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. 2015 Sep;89(17):8816-27.
doi: 10.1128/JVI.03737-14. Epub 2015 Jun 17.

Identification of the Receptor-Binding Domain of the Spike Glycoprotein of Human Betacoronavirus HKU1

Zhaohui Qian  1 , Xiuyuan Ou  2 , Luiz Gustavo Bentim Góes  3 , Christina Osborne  3 , Anna Castano  4 , Kathryn V Holmes  4 , Samuel R Dominguez  5
Affiliations

Identification of the Receptor-Binding Domain of the Spike Glycoprotein of Human Betacoronavirus HKU1

Zhaohui Qian et al. J Virol. 2015 Sep.

Abstract

Coronavirus spike (S) glycoproteins mediate receptor binding, membrane fusion, and virus entry and determine host range. Murine betacoronavirus (β-CoV) in group A uses the N-terminal domain (NTD) of S protein to bind to its receptor, whereas the β-CoVs severe acute respiratory syndrome CoV in group B and Middle East respiratory syndrome CoV in group C and several α-CoVs use the downstream C domain in their S proteins to recognize their receptor proteins. To identify the receptor-binding domain in the spike of human β-CoV HKU1 in group A, we generated and mapped a panel of monoclonal antibodies (MAbs) to the ectodomain of HKU1 spike protein. They did not cross-react with S proteins of any other CoV tested. Most of the HKU1 spike MAbs recognized epitopes in the C domain between amino acids 535 and 673, indicating that this region is immunodominant. Two of the MAbs blocked HKU1 virus infection of primary human tracheal-bronchial epithelial (HTBE) cells. Preincubation of HTBE cells with a truncated HKU1 S protein that includes the C domain blocked infection with HKU1 virus, but preincubation of cells with truncated S protein containing only the NTD did not block infection. These data suggest that the receptor-binding domain (RBD) of HKU1 spike protein is located in the C domain, where the spike proteins of α-CoVs and β-CoVs in groups B and C bind to their specific receptor proteins. Thus, two β-CoVs in group A, HKU1 and murine CoV, have evolved to use different regions of their spike glycoproteins to recognize their respective receptor proteins.

Importance: Mouse hepatitis virus, a β-CoV in group A, uses the galectin-like NTD in its spike protein to bind its receptor protein, while HCoV-OC43, another β-CoV in group A, uses the NTD to bind to its sialic-acid containing receptor. In marked contrast, the NTD of the spike glycoprotein of human respiratory β-CoV HKU1, which is also in group A, does not bind sugar. In this study, we showed that for the spike protein of HKU1, the purified C domain, downstream of the NTD, could block HKU1 virus infection of human respiratory epithelial cells, and that several monoclonal antibodies that mapped to the C domain neutralized virus infectivity. Thus, the receptor-binding domain of HKU1 spike glycoprotein is located in the C domain. Surprisingly, two β-CoVs in group A, mouse hepatitis virus and HKU1, have evolved to use different regions of their spike glycoproteins to recognize their respective receptors.

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Figures

FIG 1
FIG 1
Binding of mouse monoclonal antibodies to HKU1 S. (A) IFA. HEK 293T cells expressing HKU1 S protein were fixed and stained with the indicated MAbs (undiluted hybridoma supernatants), followed by FITC-conjugated goat anti-mouse IgG. The concentration of antibody in each undiluted hybridoma supernatant was the following: mHKUS-1, 25 μg/ml; mHKUS-2, 6.0 μg/ml; mHKUS-3, 4.0 μg/ml; mHKUS-4, 12.4 μg/ml; mHKUS-5, 25 μg/ml; mHKUS-6, 6.8 μg/ml. The experiment was repeated at least 3 times. CTRL, control. (B) Western blot analysis. Lane 1, 1 μg of purified Fc-tagged S14-294aa protein; lane 2, 1 μg of purified Fc-tagged S295-755aa protein. The blots were probed with either HRP-conjugated goat anti-human IgG or the indicated MAbs (undiluted hybridoma supernatants), followed by HRP-conjugated goat anti-mouse IgG. The experiments were done twice.
FIG 2
FIG 2
Inhibition of HKU1 virus entry by mouse MAbs to HKU1 S protein. The HKU1 virus was incubated with either human IvIg (10 mg/ml) or undiluted hybridoma supernatants of the indicated mouse MAbs for 30 min at 37°C, and then virus-antibody mixtures were incubated on apical surfaces of differentiated HTBE cells for 4 h. After removing the inocula, cells were washed and then incubated for another 48 h. Infected cells were detected by IFA with polyclonal rabbit 1814 anti-HKU1 S antibody (A), and RNA from released viruses from apical washes at 24 and 48 h postinoculation were analyzed by real-time PCR (B). No Ab, 1% BSA but no antibody; IvIg, human IvIg at 10 mg/ml; mHKUS-1 to mHKUS-6, hybridoma supernatant antibody. ND, not detected. *, P < 0.05. Experiments were done at least twice; one representative is shown.
FIG 3
FIG 3
Dose-dependent inhibition of HKU1 virus entry by antibodies mHKUS-2 and mHKUS-3. Inhibition experiments were performed as described for Fig. 2 using the given amount of purified antibodies. (A and C) IFA; (B and D) real-time PCR analysis. (A and B) Viruses from passage 1; (C and D) viruses from passage 3. No antibody, 1% BSA but no antibody. mHKUS-1 and mHKUS-5 served as negative controls at concentrations of 100 μg/ml. Experiments were repeated at least twice, and one representative is shown. ND, not detected.
FIG 4
FIG 4
Western blot analysis of N-, C-, or both terminal truncations of HKU1 S proteins. (A) Schematic diagram of N-, C-, or both terminal truncations of HKU1 S proteins. The truncated proteins with C-terminal Fc tags were purified and detected by HRP-conjugated goat anti-human IgG. The amino acid positions are indicated relative to those of wild-type HKU1 S protein. NTD, N-terminal domain; CTD, C-terminal domain; TM, transmembrane domain; linker GGGGS, flexible linker gly-gly-gly-gly-ser; FLAG tag, DYKDDDDK. (B) Western blot analysis of truncated HKU1 S protein expression. Truncated protein was separated in a 4 to 15% SDS-PAGE and transferred to nitrocellulose membranes. The blots were probed with HRP-conjugated goat anti-human IgG antibody. Lane 1, S14-755; lane 2, S14-673; lane 3, S14-534; lane 4, S14-443; lane 5, S14-294; lane 6, S295-755; lane 7, S310-673; lane 8, mock-transfected control. Lanes 1 to 6 were blotted with undiluted hybridoma supernatants (Sups), and lane 7 was blotted with a 10-fold concentrated hybridoma supernatant. Experiments were done three times, and one representative is shown.
FIG 5
FIG 5
Mapping of epitopes of MAbs to HKU1 S protein. (A) ELISAs were performed using supernatant containing the indicated proteins. Ctrl Ab, negative-control antibody; 2nd Ab control, no primary antibody but with 2nd Ab. Experiments were done twice, and one representative is shown. (B) ELISAs were performed using purified proteins and purified antibodies. The experiments were done twice, and one representative is shown.
FIG 6
FIG 6
Inhibition of HKU1 virus entry by CTD of HKU1 S protein. Differentiated HTBE cells were incubated with the indicated amount of S1, NTD, or CTD proteins at 37°C for 1 h. HKU1 viruses were diluted into the same amount of proteins and added onto the HTBE cells for 4 h. After being washed, cells were fixed and stained with polyclonal rabbit anti-HKU1 S antibodies at 48 h postinoculation (A), and released viruses from apical washes at 4 h, 24, and 48 h of postinoculation were analyzed by real-time PCR (B). ND, not detected.
FIG 7
FIG 7
Coronavirus spike proteins. (A) Diagram of coronavirus spike proteins. NTD, N-terminal domain; HRN, N-terminal heptad repeat; HRC, C-terminal heptad repeat. Group indicates the CoV genus and group. (B) Amino acid sequence alignment of S1 subunits of different betacoronaviruses. The letters with red underlining and red arrows are the contacting residues of MHV S protein with mouse CEACAM1a, while the letters with black underlining and black arrows are the sugar binding residues of BCoV S protein. The similarity of S1 among group A betacoronaviruses is about 71.9%, whereas the similarity of S1 among different group betacoronaviruses is about 29.4%.
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