INTRODUCTION
Human coronaviruses (hCoVs) are enveloped RNA viruses. They are usually associated with mild to moderate respiratory tract illnesses but can also cause severe and highly lethal disease, depending on the virus strain (
1). Six hCoV strains have been identified to date and belong to four different groups, including hCoV-229E and hCoV-NL63 in the alphacoronaviruses (group 1); hCoV-OC43 and hCoV-HKU1 in the group a betacoronaviruses (group 2a); severe acute respiratory syndrome CoV (SARS-CoV) in the group b betacoronaviruses (group 2b); and Middle East respiratory syndrome CoV (MERS-CoV) in the group c betacoronaviruses (group 2c). Infections by viruses in groups 1 and 2a are common worldwide and can also cause severe disease in young children or immunocompromised adults. SARS-CoV (
2–4) and MERS-CoV (
5,
6) are two highly virulent hCoVs causing severe respiratory diseases with high morbidity and mortality (
7); the latter strain is still circulating in human populations.
Cellular receptor specificity plays an important role in viral cell and tissue tropism, pathogenesis, interspecies transmission, and adaptation. The CoV Spike (S) glycoprotein is generally responsible for binding to cellular receptors and mediating viral entry. The S protein is a large type I transmembrane glycoprotein that exists as a trimer protruding from the surface of virions (
8). S proteins have an amino-terminal (NT) S1 domain that mediates binding with cellular receptors and a carboxy-terminal (CT) S2 domain that mediates subsequent virus-cell membrane fusions. A wide range of diverse cellular receptors specifically recognized by the S1 domains have been identified for all the aforementioned hCoVs except hCoV-HKU1. Human aminopeptidase N (CD13) is the cellular receptor for hCoV-229E (
9). 9-
O-Acetylated sialic acid (9-
O-Ac-Sia) is the cellular receptor determinant for hCoV-OC43 (
10). hCoV-NL63 and SARS-CoV both employ human angiotensin-converting enzyme 2 (ACE2) to mediate cellular entry (
11,
12), while hCoV-NL63 utilizes heparan sulfate proteoglycans for attachment to target cells (
13). MERS-CoV utilizes dipeptidyl peptidase 4 (DPP4 or CD26) receptor to enter host cells (
14).
hCoV-HKU1 was initially identified in 2005 from a pneumonia patient in Hong Kong (
15). It was subsequently found to be as common and widespread as previously known hCoVs, namely, hCoV-229E, hCoV-OC43, and hCoV-NL63 (
16–19). Characterization of hCoV-HKU1 has been challenging due to the lack of a convenient cell line-based culture system. It was recently demonstrated that hCoV-HKU1 replicates to a high titer in an
in vitro culture system that uses primary human ciliated airway epithelial (HAE) cells or type II alveolar epithelial cells (
20–22); however, the functional receptor(s) of hCoV-HKU1 and other important aspects of virus-host interaction remains unknown. As a member of group 2a CoVs, HKU1-CoVs also carry another viral surface protein hemagglutinin-esterase (HE)-encoding gene that is present exclusively in this group of CoV genomes (
23). The HE protein is also a type I transmembrane glycoprotein comprised of two functional domains: an
O-acetylated sialic acid binding domain and a corresponding sialate
O-acetylesterase domain (
24). HE protein functions primarily as a receptor-destroying enzyme (RDE) for CoVs, e.g., hCoV-OC43 and its proposed zoonotic ancestor, bovine coronavirus (BCoV) (
25). Both viruses bind to receptor 9-
O-Ac-Sia via their S proteins, and their HE proteins mediate RDE activity late in the infection cycle via the sialate-9-
O-acetylesterase domain to facilitate the release of viral progeny and escape from attachment on nonpermissive host cells (
23,
26). In contrast, mouse hepatitis virus (MHV), another member of group 2a CoVs, infects cells via the interaction of S protein with its principal receptor, the carcinoembryonic antigen-related cell adhesion molecule (CEACAM1a), while the MHV HE protein functions at very early viral attachment steps through the concerted action of its
O-acetylated sialic acid binding and RDE activities (
27,
28). To date, the function and role of the hCoV-HKU1 HE protein have remained undefined.
In this study, we found that the hCoV-HKU1 S protein mediated viral attachment by utilizing O-acetylated sialic acids on glycoprotein(s) as a receptor determinant or as initial attachment factors. The HE protein of hCoV-HKU1 did not exhibit sialic acid binding activity but instead mediated sialate-O-acetylesterase RDE activity specific to the O-acetylated sialic acids recognized by the S protein. Interestingly, HKU1-HE protein displayed sialate-9-O-acetylesterase RDE activity similar to that seen with OC43-HE and BCoV-HE. In the hCoV-HKU1 in vitro replication model, we further demonstrated that the HE protein but not an enzymatically inactive HE mutant acted as a RDE and completely blocked or greatly reduced infection, depending on the dose of inoculating hCoV-HKU1. These findings revealed that early viral entry steps for hCoV-HKU1 are similar to but also distinct from those for other members of group 2a CoVs. Like hCoV-OC43 and BCoV, hCoV-HKU1 employs O-acetylated sialic acids as a primary receptor determinant or attachment factor and its HE protein as a corresponding RDE; however, hCoV-HKU1 also uniquely requires additional receptor determinants beyond those required by hCoV-OC43 and BCoV.
MATERIALS AND METHODS
Construction of expression plasmids.
A synthetic codon-optimized sequence for the HKU1-S1 gene (GenBank accession number NC_006577.2) encoding amino acids (aa) 15 to 600 was cloned into a mammalian expression vector containing a CD5 signal peptide and a C-terminal Fc tag from mouse IgG2a (mFc). The expression cassette was under the control of a cytomegalovirus (CMV) early enhancer/chicken β actin (CAG) promoter. The resulting construct, pCAGGS-HKU1-S1(600)-mFc, encodes a chimeric S1 protein with an N-terminal CD5 signal peptide and mFc at its C terminus. Similarly, plasmids encoding other proteins, including the NT domain of HKU1-S1 (aa 15 to 268), the NT domain of hCoV-OC43-S1 (aa 15 to 268) (ATCC VR-759 strain; GenBank accession no. AAT84354), and the NT domain of S1 of CoV-HKU3 (aa 16 to 323) (DQ022305), were constructed. The extracellular domains of HE proteins from different CoVs, including HKU1-HE (aa 14 to 358; GenBank accession no. NC_006577.2), hCoV-OC43-HE protein (aa 19 to 376; AAX85668.1), BCoV-HE (aa 19 to 377; AAA92991.1), and MHV-S-HE (aa 25 to 393; AAX08110.1), were similarly constructed. Plasmids encoding mutants of HKU1-HE were generated by the site-directed QuikChange mutagenesis method (Stratagene). All mutations were confirmed by DNA sequencing, in which the codon for esterase-catalytic residue Ser40 was replaced by Ala (S40A mutant) or the catalytic triad S40, H329, and D326 were all replaced by Ala (S40A/H329A/D326A).
Expression and purification of recombinant proteins.
HEK293T cells were transiently transfected with the expression plasmids using polyethyleneimine (Polysciences). At 12 h after transfection, the medium was replaced by 293 SFM II expression medium (Life Technology). Tissue culture supernatants were harvested 3 days after transfection, and the recombinant proteins were purified by protein A-based affinity chromatography.
Flow cytometry FACS analysis.
HKU1-S1(600)-mFc or other proteins at different concentrations were diluted in fluorescence-activated cell sorter (FACS) buffer (phosphate-buffered saline [PBS] containing 0.5% bovine serum albumin [BSA] and 0.1% NaN3) and then incubated with 0.5 × 106 to 1 × 106 RD cells or red blood cells (RBCs) from mouse or rat blood samples at 4°C for 0.5 to 1 h. Cells were then washed three times with FACS buffer followed by incubation with fluorescein isothiocyanate (FITC)-labeled anti-mouse Fc antibody at a dilution following the instructions of the manufacturer (Sigma or Pierce) at 4°C for 30 min. Cells were washed as described above, and the binding of proteins to cells was analyzed by the use of a BD FACS LSRII (Becton Dickinson) flow cytometer and FCS Express software (De Novo Software). For FACS analysis to examine the inhibition of HKU1-S1 binding to RD cells, the cells were pretreated with the indicated HE proteins or enzymes at different concentrations and then incubated with HKU1-S1, after which binding was analyzed as described above. The neuraminidase (NA) was from Clostridium perfringens (Sigma), and the bovine pancreas-derived trypsin treated with N-tosyl-l-phenylalanyl chloromethyl ketone (TPCK) was purchased from Sigma. For both NA and trypsin, the pretreatment was carried out at 37°C for 1 h; for the HE proteins, the pretreatment was carried out at 4°C for 1 h.
Indirect immunofluorescence.
RD cells were seeded on glass coverslips 1 day before staining. Cells were washed three times with PBS, blocked with 0.5%BSA–PBS at 37°C for 30 min, incubated with HKU1-600-mFc or HKU3-323-mFc at 20 μg/ml in PBS at 4°C for 1 h followed by washing three times with PBS, and then incubated with FITC-goat anti-mouse Fc antibody (Sigma) at 4°C for 1 h. Cells were washed three times and then incubated with 5 μg/ml Hoechst 33258 at 37°C for 10 min, followed by three additional washes, and finally incubated with 5 μg/ml FM-4-64 on ice for 1 min. Cells were analyzed and imaged with a 63× oil objective using an Zeiss LSM510 Meta confocal microscope. Representative images are shown.
HA assay.
RBCs in an approximately 0.25% to 0.5% suspension prepared from mouse (BALB/c) or rat (Sprague Dawley) blood were added to a round-bottom 96-well plate at 50 μl/well. S1 proteins were 2-fold serial diluted with 0.5%BSA–PBS and added at 50 μl/well to the wells containing RBCs. For the HE protein inhibition assay, RBCs were first pretreated with HE (2-fold serially diluted) and washed by the use of PBS followed by addition of 50 μl/well of 10 μg/ml of hCoV-OC43-S1 to the wells containing the HE-pretreated and washed RBCs. The plates were left at room temperature for 60 min or longer until hemagglutination (HA) developed or the RBCs gradually settled. Positive hemagglutination resulted in the formation of a uniform reddish color across the well, whereas negative results appeared as dots in the center of round-bottomed plates due to the sedimentation of RBCs.
ELISA.
The binding of S1 proteins to bovine submaxillary mucin (BSM) was determined by an enzyme-linked immunosorbent assay (ELISA) as previously described (
28) with modification. Maxisorp 96-well plates (Nunc) were coated overnight at 4°C with BSM (Sigma) at 10 μg/ml and at 100 μl/well. The wells were washed with washing buffer (PBST [0.05% Tween 20–PBS]) and treated with blocking buffer (PBS, 0.05% Tween 20, 2% nonfat milk) for 1 h at room temperature. Serially diluted S1 proteins were prepared in blocking buffer (starting concentration, 20 μg/ml) and then added to the BSM-coated wells at 100 μl/well. Incubation was continued for 1 h followed by washing with PBST six times. Binding was detected using an horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Pierce) (1:10,000 in blocking buffer) followed by washing again. The optical density at 450 nm (OD
450) was measured after incubation of the peroxidase tetramethylbenzidine (TMB) substrate and stop solution.
Acetylesterase activity assay.
Chromogenic p-nitrophenyl acetate (pNPA; Sigma) substrate was 2-fold serially diluted and then incubated with 1 or 2 μg/ml HE-mFc protein or its mutants in a 100-μl volume in PBS (pH 7.4) at 37°C for different time periods as indicated. The acetylesterase activity was determined by measuring the release of para-nitrophenol (OD450) at the end of each reaction in microtiter plates with a microplate spectrophotometer (Bio-Rad). An unrelated protein was used as a control in the enzymatic assay, and the OD405 for HE protein or mutants was subtracted from that of this control. The Km value of HE protein was calculated from the Michaelis-Menten enzyme kinetics curve using Graphpad Prism 5 software.
Neuraminidase activity assay.
An Amplex red neuraminidase assay kit (Molecular Probes/Invitrogen) was used to measure NA activity. Briefly, 25 μg/ml of HKU1-HE protein was serially diluted in 50 μl of 1× reaction buffer followed by addition of 50 μl of a 2× working solution containing 100 μM Ample Red reagent, 0.2 U/ml of HRP, and 4 U/ml of galactose oxidase, and the fetuin substrate was serially diluted 100-fold from 2.5 mg/ml to 2.5 pg/ml. The mixture was incubated at 37°C for 10 min under dark conditions, the fluorescence signal was then measured at a wavelength of 595 nm, and the measured values were used to indicate relative NA activity levels.
HKU1 infection of HAE cells.
The HAE cell culture system has been described previously (
20). Briefly, the apical surface of HAE cells was washed three times
in situ with phosphate-buffered saline (PBS) and then treated with testing reagents or controls by incubation at 32°C for 1 h followed by washing with PBS to remove the testing reagents. The treatment and washing were repeated two more times. HAE cells were then inoculated with 100 μl of viral stock. Following incubation for 2 h at 32°C, the unbound virus was removed by washing with 500 μl for 10 min at 32°C for three washes, and the HAE cells were maintained at an air-liquid interface for the remainder of the experiment at 32°C. HKU1 replication kinetics were determined at specific time points postinoculation as indicated, 120 μl of PBS was applied to the apical surface of HAE cells, and the apical sample was harvested for RNA isolation after 10 min of incubation at 32°C. The RNA was then analyzed by real-time reverse transcriptase (RT)-PCR to determine viral genomic mRNA copy numbers (
20).
DISCUSSION
Sialic acid, a 9-carbon monosaccharide, includes a large number of derivatives arising from differential modifications of the parental molecule as well as various glycosidic linkages (e.g., α2,3 and α2,6) to the subterminal residue of a sugar chain.
O-Acetylation is one of the most common types of sialic acid modification. It can occur at all the four hydroxyl groups of sialic acids at positions of C4, C7, C8, and C9 and generates mostly mono-
O-acetylated but also oligo-
O-acetylated sialic acids at more than one position.
O-Ac-Sia plays fundamental roles in many biological and pathophysiological events (
40). The 9-
O-Ac-Sia serves as a receptor determinant for several members of group 2a CoVs, including the closely related BCoV, hCoV-OC43, and porcine hemagglutinating encephalomyelitis virus (PHEV) (
10,
30,
41). The binding with 9-
O-Ac-Sia is essential for these viruses to initiate infection, and their S protein is the major viral protein responsible for the binding. In this study, we found that the S protein of hCoV-HKU1 can also recognize
O-Ac-Sia but only those presented on RD cells among the many cell lines tested. Differently from the aforementioned CoVs in the same group, no binding of hCoV-HKU1 S1 with 9-
O-Ac-Sia-containing BSM as well as RBCs from mouse and rat could be detected. In addition, a previous study (
42) and our data (
Fig. 1E) both demonstrated that the NT of HKU1-S1 was unable to bind with carbohydrate moieties. In contrast, the carbohydrate receptor binding domains for hCoV-OC43 and BCoV were located in the NT of S1 (
Fig. 2) (
42). On the other hand, for hCoV-HKU1, as well as for other 9-
O-Ac-Sia recognition-dependent CoVs, the issue remains of whether, in addition to
O-Ac-Sia, they also interact with a protein receptor during the entry process.
O-Ac-Sia on RD cells can be recognized by hCoV-HKU1 S1 protein; however, the cells are not permissive for viral infection. One explanation for this is the lack of a protein receptor for hCoV-HKU1 on RD cells. In line with this, our attempts to use HKU1-S1(600) as a viral ligand protein for immunoprecipitation combined with mass spectrometric identification did not find a protein(s) specifically binding to HKU1-S1 (data not shown). It is also possible that there is another molecule(s) present only in HAE cell cultures but not on RD cells, which are important for viral infection at a later stage, e.g., for membrane fusion or viral replication, or that a restriction factor(s) may exist in RD cells to limit viral infection.
Among CoVs, HE protein is present only in members of group 2a CoVs. Sequence and structural similarities suggest that CoV HE evolved from the HEF protein of influenza C virus (
24). Although the dual function of HEF (
O-Ac-Sia receptor binding and sialate-
O-acetylesterase activity) was maintained in some CoVs, the HE appears to mainly function as a RDE in these CoVs. Comparing to the essential role of S protein in Sia-receptor binding and mediating viral entry, the Sia binding activity of HE seems to be an accessory function and its affinity and Sia preference characteristics differ among CoV strains (
23,
43). HEs of two closely related MHV field strains, MHV-DVIM and MHV-S, recognize two different types of
O-Ac-Sia, 9-
O-Ac-Sia and 4-
O-Ac-Sia, respectively, whereas many MHV laboratory strains carry defective HE genes (
27,
28). hCoV-OC43 HE lost its Sia binding activity although it has high (97%) sequence identity with the HE of BCoV (Mebus stain), which exhibits high Sia binding affinity (
43). Similarly, we did not find that HKU1-HE has
O-Ac-Sia binding activity. This is consistent with the observation described previously by Langereis et al. that the HE of hCoV-HKU1 failed to hemagglutinate erythrocytes and bind to
O-Ac-Sia (
43). Sequence comparison of HKU1-HE with HEF of influenza C virus and HEs of CoVs with known protein structures demonstrated that HKU1-HE was the most divergent one at the Sia binding loops (
23,
43), whereas the Sia-
O-acetylesterase domain is highly conserved among them. The key residues contributing to the catalytic activity of HEF and other CoV HEs, including the Ser-His-Asp catalytic triad, the oxyanion hole-contributing residues Gly
85 and Asn
117 in HEF, and an Arg
322 residue in HEF important for Sia substrate binding, are completely conserved in hCoV-HKU1 (
23). In our study, HKU1-HE indeed showed strong
O-AC-esterase activity with pNPA substrate, and the Ser-His-Asp catalytic triad mutant HEs completely lost this activity. We demonstrated that the sialic acids expressed by RD cells not only are specifically recognized by the HKU1-S1 protein but also are substrates for HE protein. Wild-type but not mutant HE treatment of RD cells showed effects similar to those seen with neuraminidase and abolished the subsequent binding of S1 to RD cells. These results indicate that the S1 protein binds with
O-Ac-Sia and that the HE has matching or even broader esterase activity.
Langereis et al. (
43) reported that the HKU1-HE, like the BCoV-HE, displayed Sia-9-
O-Ac-esterase activity using a synthetic 4,9-di-
O-Ac-Sia substrate analogue. Consistently, we demonstrated that HKU1-HE had RDE activity similar to that seen with OC43-HE and BCoV-HE with respect to removal of the binding moiety from RD cells for S1 proteins of both HKU1 and OC43. These results strongly support the idea that HKU1-HE has Sia-9-
O-Ac-esterase activity similar to that of OC43-HE and BCoV-HE. However, unlike the results seen with S1 of OC43, Sia-9-
O-AC binding activity was not detected for the S1 of hCoV-HKU1 by BSM binding and erythrocyte hemagglutination assays, which are standard methods for examining the usage of Sia-9-
O-AC as a receptor by other CoVs (
Fig. 2C to
E). Thus, it is likely that HKU1 is different from OC43 and BCoV in terms of the use of Sia-9-
O-AC as a receptor via its Spike protein. Sia-9-
O-AC may be required for but not sufficient to support the HKU1-S1 binding. In addition of Sia-9-
O-AC, RD cells may express other types of
O-acetylated sialic acid that are lacking or at a lower level in BSM and at the erythrocyte cell surface but required for HKU1-S1 recognition. One may also speculate that certain examples of di-
O-Ac-Sia, tri-
O-Ac-Sia, or oligo-
O-acetylated Sia in which all have an acetyl group at C9 (9-
O-AC) in common or a certain particular sugar chain core structure(s) to which Sia-9-
O-Ac is attached or the linkage of sialic acid to the penultimate residue of a sugar chain may also be required.
Finally, in HAE cultures, we demonstrated that NA and HKU1-HE but not an enzymatically inactive HE mutant dramatically reduced virus infection or completely blocked infection when a lower viral challenge dose was applied. The results of treatment of HAE cells with NA and HKU1-HE prior to infection strongly suggested that the Sia-9-
O-AC-esterase activity of HE acted as a RDE and removed the critical receptor binding moieties so that the early viral entry was impaired. Considering that the HAE cells continuously secrete a large amount of mucus, the effect of inhibition of viral infection by pretreatment of the cells with NA and HE is remarkable and suggests an essential role for sialic acids in the initiation of infection. Our study results also suggest that acetyl modification at 9-O of sialic acid may be a necessary but not sufficient receptor or attachment factor determinant and warrant further investigation to determine the fine specificity and preference of sialic acids recognized by HKU1-S protein. Nevertheless, for the first time, this study provided experimental evidence to support the idea that
O-Ac-Sia, by interacting with S1 of hCoV-HKU1, serves as an essential determinant for viral attachment during the early entry step and that HE possesses 9-
O-AC-esterase or even broader activity and primarily acts as a RDE for hCoV-HKU1 infection. hCoV-HKU1 is similar to BCoV and hCoV-OC43, employing its two surface proteins, S and HE, to complete the viral infection cycle in a concerted manner, with S protein mediating receptor binding and entry and HE protein mediating RDE activity late in the infection cycle to facilitate viral progeny release and achieve efficient virus dissemination (
23,
26).