Human Coronavirus HKU1 Spike Protein Uses O-Acetylated Sialic Acid as an Attachment Receptor Determinant and Employs Hemagglutinin-Esterase Protein as a Receptor-Destroying Enzyme

doi:10.1128/JVI.00854-15

. 2015 Jul;89(14):7202-13.

doi: 10.1128/JVI.00854-15. Epub 2015 Apr 29.

Human Coronavirus HKU1 Spike Protein Uses O-Acetylated Sialic Acid as an Attachment Receptor Determinant and Employs Hemagglutinin-Esterase Protein as a Receptor-Destroying Enzyme

Xingchuan Huang¹, Wenjuan Dong², Aleksandra Milewska³, Anna Golda³, Yonghe Qi⁴, Quan K Zhu⁵, Wayne A Marasco⁵, Ralph S Baric⁶, Amy C Sims⁷, Krzysztof Pyrc⁸, Wenhui Li⁹, Jianhua Sui⁹

Affiliations

¹ National Institute of Biological Sciences, Changping, Beijing, China.
² National Institute of Biological Sciences, Changping, Beijing, China China Agricultural University Graduate Program, National Institute of Biological Sciences, Beijing, China.
³ Microbiology Department, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland.
⁴ National Institute of Biological Sciences, Changping, Beijing, China Beijing Normal University Graduate Program, National Institute of Biological Sciences, Beijing, China.
⁵ Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, and Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA.
⁶ Department of Microbiology and Immunology, University of North Carolina, Chapel Hill, North Carolina, USA Department of Epidemiology, University of North Carolina, Chapel Hill, North Carolina, USA.
⁷ Department of Epidemiology, University of North Carolina, Chapel Hill, North Carolina, USA.
⁸ Microbiology Department, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland.
⁹ National Institute of Biological Sciences, Changping, Beijing, China liwenhui@nibs.ac.cn suijianhua@nibs.ac.cn.

PMID: 25926653
PMCID: PMC4473545
DOI: 10.1128/JVI.00854-15

Human Coronavirus HKU1 Spike Protein Uses O-Acetylated Sialic Acid as an Attachment Receptor Determinant and Employs Hemagglutinin-Esterase Protein as a Receptor-Destroying Enzyme

Xingchuan Huang et al. J Virol. 2015 Jul.

. 2015 Jul;89(14):7202-13.

doi: 10.1128/JVI.00854-15. Epub 2015 Apr 29.

Authors

Xingchuan Huang¹, Wenjuan Dong², Aleksandra Milewska³, Anna Golda³, Yonghe Qi⁴, Quan K Zhu⁵, Wayne A Marasco⁵, Ralph S Baric⁶, Amy C Sims⁷, Krzysztof Pyrc⁸, Wenhui Li⁹, Jianhua Sui⁹

Affiliations

¹ National Institute of Biological Sciences, Changping, Beijing, China.
² National Institute of Biological Sciences, Changping, Beijing, China China Agricultural University Graduate Program, National Institute of Biological Sciences, Beijing, China.
³ Microbiology Department, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland.
⁴ National Institute of Biological Sciences, Changping, Beijing, China Beijing Normal University Graduate Program, National Institute of Biological Sciences, Beijing, China.
⁵ Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, and Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA.
⁶ Department of Microbiology and Immunology, University of North Carolina, Chapel Hill, North Carolina, USA Department of Epidemiology, University of North Carolina, Chapel Hill, North Carolina, USA.
⁷ Department of Epidemiology, University of North Carolina, Chapel Hill, North Carolina, USA.
⁸ Microbiology Department, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland.
⁹ National Institute of Biological Sciences, Changping, Beijing, China liwenhui@nibs.ac.cn suijianhua@nibs.ac.cn.

PMID: 25926653
PMCID: PMC4473545
DOI: 10.1128/JVI.00854-15

Abstract

Human coronavirus (hCoV) HKU1 is one of six hCoVs identified to date and the only one with an unidentified cellular receptor. hCoV-HKU1 encodes a hemagglutinin-esterase (HE) protein that is unique to the group a betacoronaviruses (group 2a). The function of HKU1-HE remains largely undetermined. In this study, we examined binding of the S1 domain of hCoV-HKU1 spike to a panel of cells and found that the S1 could specifically bind on the cell surface of a human rhabdomyosarcoma cell line, RD. Pretreatment of RD cells with neuraminidase (NA) and trypsin greatly reduced the binding, suggesting that the binding was mediated by sialic acids on glycoproteins. However, unlike other group 2a CoVs, e.g., hCoV-OC43, for which 9-O-acetylated sialic acid (9-O-Ac-Sia) serves as a receptor determinant, HKU1-S1 bound with neither 9-O-Ac-Sia-containing glycoprotein(s) nor rat and mouse erythrocytes. Nonetheless, the HKU1-HE was similar to OC43-HE, also possessed sialate-O-acetylesterase activity, and acted as a receptor-destroying enzyme (RDE) capable of eliminating the binding of HKU1-S1 to RD cells, whereas the O-acetylesterase-inactive HKU1-HE mutant lost this capacity. Using primary human ciliated airway epithelial (HAE) cell cultures, the only in vitro replication model for hCoV-HKU1 infection, we confirmed that pretreatment of HAE cells with HE but not the enzymatically inactive mutant blocked hCoV-HKU1 infection. These results demonstrate that hCoV-HKU1 exploits O-Ac-Sia as a cellular attachment receptor determinant to initiate the infection of host cells and that its HE protein possesses the corresponding sialate-O-acetylesterase RDE activity.

Importance: Human coronaviruses (hCoV) are important human respiratory pathogens. Among the six hCoVs identified to date, only hCoV-HKU1 has no defined cellular receptor. It is also unclear whether hemagglutinin-esterase (HE) protein plays a role in viral entry. In this study, we found that, similarly to other members of the group 2a CoVs, sialic acid moieties on glycoproteins are critical receptor determinants for the hCoV-HKU1 infection. Interestingly, the virus seems to employ a type of sialic acid different from those employed by other group 2a CoVs. In addition, we determined that the HKU1-HE protein is an O-acetylesterase and acts as a receptor-destroying enzyme (RDE) for hCoV-HKU1. This is the first study to demonstrate that hCoV-HKU1 uses certain types of O-acetylated sialic acid residues on glycoproteins to initiate the infection of host cells and that the HKU1-HE protein possesses sialate-O-acetylesterase RDE activity.

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Figures

**FIG 1**
Specific binding of HKU1-S1 to RD cells. (A) SDS-PAGE of expressed recombinant S1 or control (Ctrl.) proteins. All proteins were expressed in 293T cells and purified by the use of protein A Sepharose beads. Purified proteins were run on SDS-PAGE and stained by the use of Coomassie blue. (B) FACS analysis of HKU1-S1(600)-mFc (5 μg/ml) binding to RD and HeLa cells. HKU3-323-mFc was used as a negative-control protein. (C) HKU1-S1(600)-mFc binding to a molecule(s) located on the RD cell surface. Results of immunofluorescence microscopy imaging of RD cells stained by HKU1-S1(600)-mFc or the control protein, HKU3-323-mFc, are shown. Cell membranes were stained with FM-4-64 (red), the nuclei were stained with Hoechst dye 33258 (blue), and the HKU1-S1 staining was detected by an 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) FACS analysis of the N termini of HKU1-S1 and OC43-S1 binding to RD cells. HKU3-323-mFc at 10 μg/ml was used as a control (Ctrl.) in panels D and E. The graphs shown in panels B to E are representative of the results of at least two independent experiments for each panel.

**FIG 2**
Characterization of the binding receptor of HKU1-S1 protein on RD cells by FACS. (A) Pretreatment of RD cells with neuraminidase (NA) greatly reduced HKU1-S1 binding in a dose-dependent manner. RD cells were pretreated with NA at different concentrations as indicated and then stained with 5 μg/ml of HKU1-S1(600)-mFc, OC43 (268)-mFc, or SIRPα-mFc control protein followed by detection with an FITC-labeled anti-mouse Fc antibody. (B) Pretreatment of RD cells with TPCK-treated trypsin reduced HKU1-S1(600) and OC43-S1(268) binding to RD cells. The FACS assay method was similar to that described for panel A. “Ctrl.” indicates RD cells that were stained with HKU1-S1(268)-mFc only in panels A and B. (C) HKU1-S1(600)-mFc did not bind to BSM (containing 9-O-Ac-sia) directly coated on an ELISA plate. OC43-S1 served as a positive control. (D) HKU-S1 did not hemagglutinate mouse RBCs (mRBCs) or rat RBCs (rRBCs). OC43-S1(268)-mFc served as a positive control and showed HA activity in a dose-dependent manner. (E) Binding of HKU1-S1 and OC43-S1 to rat RBCs and mouse RBCs determined by FACS analysis. Data shown are representative of the results of at least two independent experiments for each panel.

**FIG 3**
HKU1-HE is an O-acetylesterase and RDE for HKU1-S1 binding to RD cells. (A) SDS-PAGE of expressed recombinant HE proteins. All proteins were expressed in 293T cells and purified by the use of protein A Sepharose beads. Purified proteins were run on SDS-PAGE and stained by the use of Coomassie blue. (B) HE-mFc protein itself did not bind to RD cells. (C) HKU1-HE acted as a RDE on RD cells for HKU1- and OC43-S1 protein. Pretreatment of RD cells with HKU1-HE as well as with OC43- and BCoV-HE greatly reduced binding of HKU1-S1 or OC43-S1 (at 5 μg/ml) to RD cells in a dose-dependent manner. MHV-S-HE showed no effect. The graphs shown in panels B and C are representative of the results of at least two independent experiments. (D) HKU1-HE acted as a RDE on RBCs for OC43-S1 protein. Pretreatment of rat (r) or mouse (m) RBCs with HKU1-, OC43-, or BCoV-HE inhibited OC43-S1-mediated hemagglutination activity, whereas MHV-S-HE had no effect. Data or images representative of the results of at least two independent experiments are shown for panels A to D. (E) HKU1-HE is an acetylesterase. HEs of HKU1, OC43, BCoV, and MHV-S at 2 μg/ml were used to hydrolyze pNPA (2-fold serially diluted) at room temperature for 15 min. Enzyme activity was assessed by measuring optical density at 405 nm (OD₄₀₅). The *K_m* and V_max values were calculated from the Michaelis-Menten enzyme kinetics curve fitting of two independent repeats.

**FIG 4**
Treatment of HAE cells with HE or NA but not the enzymatically inactive HE mutant inhibited HKU1 infection. (A) Esterase-catalytic active-site residues in HE. Sequence alignment of amino acids around the catalytically active site (in red) is shown. The BCoV-HE amino acid numbering scheme was used (22). The 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, and AJ872181, respectively. For HE proteins of MHV-S, bovine torovirus (BToV), and porcine torovirus (PToV) strain p10, the Uniprot accession numbers are P31614, P0C0V9, and Q70KP1, respectively. (B) HKU1-HE proteins with substitutions at catalytically active sites are enzymatically inactive. HKU1-HE or the mutants at 1 μg/ml were incubated with 2-fold serially diluted pNPA at room temperature for 15 min prior to measuring OD₄₀₅. Each data point represents the OD₄₀₅ value for HE or its mutants subtracted from that of a negative control. (C) Esterase inactive mutants of HE did not block HKU1-S1 protein binding to RD cells. RD cells were treated with 10 μg/ml of HE or HE mutant proteins prior to HKU1-S1 staining (5 μg/ml). (D) HE and NA pretreatment of HAE cells inhibited HKU1 infection. Prior to HKU1 virus inoculation, HAE cells were pretreated with HE proteins or NA at different concentrations as indicated for 1 h, followed by HKU1 virus inoculation at a high dose as indicated. The replication kinetics of HKU1 virus was assessed in apical washes from infected HAE cultures by real-time RT-PCR. Virus yield is presented as the number of virus RNA copies/ml. (E) HE pretreatment blocked HKU1 infection of HAE cells. HAE cells were pretreated with HE protein or enzymatically inactive HE mutant (S40A) for 1 h prior to viral challenge at a lower dose than that used for the experiment whose results are shown in panel D. The replication kinetics of HKU1 virus was assessed as described for panel D. The dotted line indicates the detection limit of the assay for panels D and E.

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References

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1. Assiri A, McGeer A, Perl TM, Price CS, Al Rabeeah AA, Cummings DA, Alabdullatif ZN, Assad M, Almulhim A, Makhdoom H, Madani H, Alhakeem R, Al-Tawfiq JA, Cotten M, Watson SJ, Kellam P, Zumla AI, Memish ZA. 2013. Hospital outbreak of Middle East respiratory syndrome coronavirus. N Engl J Med 369:407–416. doi:10.1056/NEJMoa1306742. - DOI - PMC - PubMed

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[1] Graham RL, Donaldson EF, Baric RS. 2013. A decade after SARS: strategies for controlling emerging coronaviruses. Nat Rev Microbiol 11:836–848. doi:10.1038/nrmicro3143. - DOI - PMC - PubMed

[2] Graham RL, Donaldson EF, Baric RS. 2013. A decade after SARS: strategies for controlling emerging coronaviruses. Nat Rev Microbiol 11:836–848. doi:10.1038/nrmicro3143. - DOI - PMC - PubMed

[3] Rota PA, Oberste MS, Monroe SS, Nix WA, Campagnoli R, Icenogle JP, Penaranda S, Bankamp B, Maher K, Chen M-h, Tong S, Tamin A, Lowe L, Frace M, DeRisi JL, Chen Q, Wang D, Erdman DD, Peret TCT, Burns C, Ksiazek TG, Rollin PE, Sanchez A, Liffick S, Holloway B, Limor J, McCaustland K, Olsen-Rasmussen M, Fouchier R, Gunther S, Osterhaus ADME, Drosten C, Pallansch MA, Anderson LJ, Bellini WJ. 2003. Characterization of a novel coronavirus associated with severe acute respiratory syndrome. Science 300:1394–1399. doi:10.1126/science.1085952. - DOI - PubMed

[4] Rota PA, Oberste MS, Monroe SS, Nix WA, Campagnoli R, Icenogle JP, Penaranda S, Bankamp B, Maher K, Chen M-h, Tong S, Tamin A, Lowe L, Frace M, DeRisi JL, Chen Q, Wang D, Erdman DD, Peret TCT, Burns C, Ksiazek TG, Rollin PE, Sanchez A, Liffick S, Holloway B, Limor J, McCaustland K, Olsen-Rasmussen M, Fouchier R, Gunther S, Osterhaus ADME, Drosten C, Pallansch MA, Anderson LJ, Bellini WJ. 2003. Characterization of a novel coronavirus associated with severe acute respiratory syndrome. Science 300:1394–1399. doi:10.1126/science.1085952. - DOI - PubMed

[5] Ksiazek TG, Erdman D, Goldsmith CS, Zaki SR, Peret T, Emery S, Tong S, Urbani C, Comer JA, Lim W, Rollin PE, Dowell SF, Ling AE, Humphrey CD, Shieh WJ, Guarner J, Paddock CD, Rota P, Fields B, DeRisi J, Yang JY, Cox N, Hughes JM, LeDuc JW, Bellini WJ, Anderson LJ; SARS Working Group . 2003. A novel coronavirus associated with severe acute respiratory syndrome. N Engl J Med 348:1953–1966. doi:10.1056/NEJMoa030781. - DOI - PubMed

[6] Ksiazek TG, Erdman D, Goldsmith CS, Zaki SR, Peret T, Emery S, Tong S, Urbani C, Comer JA, Lim W, Rollin PE, Dowell SF, Ling AE, Humphrey CD, Shieh WJ, Guarner J, Paddock CD, Rota P, Fields B, DeRisi J, Yang JY, Cox N, Hughes JM, LeDuc JW, Bellini WJ, Anderson LJ; SARS Working Group . 2003. A novel coronavirus associated with severe acute respiratory syndrome. N Engl J Med 348:1953–1966. doi:10.1056/NEJMoa030781. - DOI - PubMed

[7] Anderson LJ, Tong S. 2010. Update on SARS research and other possibly zoonotic coronaviruses. Int J Antimicrob Agents 36(Suppl 1):S21–S25. doi:10.1016/j.ijantimicag.2010.06.016. - DOI - PMC - PubMed

[8] Anderson LJ, Tong S. 2010. Update on SARS research and other possibly zoonotic coronaviruses. Int J Antimicrob Agents 36(Suppl 1):S21–S25. doi:10.1016/j.ijantimicag.2010.06.016. - DOI - PMC - PubMed

[9] Assiri A, McGeer A, Perl TM, Price CS, Al Rabeeah AA, Cummings DA, Alabdullatif ZN, Assad M, Almulhim A, Makhdoom H, Madani H, Alhakeem R, Al-Tawfiq JA, Cotten M, Watson SJ, Kellam P, Zumla AI, Memish ZA. 2013. Hospital outbreak of Middle East respiratory syndrome coronavirus. N Engl J Med 369:407–416. doi:10.1056/NEJMoa1306742. - DOI - PMC - PubMed

[10] Assiri A, McGeer A, Perl TM, Price CS, Al Rabeeah AA, Cummings DA, Alabdullatif ZN, Assad M, Almulhim A, Makhdoom H, Madani H, Alhakeem R, Al-Tawfiq JA, Cotten M, Watson SJ, Kellam P, Zumla AI, Memish ZA. 2013. Hospital outbreak of Middle East respiratory syndrome coronavirus. N Engl J Med 369:407–416. doi:10.1056/NEJMoa1306742. - DOI - PMC - PubMed

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Human Coronavirus HKU1 Spike Protein Uses O-Acetylated Sialic Acid as an Attachment Receptor Determinant and Employs Hemagglutinin-Esterase Protein as a Receptor-Destroying Enzyme

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Human Coronavirus HKU1 Spike Protein Uses O-Acetylated Sialic Acid as an Attachment Receptor Determinant and Employs Hemagglutinin-Esterase Protein as a Receptor-Destroying Enzyme

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