV-OC43 and BCoV in using 9-O-Ac-sia as a 330

binding determinant, 9-O-Ac-sia alone at least is not sufficient to mediate entry for 331

hCoV-HKU1. 332

333

HKU1-HE is an O-acetylesterase and possesses RDE activity 334

By sequence similarity analysis, the HE protein of HKU1 was predicted to have a 335

hemagglutinin domain and a putative sia-O-acetyl-esterase active site (15). However there is 336

only 50-57% amino acid conservation between the HKU1-HE and those of other group 2a 337

CoVs. Currently no function for HKU1-HE during infection has been demonstrated. 338

Sia-O-acetylesterases and NAs are two types of viral RDEs identified so far. 339

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Sia-O-acetylesterases, e.g. 9-O-acetylesterase, which was originally found in influenza C 340

viruses and also represented by the HE proteins of CoVs: hCoV-OC43, BCoV, and porcine 341

Toroviruses (23); NAs are present in influenza A and B viruses. NA removes both free and 342

modified terminal sialic acids from a sugar chain, whereas sia-O-acetylesterases removes the 343

O-acetyl modifications from sailic acids. To investigate whether HKU1-HE has similar 344

functions as other CoVs, acting as lectin and/or RDE, we first expressed the extracellular 345

domain of HKU1 HE protein (aa14-358) and fused it to the Fc domain from murine IgG2a 346

(HKU1-HE-mFc) (Fig. 3A). When HKU1-HE-mFc protein was incubated with RD cells, no 347

direct binding was detected (Fig. 3B). This result suggests that HKU1-HE is unlikely to 348

mediate viral attachment. To determine if the HKU1-HE protein has RDE activity, RD cells 349

were pretreated with HKU1-HE-mFc protein, and then incubated with HKU1-S1(600)-mFc, 350

followed by FACS to determine how much HKU1-S1(600) could still bind to the RD cells. 351

Remarkably, pretreatment of RD cells with HKU1-HE dramatically reduced the binding of S1 352

to RD cells in a dose-dependent manner (Fig. 3C), similar to the reduction seen followed NA 353

pretreatment. This indicates that the HKU1-HE serves as a RDE for hCoV-HKU1, possessing 354

enzymatic activities capable of cleaving off the binding determinants for the S protein of HKU1 355

from the surface of the host cell. We next expressed OC43-HE, BCoV-HE and MHV-S-HE 356

proteins (Fig. 3A) and compared their RDE activities on RD cells to HKU1-HE’s activity in 357

eliminating HKU1-S1 or OC43-S1 binding to the cells. Similar to HKU1-HE, these HE proteins 358

showed no direct binding to RD cells by FACS analysis (Data not shown). Interestingly, 359

HKU1-HE pretreatment also reduced OC43-S1 binding to RD cells; and vice versa 360

pretreatment of cells with OC43-HE not only blocked OC43-S1 binding but also the binding of 361

HKU1-S1 (Fig. 3C); BCoV-HE had the same activities as OC43 in acting as a RDE for both 362

HKU1-S1 and OC43-S1 on RD cells; whereas pretreatment of RD cells with MHV-S-HE, 363

which is a 4-O-acetylesterase, had no effects on the binding of both HKU1-S1 and OC43-S1 364

(Fig. 3C). Furthermore the HA activity of OC43-S1 on rat and mouse RBCs were not only 365

inhibited by OC43- and BCoV-HE but also HKU1-HE, whereas MHV-S-HE had no effect (Fig. 366

3D). These results suggest that HKU1-HE has similar 9-O-acetylesterase activity as 367

OC43-HE and BCoV-HE, but different from MHV-S-HE. 368

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To further confirm that HKU1-HE indeed acts as a RDE solely by its sia-O-acetylesterase 369

activity, we first determined if HKU1-HE had any neuraminidase activity using an Amplex red 370

neuraminidase assay kit. As expected HKU1-HE had no detectable NA activity (data not 371

shown). On the other hand, HKU1-HE protein showed strong acetylesterase activity as 372

measured by using chromogenic p-nitrophenyl acetate (pNPA) as substrate (Fig. 3E). 373

HKU1-HE hydrolyzed pNPA, released para-nitrophenol (pNP) product with a Km=0.28±0.1 374

mM, Vmax=0.77±0.03 in the presence of 2 μg/mL of HKU1-HE protein. Similarly, OC43-HE, 375

BCoV-HE and MHV-S-HE were measured for acetylesterase activity, they all showed stronger 376

acetylesterase activity than HKU1-HE (Fig. 3E) with parameters Km=1.20 ± 0.06mM, 377

Vmax=3.32±0.00 for OC43-HE, Km=1.50±0.09mM, Vmax=3.42±0.04 for BCoV-HE and 378

Km=1.25 ± 0.01mM, Vmax=3.34 ± 0.04 for MHV-S-HE, respectively. Considering that 379

HKU1-HE treatment has the same effect as NA treatment of RD cells on the HKU1-S1 binding 380

and it has no NA activity, it is conceivable that the HKU1-HE mediates RDE through its 381

sialate-9-O-acetylesterase activity similar to BCoV- and OC43-HE. 382

Sequence alignment of HKU1-HE protein against HEF of influenza C, HEs of hCoV-OC43, 383

BCoV, MHV and Toroviruses revealed that the conserved sialate-O-acetylesterase catalytic 384

active sites (S40, H329 and D326 catalytic triad (24)) are also present in HE protein of HKU1 385

(Fig. 4A). BCoV-HE mutant protein containing S40A substitution has been demonstrated to 386

be enzymatically inactive (24). To determine if the three amino acids were critical for the 387

acetylesterase activity of HKU1-HE, HE mutants containing single S40A or triple 388

S40A/H329A/D326A mutations were expressed and tested for their capacity to hydrolyze 389

pNPA substrate. As shown in Fig. 4B, both HE mutants completely lost esterase activity. 390

Furthermore, unlike the wild type HE protein, pretreatment of RD cells with these 391

acetylesterase inactive mutant HEs had no effect on HKU1-S1 protein binding to RD cells (Fig. 392

4C). This result suggests that HKU1-S1 binding required specific type(s) of O-acetylated sialic 393

acids to be present on the cell surface, which corresponds to the sialic acid acetylesterase 394

specificity of the HKU1-HE. Considering that HKU1-S1 did not bind to 9-O-Ac containing BSM 395

and RBCs, combined with the result that HKU1-HE and OC43-HE mutually served as RDE for 396

their S1 proteins binding to RD cells, it is very likely that the acetyl modification at 9-O position 397

15 / 25

of sialic acid is a necessary but not sufficient binding determinant for HKU1-S protein. 398

Accordingly, HKU1-HE protein possesses sialate-9-O-acetylesterase activity or even broader 399

sialate-O-acetylesterase activity. 400

401

HKU1-HE inhibited hCoV-HKU1 infection in human tracheobronchial epithelial cultures 402

(HAE) via its RDE activity. 403

Though HKU1-S protein can bind to RD cells, the lentivirus-based HKU1 spike protein 404

pseudovirus was not able to enter RD cells (data not shown), and previous attempts to culture 405

clinical isolates of HKU1 in RD cells also failed (20). To date no cell line has been found to be 406

permissive for hCoV-HKU1 infection. Only the HAE primary cell culture system utilizing 407

well-differentiated human bronchial epithelial cells has been demonstrated to be a robust in 408

vitro model for hCoV-HKU1 infection and propagation (20). HAE cultures have also been 409

successfully used as a model system for studying SARS-CoV, hCoV-NL63, hCoV-229E, 410

MERS-CoV and hCoV-OC43 (22, 37-39). The infection and propagation of hCoV-HKU1 in the 411

HAE cultures models natural infection of the human upper respiratory tract. To determine 412

whether sialic acids are important during hCoV-HKU1 infection and whether HKU1-HE protein 413

possesses RDE activity in a natural infection model, HAE were incubated with NA, HKU1-HE 414

or control proteins for 1 hr and removed by washing prior to hCoV-HKU1 virus inoculation. 415

Apical washes of infected cultures were collected over time until 96 hrs post inoculation for 416

RNA isolation and the number of viral genomic RNA copies was analyzed by real-time 417

RT-PCR to determine the level of viral infection. As shown in Fig. 4D, both NA and HKU1-HE 418

pretreatment of HAE markedly inhibited hCoV-HKU1 infection. HKU1-HE at 100 μg/mL 419

reduced viral titers by 2-3 logs at 48, 72 and 96 hrs post viral inoculation. NA also showed 420

dose-dependent inhibition of viral infection though not as efficient as HE protein. 421

Pre-incubation of HAE with HKU1-HE protein inhibited viral replication suggesting that 422

HKU1-HE can destroy sialic acid moieties required for hCoV-HKU1 entry. When a lower titer 423

of HKU1 virus inocula was used, HKU1-HE protein at 100 μg/mL completely blocked 424

hCoV-HKU1 replication in HAE, whereas an enzymatic inactive HE variant HKU1-HE-S40A 425

showed no inhibition activity (Fig. 4E). These results demonstrate that hCoV-HKU1 uses 426

16 / 25

O-acytylated sialic acids as an attachment factor and that this interaction is required for 427

efficient infection of the target cell; HKU1-HE protein possesses sialate-O-acetylesterase 428

RDE activity. 429

430

Discussion 431

Sialic acid, a 9-carbon monosaccharide, includes a large number of derivatives arising 432

from differential modifications of the parental molecule as well as various glycosidic linkage 433

(e.g., α2,3 or α2,6) to the subterminal residue of a sugar chain. O-acetylation is one of the 434

most common types of sialic acid modification. It can occur at all the four hydroxyl groups of 435

sialic acids at positions of C4, C7, C8 and C9, generates mostly mono-O-acetylated but also 436

oligo-O-acetylated sialic acids at more than one position. O-AC-Sia plays fundamental roles in 437

many biological and pathophysiological events (40). The 9-O-Ac-Sia serves as a receptor 438

determinant for several members of Group 2a CoVs, including the closely related BCoV, 439

hCoV-OC43, and porcine hemagglutinating encephalomyelitis virus (PHEV) (10, 30, 41). The 440

binding with 9-O-Ac-Sia is essential for these viruses to initiate infection and their S protein is 441

the major viral protein responsible for the binding. In this study, we found that the S protein of 442

hCoV-HKU1 can also recognize O-Ac-Sia but only those presented on RD cells among the 443

many cell lines tested. Different from the aforementioned CoVs in the same group, no binding 444

of hCoV-HKU1 S1 with 9-O-Ac-Sia containing BSM as well as RBCs from mouse and rat 445

could be detected. In addition, a previous study (42) and our data (Fig. 1E) both demonstrated 446

that the NT of HKU1-S1 was unable to bind with carbohydrate moieties, in contrast the 447

carbohydrate receptor binding domains for hCoV-OC43 and BCoV were located in the NT of 448

S1 (Fig. 2) (42). On the other hand, for both hCoV-HKU1 and other 9-O-Ac-Sia-recognition 449

dependent CoVs, the question remains as to whether in addition to O-Ac-Sia, they also 450

interact with a protein receptor during the entry process. O-Ac-Sia on RD cells can be 451

recognized by hCoV-HKU1 S1 protein, however the cells are not permissive for viral infection. 452

One explanation for this is the lack of a protein receptor for hCoV-HKU1 on RD cells. In line 453

with this, our attempts using HKU1-S1(600) as a viral ligand protein for immunoprecipitation 454

and combining with mass spectrometric identification did not find a protein(s) specific binding 455

17 / 25

to HKU1-S1 (data not shown). It is also possible that there are other molecule(s) present only 456

in HAE cultures but not on RD cells, which are important for viral infection at a later stage, e.g. 457

membrane fusion or viral replication; or a restriction factor(s) that may exist in RD cells to limit 458

viral infection. 459

460

Among CoVs, HE protein is only present in members of group 2a CoVs. Sequence and 461

structural similarity suggest that CoV HE evolved from the HEF protein of influenza C virus 462

(24). Although the dual function of HEF (O-Ac-Sia receptor binding and 463

sialate-O-acetylesterase activity) was maintained in some CoVs, the HE appears to mainly 464

function as a RDE in these CoVs. Comparing to the essential role of S protein in sia-receptor 465

binding and mediating viral entry, the sia-binding activity of HE seems to be an accessory 466

function and varies in its affinity and sia-preference among CoV strains (23, 43). HEs of two 467

closely related MHV field strains, MHV-DVIM and MHV-S, recognize two different types of 468

O-Ac-Sia, 9-O-Ac-Sia and 4-O-Ac-Sia, respectively, whereas many MHV laboratory strains 469

carry defective HE genes (27, 28). HCoV-OC43 HE lost its sia-binding activity although it has 470

high sequence identity (97%) with the HE of BCoV (Mebus stain) which exhibits high 471

sia-binding affinity (43). Similarly, we did not find that HKU1-HE has O-Ac-Sia binding 472

activity. This is consistent with the observation described previously by Langereis et al. that 473

the HE of hCoV-HKU1 failed to hemagglutinate erythrocytes and bind to O-Ac-Sia (43). 474

Sequence comparison of HKU1-HE with influenza C’s HEF and HEs of CoVs with known 475

protein structures demonstrated that HKU1-HE was the most divergent one at the Sia-binding 476

loops (23, 43), whereas the Sia-O-acetylesterase domain is highly conserved among them. 477

The key residues contributing to the catalytic activity of HEF and other CoV HEs are 478

completely conserved in hCoV-HKU1, including the Ser-His-Asp catalytic triad, the oxyanion 479

hole contributing residues Gly85 and Asn117 in HEF, and an Arg322 residue in HEF important for 480

Sia substrate binding (23). In our study, HKU1-HE indeed showed strong O-AC-esterase 481

activity with pNPA substrate, and the Ser-His-Asp catalytic triad mutant HEs completely lost 482

this activity. We demonstrated that the sialic acids expressed by RD cells are not only 483

specifically recognized by the HKU1-S1 protein but also are substrates for HE protein. 484

18 / 25

Wild-type but not mutant HE treatment of RD cells showed similar effect as neuraminidase 485

and abolished the subsequent binding of S1 to RD cells. These results indicate that the S1 486

protein binds with O-Ac-Sia and the HE has the matching Sia-O-Ac or even broader esterase 487

activity. 488

489

Langereis et al (43) reported that the HKU1-HE like the BCoV-HE displayed 490

Sia-9-O-Ac-esterase activity using a synthetic 4,9-di-O-Ac-Sia substrate analogue. 491

Consistently, we demonstrated that HKU1-HE had similar RDE activity as OC43-HE and 492

BCoV-HE to remove the binding moiety from RD cells for S1 proteins of both HKU1 and OC43. 493

These results strongly support that HKU1-HE has similar Sia-9-O-Ac-esterase activity as 494

OC43-HE and BCoV-HE. However, unlike the S1 of OC43, Sia-9-O-AC binding activity was 495

not detected for the S1 of hCoV-HKU1 by BSM binding and erythrocytes hemagglutination 496

assays which are standard methods for examining the usage of Sia-9-O-AC as a receptor by 497

other CoVs (Fig. 2C-E). Thus it is likely that HKU1 is different than OC43 and BCoV in terms 498

of using Sia-9-O-AC as a receptor via its Spike. Sia-9-O-AC may be required but not sufficient 499

to support the HKU1-S1 binding. In addition of Sia-9-O-AC, RD cells may express other type 500

of O-Acetylated sialic acid that is lacking or at lower level in BSM and erythrocyte cell surface 501

but required for HKU1-S1 recognition. One may also speculate that certain di-O-Ac-Sia, 502

tri-O-Ac-Sia or oligo-O-acetylated Sia in which all have an acetyl group at C9 (9-O-AC) in 503

common; or certain particular sugar chain core structure(s) to which Sia-9-O-Ac attached or 504

the linkage of sialic acid to the penultimate residue of a sugar chain may also be required. 505

506

Finally, in HAE cultures, we demonstrated that NA and HKU1-HE but not enzymatic 507

inactive HE mutant dramatically reduced virus infection or completely blocked infection when 508

lower viral challenge dose was applied. Treatment of HAEs with NA and HKU1-HE prior to 509

infection strongly suggests that the sia-9-O-AC-esterase activity of HE acted as a RDE and 510

removed the critical receptor binding moieties so that the early viral entry was impaired. 511

Considering the HAE cells continuously secret large amount of mucus, the effect of inhibition 512

of viral infection by pretreatment of the cells with NA and HE is remarkable and suggests an 513

19 / 25

essential role for sialic acids to initiate infection. Our study also suggests that acetyl 514

modification at 9-O of sialic acid may be a necessary but not sufficient receptor or attachment 515

factor determinant, and warrants further investigation to determine the fine specificity and 516

preference of sialic acids recognized by HKU1-S protein. Nevertheless, for the first time this 517

study provided experimental evidence to support O-Ac-Sia, by interacting with S1 of 518

hCoV-HKU1, serves as an essential determinant for viral attachment during the early entry 519

step, and HE possesses 9-O-AC-esterase or even broader activity and primarily acts as a 520

RDE for hCoV-HKU1 infection. HCoV-HKU1 is similar to BCoV and hCoV-OC43 employing its 521

two surface proteins S and HE to complete the viral infection cycle in a concerted manner with 522

S protein mediating receptor-binding and entry, HE protein mediating RDE activity late in the 523

infection cycle to facilitate viral progeny release and achieve efficient virus dissemination (23, 524

26). 525

526

Figure Legends 527

528

Fig. 1. Specific binding of HKU1-S1 to RD cells. (A) SDS-PAGE of expressed 529

recombinant S1 or control proteins. All proteins were expressed in 293T cells and purified by 530

protein A sepharose beads. Purified proteins were run on SDS-PAGE and stained by 531

coomassie blue. (B) FACS analysis of HKU1-S1(600)-mFc (5 μg/mL) binding to RD and Hela 532

cells. HKU3-323-mFc was used a negative control protein. (C) HKU1-S1(600)-mFc 533

binding to a molecule(s) located on RD cell surface. Immunofluorescence microscopy imaging 534

of RD cells stained by HKU1-S1(600)-mFc or control protein, HKU3-323-mFc. Cells 535

membrane was stained with FM-4-64 in red, Hoechst dye 33258 stained the nuclei (blue), and 536

the HKU1-S1 staining was detected by a FITC-labeled anti-mouse Fc antibody. (D) 537

HKU1-S1(600)-mFc binding to RD cells in a dose dependent manner in a FACS analysis. (E) 538

FACS analysis of the N-terminals of HKU1-S1 and OC43-S1 binding to RD cells. 539

HKU3-323-mFc at 10 μg/mL was used as a control (labeled as Ctrl.) in panels D and E. 540

Graphs shown in panel B-E are reprehensive of at least two independent experiments for 541

each panel. 542

20 / 25

543

Fig. 2. Characterization of the binding receptor of HKU1-S1 protein on RD cells by 544

FACS. (A) Pretreatment of RD cells with Neuramidase (NA) greatly reduced HKU1-S1 545

binding in a dose-dependent manner. RD cells were pretreated with NA at different 546

concentrations as indicated and then stained with 5 μg/mL of HKU1-S1(600)-mFc, 547

OC43(268)-mFc or SIRPα-mFc control protein, respectively, then followed by detecting with a 548

FITC-labeled anti-mouse Fc antibody. (B) Pretreatment of RD cells with TPCK-treated Trypsin 549

reduced HKU1-S1(600) and OC43-S1(268) binding to RD cells. The FACS assay method was 550

similar as described in panel A. Ctrl. indicates RD cells were stained with HKU1-S1(268)-mFc 551

only in panel A and B. (C) HKU1-S1(600)-mFc did not bind to BSM (containing 9-O-Ac-sia) 552

directly coated on ELISA plate. OC43-S1 served as a positive control. (D) HKU-S1 did not 553

hemagglutinate mouse or rat RBCs. OC43-S1(268)-mFc served as a positive control and 554

showed HA activity dose-dependently. (E) Binding of HKU1-S1 and OC43-S1 to rat and 555

mouse RBCs by FACS analysis. Data shown are reprehensive of at least two independent 556

experiments for each panel. 557

558

Fig. 3. HKU1-HE is an O-acetylesterase and RDE for HKU1-S1 binding to RD cells. (A) 559

SDS-PAGE of expressed recombinant HE proteins. All proteins were expressed in 293T cells 560

and purified by protein A sepharose beads. Purified proteins were run on SDS-PAGE and 561

stained by coomassie blue. (B) HE-mFc protein itself did not bind to RD cells. (C) HKU1-HE 562

acted as a RDE on RD cells for HKU1- and OC43-S1 protein. Pretreatment of RD cells with 563

HKU1-HE as well as OC43- and BCoV-HE greatly reduced HKU1-S1 or OC43-S1 (at 5 μg/mL) 564

binding to RD cells in a dose-dependent manner. MHV-S-HE showed no effect. Graphs 565

shown in panel B-C are reprehensive of at least two independent experiments. (D) HKU1-HE 566

acted as a RDE on RBCs for OC43-S1 protein. Pretreatment of rat or mouse RBCs with 567

HKU1-, OC43-, BCoV-HE inhibited OC43-S1 mediated hemagglutination activity, whereas 568

MHV-S-HE had no effect. Representative data or images of at least two independent 569

experiments were shown for panels A-D. (E) HKU1-HE is an acetylesterase. HEs of HKU1, 570

OC43, BCoV and MHV-S at 2 μg/mL were used to hydrolyze pNPA (2-fold serially diluted) at 571

21 / 25

room temperature for 15 mins. Enzyme activity was assessed by measuring optical density at 572

405 nm (OD405). The Km and Vmax values were calculated from the Michaelis-Menten 573

Enzyme Kinetics curve fitting of the two independent repeats. 574

575

Fig. 4. Treatment of HAE with HE or NA but not the enzymatic inactive HE mutant 576

inhibited HKU1 infection. (A) Esterase catalytic active site residues in HE. Sequence 577

alignment of amino acids around the catalytic active site (in red) was shown. BCoV-HE amino 578

acid numbering scheme was used (22). GenBank accession numbers of HE proteins of BCoV, 579

OC43, HKU1, MHV-DVIM and influenza C virus are AAA92991.1, AAX85668.1, NC_006577.2, 580

AAC63044.1 and AJ872181, respectively. For HE proteins of MHV-S, bovine Torovirus (BToV), 581

and porcine Torovirus (PToV) stain p10, the Uniprot accession numbers are P31614, P0C0V9, 582

and Q70KP1, respectively. (B) HKU1-HE proteins with substitutions at catalytic active sites 583

are enzymatic inactive. HKU1-HE or the mutants at 1 μg/mL were incubated with 2-fold 584

serially diluted pNPA at room temperature for 15 mins prior to measuring OD405. Each data 585

points represent OD405 of HE or its mutants subtracted with that of a negative control. (C) 586

Esterase inactive mutants of HE did not block HKU1-S1 protein binding to RD cells. RD cells 587

were treated with 10 μg/mL of HE or HE mutant proteins prior to HKU1-S1 staining (5 μg/mL). 588

(D) HE and NA pretreatment of HAE inhibited HKU1 infection. Prior to HKU1 virus inoculation, 589

HAE cells were pretreated with HE proteins or NA at different concentrations as indicated for 1 590

hr, followed by HKU1 virus inoculation at high dose as indicated. Replication kinetics of HKU1 591

virus was assessed in apical washes from infected HAE cultures by real-time RT-PCR. Virus 592

yield is presented as number of virus RNA copies/mL. (E) HE pretreatment blocked HKU1 593

infection of HAE cells. HAE were pretreated with HE protein or enzymatic inactive HE mutant 594

(S40A) for 1 hour prior to viral challenge at lower dose than used panel D. Replication kinetics 595

of HKU1 virus was assessed as in panel D. Dotted line indicates the detection limit of the 596

assay for panel D-E. 597

598

Acknowledgement: 599

This work was supported by the Ministry of Science and Technology, China to JS 600

22 / 25

(2012CB837602), to WL (2010CB530101) and the Science and Technology Bureau of Beijing 601

Municipal Government to WL and JS, and by the Thousand Young Talents Program, China to 602

JS, the National Centre for Research and Development, Poland (Lider/27/55/L-2/10/2011) 603

and the National Science Center, Poland (UMO-2012/07/E/NZ6/01712) to KP, and NIH (U19 604

AI109761) to RSB and NIH (AI085524) to WAM and RSB. 605

606

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28. Langereis MA, Zeng Q, Heesters BA, Huizinga EG, de Groot RJ. 2012. The murine coronavirus 688 hemagglutinin-esterase receptor-binding site: a major shift in ligand specificity through modest 689 changes in architecture. PLoS pathogens 8:e1002492. 690

29. Lau SK, Woo PC, Li KS, Huang Y, Tsoi HW, Wong BH, Wong SS, Leung SY, Chan KH, Yuen KY. 2005. 691 Severe acute respiratory syndrome coronavirus-like virus in Chinese horseshoe bats. Proceedings of the 692 National Academy of Sciences of the United States of America 102:14040-14045. 693

30. Schwegmann-Wessels C, Herrler G. 2006. Sialic acids as receptor determinants for coronaviruses. 694 Glycoconjugate journal 23:51-58. 695

31. Stroh LJ, Stehle T. 2014. Glycan Engagement by Viruses:Receptor Switches and Specificity. Annu. Rev. 696 Virol. 1:285-306. 697

32. Barclay AN, Van den Berg TK. 2014. The interaction between signal regulatory protein alpha 698 (SIRPalpha) and CD47: structure, function, and therapeutic target. Annual review of immunology 699 32:25-50. 700

33. Ravindranath MH, Higa HH, Cooper EL, Paulson JC. 1985. Purification and characterization of an 701 O-acetylsialic acid-specific lectin from a marine crab Cancer antennarius. The Journal of biological 702 chemistry 260:8850-8856. 703

34. Traving C, Schauer R. 1998. Structure, function and metabolism of sialic acids. Cellular and molecular 704 life sciences : CMLS 54:1330-1349. 705

35. Rogers GN, Herrler G, Paulson JC, Klenk HD. 1986. Influenza C virus uses 706 9-O-acetyl-N-acetylneuraminic acid as a high affinity receptor determinant for attachment to cells. The 707 Journal of biological chemistry 261:5947-5951. 708

36. Bulai T, Bratosin D, Pons A, Montreuil J, Zanetta JP. 2003. Diversity of the human erythrocyte 709 membrane sialic acids in relation with blood groups. FEBS letters 534:185-189. 710

37. B SB, Orenstein JM, Fox LM, Randell SH, Rowley AH, Baker SC. 2009. Human airway epithelial cell 711 culture to identify new respiratory viruses: coronavirus NL63 as a model. Journal of virological methods 712 156:19-26. 713

38. Mossel EC, Wang J, Jeffers S, Edeen KE, Wang S, Cosgrove GP, Funk CJ, Manzer R, Miura TA, Pearson 714 LD, Holmes KV, Mason RJ. 2008. SARS-CoV replicates in primary human alveolar type II cell cultures 715 but not in type I-like cells. Virology 372:127-135. 716

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43. Langereis MA. 2011. Viral hemagglutinin-esterases; mediators of dynamic virion-glycan interactions. 730 Ph.D. thesis. University of Utrecht, Utrecht. http://dspace.library.uu.nl/handle/1874/211181. 731

HK

U1-

S1(6

00)-m

Fc

Ctr

l.HK

U3-

S1-m

FcKDa130

95725543

34

Mar

ker

OC

43-S

1(26

8)-m

Fc

A B

Figure 1

D

HKU1-S1/FITC FM-4-64

Hoechst Merge

HKU3-S1/FITC FM-4-64

Hoechst Merge

C

Cou

nts

FITC

Cou

nts

1.25 µg/mLCtrl. 2.5 µg/mL 5 µg/mL 10 µg/mL0.61 µg/mL

HKU1-S1(268)-mFcHKU1-S1(600)-mFc

RD cell HeLa cell

Ctrl. HKU3-S1(323)-mFcCell only HKU1-S1(600)-mFc

Cou

nts

FITC FITC

FITC

HKU1-S1(600)-mFc Ctrl. HKU3-S1(323)-mFc

EOC43-S1(268)-mFc

HK

U1-

S1(2

68)-m

Fc

Ctr

l.SI

RPαα

-mFc

26

17

FITC

Fig. 1. Specific binding of HKU1-S1 to RD cells. (A) SDS-PAGE of expressed recombinant S1 or control proteins. All proteins were expressed in293T cells and purified by protein A sepharose beads. Purified proteins were run on SDS-PAGE and stained by coomassie blue. (B) FACS analysis ofHKU1-S1(600)-mFc (5 µg/mL) binding to RD and Hela cells. HKU3-323-mFc was used a negative control protein. (C) HKU1-S1(600)-mFc binding toa molecule(s) located on RD cell surface. Immunofluorescence microscopy imaging of RD cells stained by HKU1-S1(600)-mFc or control protein,HKU3-323-mFc. Cells membrane was stained with FM-4-64 in red, Hoechst dye 33258 stained the nuclei (blue), and the HKU1-S1 staining was detect-ed by a FITC-labeled anti-mouse Fc antibody. (D) HKU1-S1(600)-mFc binding to RD cells in a dose dependent manner in a FACS analysis. (E) FACSanalysis of the N-terminals of HKU1-S1 and OC43-S1 binding to RD cells. HKU3-323-mFc at 10 µg/mL was used as a control (labeled as Ctrl.) in pan-els D and E. Graphs shown in panel B-E are reprehensive of at least two independent experiments for each panel.

Figure 2

NA treatment/HKU1-S1 binding

FITC

Cou

nts

FITC

20

500

Ctrl.

100

[NA

]mU

/mL

NA treatment/SIRPα binding

NA treatment/OC43-S1 binding

FITC

SIRPα only

A

B

D

CTrypsin treatment /HKU1-S1 binding

5.020Ctrl.

1.25 [Try

psin

]µg/

mL

HKU1-S1 only

Cou

nts

Trypsin treatment /OC43-S1 binding

5.020Ctrl.

1.25 [Try

psin

]µg/

mL

OC43-S1 only

FITCFITC

20

40

HK

U1-

S1

OC

43-S

1

rRBCs

10

5

2.5

1.25

0.61

0[S1]µg/mL

mRBCs

HK

U1-

S1

OC

43-S

1

HKU1-S1 only OC43-S1 only

0.31

0.62

1.25 2.5 5 10 20

0.0

0.1

0.2

0.3

0.4

0.5

OC43-S1HKU1-S1

[S1] µg/mL

OD

450

E

0

52.51.250.6250.3130.1560.078

[S1] µg/mL

rRBCsmRBCs

HKU1-S1 HKU1-S1

OC43-S1 OC43-S1

Cou

nts

FITCFITC

Cou

nts

Fig. 2. Characterization of the binding receptor of HKU1-S1 protein on RD cells by FACS. (A) Pretreatment of RD cells withNeuramidase (NA) greatly reduced HKU1-S1 binding in a dose-dependent manner. RD cells were pretreated with NA at different con-centrations as indicated and then stained with 5 µg/mL of HKU1-S1(600)-mFc, OC43(268)-mFc or SIRPα-mFc control protein, respec-tively, then followed by detecting with a FITC-labeled anti-mouse Fc antibody. (B) Pretreatment of RD cells with TPCK-treated Trypsinreduced HKU1-S1(600) and OC43-S1(268) binding to RD cells. The FACS assay method was similar as described in panel A. Ctrl. indi-cates RD cells were stained with HKU1-S1(268)-mFc only in panel A and B. (C) HKU1-S1(600)-mFc did not bind to BSM (containing9-O-Ac-sia) directly coated on ELISA plate. OC43-S1 served as a positive control. (D) HKU-S1 did not hemagglutinate mouse or ratRBCs. OC43-S1(268)-mFc served as a positive control and showed HA activity dose-dependently. (E) Binding of HKU1-S1 and OC43-S1 to rat and mouse RBCs by FACS analysis. Data shown are reprehensive of at least two independent experiments for each panel.

Figure 3

Cou

nts

FITC

HKU1-HE binding

Ctrl.HKU1-HE only

HK

U1-

HE

Cou

nts

HKU1-S1 binding OC43-S1 binding

OC

43-H

EC

ount

sC

ount

sM

HV-

S-H

E

FITC FITC

B

C E

HK

U1-

HE

0.0

0.2

0.4

0.6

OD

405

0.8

Km=0.28 ± 0.10 mMVmax=0.77 ± 0.03

OD

405

0.0

0.5

1.0

1.5

2.0

2.5

3.0

BC

oV-H

E

Km=1.50 ± 0.09 mMVmax=3.42 ± 0.04

0 1 2 3 4 50.0

0.5

1.0

1.5

2.0

2.5

3.0

[pNPA]mM

OD

405

Repeat 1Repeat 2

0.0

0.5

1.0

1.5

2.0

2.5

3.0

OD

405

OC

43-H

E

Km=1.20 ± 0.06 mMVmax=3.32 ± 0.00

AC

ount

sB

CoV

-HE

MH

V-S

-HE

13095725543

34

MH

V-S-

HE-

mFc

OC

43-H

E-m

FcKDa M

arke

r

BC

oV-H

E-m

Fc

HK

U1-

HE-

mFc

26

17

HKU1 OC43 BCoV MHV-S

HE proteins

RBCs r m r m r m r m

OC

43-S

1,10

µµg/m

L

D

20

10

5

2.5

1.25

0

[HE]

µµg/m

L

0.61

0.31

Km=1.25 ± 0.01 mMVmax=3.34 ± 0.04

0.61

10Ctrl.

2.5

[HE]

µµg/m

L

HKU1-S1 only0.61

10Ctrl.

2.5[H

E]µµg

/mL

OC43-S1 only

Fi g . 3 . H KU1 -H E i s an O -ace ty l e s t eras e an d RDE f or H KU1-S 1 b i n d i n g to RD ce l l s . (A) S DS -PAGE of ex p res s edrecom bi nan t HE pro t e i n s . A l l p ro t e i n s were ex p res s ed i n 293T ce l l s and pu r i f i ed by p ro t e i n A s epha ros e beads . P u r i -f i ed p ro t e i n s were run on S DS -PAGE an d s t a i ned by coom as s i e b l ue . (B ) HE-m Fc pro t e i n i t s e l f d i d no t b i nd t o R Dc e l l s . ( C ) H K U 1 - H E a c t e d a s a R D E o n R D c e l l s f o r H K U 1 - a n d O C 4 3 - S 1 p ro t e i n . P re t re a t m e n t o f R D c e l l s w i t hHKU1-HE as we l l a s OC 4 3- and BC oV-HE grea t l y reduced HKU1-S 1 or OC 43-S 1 (a t 5 µg/ m L) b i nd i n g t o R D ce l l s i n ad o s e - d e p e n d e n t m a n n e r. M H V- S - H E s h o w e d n o e f f e c t . G r a p h s s h o w n i n p a n e l B- C a re re p re h e n s i v e o f a t l e a s t t w oi n d ep en den t ex pe r i m en t s . (D) HKU1 -HE ac t ed as a R DE on R BC s fo r OC 43-S 1 p ro t e i n . P re t rea t m en t o f r a t o r m ous eR BC s wi t h HKU1 - , OC 43 - , BC oV-HE i n h i b i t ed OC 43-S 1 m ed i a t ed hem aggl u t i na t i on ac t i v i t y, whereas M HV-S -HE hadn o e f f e c t . R e p re s e n t a t i v e d a t a o r i m a ge s o f a t l e a s t t w o i n d e p e n d e n t e x p e r i m e n t s w e re s h o w n f o r p a n e l s A - D . ( E )HKU1-HE i s an ace t yl e s t e r a s e . HEs o f HKU1, OC 43 , BC o V and M HV-S a t 2 µg/ m L were us ed t o hyd ro l yz e pNPA (2 -fo l d s e r i a l l y d i l u t ed ) a t room t em p era t u re fo r 15 m i ns . En z ym e ac t i v i t y was as s es s ed b y m eas u r i ng op t i ca l dens i t y a t4 05 nm (OD40 5) . The Km and Vm ax va l ues were ca l cu l a t ed f rom t he M i chae l i s -M en t en Enz ym e Ki ne t i c s cu rve f i t t i ngo f t h e t wo i n dep en d en t repea t s .

Figure 4

A

B

0 2 24 48 72 961.0××102

1.0××104

1.0××1061.0××1006

1.0××1007

1.0××1008

1.0××1009

1.0××1010

1.0××1011

1.0××1012

1.0××1013Virus onlyHKU1 S268-mFc 100 µg/mLNA 50 mU/mLNA 500 mU/mLHE-mFc 10 µg/mLHE-mFc 100 µg/mL

Hours post infection

Viru

syi

eld

[cop

ies/

mL]

0 2 24 48 72 961.0××1002

1.0××1003

1.0××1004

1.0××1005

1.0××1006

1.0××1007

1.0××1008

1.0××1009Virus only

HE-mFc 10 µg/mL

HE-mFc 100 µg/mL

HKU1 S268-mFc 100 µg/mL

NA 500mU/ml

HE-S40A-mFc 100 µg/ml

Hours post infection

Viru

syi

eld

[cop

ies/

mL]

5 10 15 20-0.2

0.0

0.2

0.4

0.6

0.8

1.0

1.2

HEHE-S40AHE-S40A/H329A/D326AHKU1-S1

[pNPA] mM

OD

405

Cou

nts

HE and mutants treatment /S1 binding

FITC

HE-S40A/H329A/D326A

HE wt

Ctrl.

HKU1-S1 only

HE-S40A

HE-S40A/H329A/D326A

HE wt

Ctrl.

HKU1-S1 only

HE-S40A

Influenza C ...FGDSR.....ADDHHGDBToV (BRV-1) ...FGDSR.....SDANHGDPToV (P10) ...FGDSR.....ADENHGDMHV-DVIM ...FGDSR.....YDSHHGDMHV-S ...FGDSR.....HDVHHGDBCoV ...FGDSR.....YDINHGDOC43 ...FGDSR.....YDINHGDHKU1 ...FGDSR.....YDINHGD

40

326

329

C

D

E

Fig. 4. Treatment of HAE with HE or NA but not the enzymatic inactive HE mutant inhibited HKU1 infection. (A)Esterase catalytic active site residues in HE. Sequence alignment of amino acids around the catalytic active site (in red) wasshown. BCoV-HE amino acid numbering scheme was used (22). GenBank accession numbers of HE proteins of BCoV,OC43, HKU1, MHV-DVIM and influenza C virus are AAA92991.1, AAX85668.1, NC_006577.2, AAC63044.1 andAJ872181, respectively. For HE proteins of MHV-S, bovine Torovirus (BToV), and porcine Torovirus (PToV) stain p10, theUniprot accession numbers are P31614, P0C0V9, and Q70KP1, respectively. (B) HKU1-HE proteins with substitutions atcatalytic active sites are enzymatic inactive. HKU1-HE or the mutants at 1 µg/mL were incubated with 2-fold serially dilut-ed pNPA at room temperature for 15 mins prior to measuring OD405. Each data points represent OD405 of HE or itsmutants subtracted with that of a negative control. (C) Esterase inactive mutants of HE did not block HKU1-S1 proteinbinding to RD cells. RD cells were treated with 10 µg/mL of HE or HE mutant proteins prior to HKU1-S1 staining (5µg/mL). (D) HE and NA pretreatment of HAE inhibited HKU1 infection. Prior to HKU1 virus inoculation, HAE cells werepretreated with HE proteins or NA at different concentrations as indicated for 1 hr, followed by HKU1 virus inoculation athigh dose as indicated. Replication kinetics of HKU1 virus was assessed in apical washes from infected HAE cultures byreal-time RT-PCR. Virus yield is presented as number of virus RNA copies/mL. (E) HE pretreatment blocked HKU1 infec-tion of HAE cells. HAE were pretreated with HE protein or enzymatic inactive HE mutant (S40A) for 1 hour prior to viralchallenge at lower dose than used panel D. Replication kinetics of HKU1 virus was assessed as in panel D. Dotted line indi-cates the detection limit of the assay for panel D-E.