Membrane Protein of Human Coronavirus NL63 Is Responsible for Interaction with the Adhesion Receptor

HCoV-NL63 VLPs composed of the M, E, N, and (optionally) S proteins were produced in insect cells, as previously described (22), with the modification that the N protein was also included. To evaluate protein expression, insect cells infected with bicistronic recombinant baculovirus (rBV) coding for M and E proteins (M+E) and monocistronic N rBV (and optionally monocistronic S rBV) were immunostained and examined under a confocal microscope. Colocalization of the M and N proteins was observed, suggesting the formation of a protein complex within the producer cells (Fig. 1A). To verify whether VLPs were effectively assembled and released, the culture medium harvested from insect cells expressing M, E, N, and S proteins was analyzed by Western blotting. The M (26-kDa), N (42-kDa), and S (150-kDa) proteins were detected in the secreted fraction when insect cells were coinfected with (M+E) rBV, N rBV, and, optionally, S rBV (Fig. 1B). In agreement with our previous studies, the M protein migrated as two bands, reflecting its two different glycosylation states. Similarly, two bands of the N protein were observed, upon electrophoresis, resulting from protein degradation under denaturing conditions (23). In contrast, the apparent molecular weight of the S protein band was higher than expected, most likely because of the glycosylation and/or incomplete denaturation of the protein trimer. Both VLPs, composed of M, E, and N (MEN) and M, E, N, and S (MENS) proteins, were concentrated and purified using a heparin column according to a previously developed protocol (22) (Fig. 1C). The analysis of purified samples by transmission electron microscopy (TEM) showed spherical particles of different diameters, which might reflect the amount of the N protein incorporated into VLPs (Fig. 1D).

Previously, we have shown that HCoV-NL63 VLPs composed of the M, E, and S proteins penetrate LLC-Mk2 cells, which are naturally permissive to HCoV-NL63 infection, and that this process is ACE2 dependent. We have also observed that VLPs composed of M and E proteins adhere to but do not enter the target cell (data not shown). In the present study, we evaluated the binding and endocytosis of MEN and MENS HCoV-NL63 VLPs.

LLC-Mk2 cells were incubated with the purified MEN and MENS VLPs, and we found that MEN VLPs localize to the cell surface, whereas MENS VLPs were present on the cell surface and in the cytoplasm (Fig. 2). This confirmed that the S protein is required for particle internalization but dispensable for VLP adhesion. To further validate this result, we blocked the virus-ACE2 interaction with anti-ACE2 antibody, which resulted in inhibition of MENS VLP internalization but not adhesion to the cell surface (Fig. 3). Interestingly, for MENS VLPs, we observed a higher number of viral particles on cells preincubated with anti-ACE2 antibodies. We believe that this is an artifact, as during entry, VLPs fuse with cellular membranes, which results in diffusion of the fluorescence signal and a decrease of the score. Blocking of the interaction with the cellular receptor hampers VLP-cell fusion, and the scored number of particles remains high. Adhesion of MEN VLPs to cells was not affected by inhibition of the VLP-ACE2 interaction. Altogether, these observations indicate that S protein of HCoV-NL63 is not involved in virus attachment to target cells.

Using flow cytometry, we next investigated whether the adhesion of MEN and MENS VLPs to the cell surface involves interaction with HSPGs, as has been shown for HCoV-NL63. For this, purified VLPs were preincubated with soluble HS and used to inoculate LLC-Mk2 cells. Identical samples were prepared using untreated VLPs. The cells were then immunostained to label the VLPs, and the signal was recorded. Native, iodixanol-concentrated HCoV-NL63 was used as a control. We observed that both MEN and MENS VLPs bound to the cell surface in the absence of HS and that HS pretreatment of VLPs hampered adhesion (Fig. 4).

MEN VLPs contain the N protein encapsulated in an envelope formed by the M and E proteins. We have a priori assumed that the M protein serves as a partner for cellular HSPG during viral or VLP adhesion, and we verified this hypothesis experimentally. First, an array of synthetic, overlapping peptides covering the complete M protein was used to test binding with the labeled heparin. The M protein regions potentially interacting with heparin were hence identified as amino acids (aa) 25 to 51, aa 93 to 119, and aa 153 to 207 (Fig. 5). Most of the peptides are arginine and lysine rich, which is in agreement with previously reported data (24, 25).

Next, we predicted the M protein topology in silico, and based on these data and previous reports, we concluded that regions spanning aa 25 to 51 and aa 93 to 119 may not be involved in the interactions with HPSGs, as they are localized inside the virion or in transmembrane domains (TMDs) (Fig. 6). This led us to the conclusion that the HSPG binding site is localized in the C-terminal region of aa 153 to 226, predicted to be exposed on the virion surface.

To further evaluate the interaction between the C-terminal domain of the M protein and HSPGs, we expressed and purified a His-tagged fragment of the M protein consisting of amino acids 153 to 226 (6×His-M_153–226) in bacteria. To investigate whether the protein adhered to cellular HSPG, we performed an in situ enzyme-linked immunosorbent assay (ELISA), with different amounts of the protein added to cells seeded in a microplate. The 6×His-M_153–226 protein and an equal molar amount of 6×His-N HKU1, as a control, were serially diluted and incubated with LLC-Mk2 cells. A signal developed using an anti-His-horseradish peroxidase (HRP) antibody was detected only with the 6×His-M_153–226 protein and was proportional to its concentration (Fig. 7A). Importantly, when the 6×His-M_153–226 protein was preincubated with soluble HS, the interaction with LLC-Mk2 cells was diminished (Fig. 7B), suggesting that the putative epitope is localized within the region from aa 153 to 226 of the M protein.

To further validate the notion that HCoV-NL63 employs the M protein to bind to the cell, we performed a “pseudoneutralization” assay using a polyclonal anti-M rabbit serum, obtained after immunizing rabbits with peptides corresponding to aa 181 to 195 and aa 211 to 226 of the M protein. Purified MEN and MENS VLPs and HCoV-NL63 were preincubated with the serum and used to inoculate LLC-Mk2 cells. Adhesion of VLPs or HCoV-NL63 to cells was evaluated by confocal microscopy. As shown in Fig. 8, VLPs and HCoV-NL63 adhere to the cells in the control sample, but this interaction was impaired in the presence of the anti-M serum.

Sf9 (Spodoptera frugiperda; ATCC CRL-1711) and HF (High Five) (Trichoplusia ni; ATCC CRL-7701) cells were cultured in ESF medium (Expression Systems, CA, USA) supplemented with 2% FBS (fetal bovine serum; Thermo Fisher Scientific, Poland), 100 μg/ml streptomycin, 100 IU/ml penicillin, 10 μg/ml gentamicin, and 0.25 μg/ml amphotericin B. The culture was maintained in a humidified incubator at 27°C. Sf9 cells were used for baculovirus (BV) generation and amplification, while HF cells were used for recombinant protein expression.

LLC-Mk2 cells (Macaca mulatta kidney epithelial cells; ATCC CCL-7) were maintained in minimal essential medium (MEM) (2 parts Hanks’ MEM and 1 part Earle’s MEM; Thermo Fisher Scientific, Poland) supplemented with 3% FBS, 100 μg/ml streptomycin, 100 IU/ml penicillin, and 5 μg/ml ciprofloxacin. The culture was maintained at 37°C under 5% CO₂.

VLPs composed of membrane (M), envelope (E), and spike (S) proteins of HCoV-NL63 were produced as described previously (22), but nucleocapsid (N) protein was additionally included for this work. For this, the N gene was subcloned from pET Duet (23) to pFastBac Dual, under the control of the polyhedrin promoter. Recombinant baculoviruses (rBVs) were generated using the Bac-to-Bac system (Thermo Fisher Scientific, Poland) and titrated using a plaque assay. Subsequently, HF cells were coinfected with (M+E) bicistronic rBV, N monocistronic rBV, and S monocistronic rBV at a multiplicity of infection (MOI) of 4 and cultured for 72 h. Secreted VLPs were then harvested by centrifugation (5,000 × g for 30 min).

For purification of VLPs, the harvested culture medium was diluted (1:1) with binding buffer (20 mM K₂HPO₄-KH₂PO₄ [pH 6.2], 70 mM NaCl) and loaded onto a 5-ml heparin HT column (GE Healthcare, Poland) using an Äkta fast-performance liquid chromatography (FPLC) system (Äkta, Sweden). Before purification, the column was equilibrated with binding buffer. Proteins were eluted with a linear NaCl gradient (50 mM to 2 M NaCl in binding buffer), and collected peak fractions were analyzed using SDS-PAGE and Western blotting.

Insect cells or culture media were harvested, resuspended in denaturing buffer to final concentrations of 1.5% SDS and 2.5% β-mercaptoethanol, and resolved by Laemmli SDS-PAGE using 4-to-20% gradient precast gels (Bio-Rad, Poland). A PageRuler Prestained Plus protein ladder (Thermo Fisher Scientific, Poland) was used in this study as a protein size marker. Gels were stained with Coomassie brilliant blue or subjected to electrotransfer in buffer containing 25 mM Tris, 192 mM glycine, and 20% methanol onto an activated polyvinylidene difluoride (PVDF) membrane. Following transfer, the membrane was blocked with 5% skim milk in Tris-buffered saline supplemented with 0.05% Tween 20 (TBS-T), followed by 1.5 h of incubation with rabbit polyclonal anti-M serum (1:15,000; kindly provided by Lia van der Hoek and generated by rabbit immunization with peptides spanning aa 180 to 195 and aa 212 to 226 of the M protein), mouse monoclonal anti-N antibody (1:1,000; Ingenansa, Spain), and mouse polyclonal anti-S serum (1:250; Eurogentec, Belgium) and 1 h of incubation with anti-rabbit (1:20,000; Dako, Denmark) and anti-mouse (1:20,000; Dako, Denmark) secondary antibodies conjugated with horseradish peroxidase (HRP), respectively. The signal was developed using the Immobilon Western chemiluminescent HRP substrate (Millipore, Poland) and visualized by exposing the membrane to an X-ray film (Thermo Fisher Scientific, Poland).

For assessment of protein colocalization in insect cells, HF cells were grown in 6-well culture plates on glass coverslips coated with 0.01% poly-l-ornithine (Sigma-Aldrich, Poland). Cells were infected with rBVs at an MOI of 1, fixed at 48 h postinfection with 4% formaldehyde, permeabilized with 0.2% Triton X-100 in phosphate-buffered saline (PBS), and blocked for 1 h, at room temperature with 5% bovine serum albumin (BSA) in PBS. Expression of M and N proteins was detected with rabbit polyclonal anti-M serum (the same as described above; 1:1,000) and mouse monoclonal anti-N antibody (the same as described above; 1:2,000), respectively, followed by detection with Alexa fluorophore secondary antibodies at a 1:400 dilution (Santa Cruz Biotechnology, USA). Cell nuclei were stained with DAPI (4′,6′-diamidino-2-phenylindole) (0.1 g/ml in PBS; Sigma-Aldrich, Poland). Coverslips were mounted on glass slides with Prolong diamond antifade mountant (Sigma-Aldrich, Poland).

For transduction and adhesion analyses of VLPs, LLC-Mk2 cells were grown to 80% confluence for 48 h in 6-well culture plates on glass coverslips. Cells were then washed with ice-cold PBS and inoculated with 1 ml of purified VLPs. Next, LLC-Mk2 cells were incubated for 2 h at 4°C and subsequently for 90 min at 32°C under 5% CO₂ and further washed three times with PBS. Subsequently, cells were fixed, permeabilized, and stained with anti-M polyclonal serum, as described above. Additionally, actin filaments were visualized with phalloidin conjugated with Alexa 647 (0.132 μM; Sigma-Aldrich, Poland).

To verify the role of the ACE2 protein during VLP entry, LLC-Mk2 cells were incubated with anti-ACE2 polyclonal antibodies (catalog number AF933; R&D Systems) or an appropriate isotype control antibody (catalog number GTX35039; GeneTex) for 1 h at 37°C (5 μg/ml). The anti-ACE2 antibody concentration was determined based on HCoV-NL63 neutralization experiments in LLC-Mk2 cells (not shown). Furthermore, cells were overlaid with purified MEN or MENS VLPs and incubated for 2 h at 4°C and subsequently for 90 min at 32°C under 5% CO₂ in the presence of antibodies. Next, cells were washed three times with PBS, fixed, permeabilized, and stained with anti-M polyclonal serum, as described above.

Fluorescent images were acquired using a Zeiss LSM 710 confocal microscope (Carl Zeiss Microscopy GmbH), deconvolved using AutoQuant X3 software, and processed in ImageJ Fiji (National Institutes of Health, Bethesda, MD, USA) (66).

The number of particles and number of cells were quantified using the built-in ImageJ Fiji tool “3D Objects Counter.” The numbers of internalized particles were counted manually from orthogonal views. For this study, the actin cortex was assumed to indicate the cell surface.

Samples were prepared as described previously (22). Briefly, purified VLPs were fixed in Karnovsky solution and loaded onto copper grids coated with a support film (Formvar 15/95E; Sigma-Aldrich, St. Louis, MO, USA). After drying, the material was stained with uranyl acetate (Polyscience, Inc., Warrington, PA, USA) and lead citrate (Sigma-Aldrich, St. Louis, MO, USA). Subsequently, the grids were washed with water and dried in air at room temperature. Ultrastructural observations were performed by using a Hitachi H500 transmission electron microscope at an accelerating voltage of 75 kV.

To evaluate adhesion of VLPs to LLC-Mk2 cells, cells were grown for 48 h to reach 100% confluence in 6-well culture plates on glass coverslips. Cells were washed twice with PBS and incubated with purified VLPs, iodixanol-concentrated HCoV-NL63 (12, 67), mock supplemented with heparan sulfate (HS) (1 mg/ml; Sigma-Aldrich, Poland), or PBS for 4 h at 4°C. The cells were then washed three times with PBS, fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS, and incubated overnight in 5% bovine serum albumin and 0.5% Tween 20 in PBS. To examine HCoV-NL63 or VLP adhesion, cells were incubated for 2 h at room temperature with a mouse monoclonal anti-N antibody (the same as described above; 1:1,000 in 2.5% BSA with 0.5% Tween 20 in PBS), followed by 1 h of incubation with an Alexa Fluor 488-labeled goat anti-mouse antibody (1:400). Cells were then washed, mechanically detached from the glass coverslips, resuspended in PBS, and analyzed by flow cytometry (FACSCalibur; Becton, Dickinson). Data were processed using CellQuest software (Becton, Dickinson) and FlowJo V10.

The region coding for the C-terminal fragment (aa 153 to 226) of the M protein was PCR amplified (5′ primer CCA gga tcc gGA TGG CCA TAA GAT TGC TAC TCG TG and 3′ primer GCA ctc gag TTA GAT TAA ATG AAG CAA CTT), digested with BamHI and XhoI enzymes (Thermo Fisher Scientific, Poland), gel purified, and cloned into the pET Duet plasmid in a manner to include the 6×His tag in frame at the N terminus. The sequence (6×His-M_153–226) was verified by sequencing. The Escherichia coli BL21 strain was transformed with the recombinant plasmid and precultured overnight at 37°C. LB medium (1 liter; BioShop-LabEmpire, Poland) was inoculated with the preculture, induced with isopropyl-β-d-thiogalactopyranoside (IPTG) (0.5 mM; BioShop-LabEmpire, Poland) at an optical density of 0.6, and harvested at 4 h postinduction. Bacterial cell pellets were subsequently resuspended in 50 ml of lysis buffer (50 mM Tris [pH 7.5], 5 mM urea, 250 mM NaCl, 5 mM dithiothreitol [DTT], 1 mM EDTA, 1% Triton X-100) and subjected to 2 cycles of Cell Disruptor (Constant Systems, UK) operation at 25 lb/in². Lysates were then centrifuged (40 min at 5,000 × g), and the supernatant was diluted (1:1) with immobilized-metal affinity chromatography (IMAC) binding buffer (20 mM K₂HPO₄-KH₂PO₄ [pH 7.4], 500 mM NaCl, 20 mM imidazole). Diluted supernatants were loaded onto a 1-ml IMAC column (GE Healthcare, Poland) charged with Ni²⁺ and connected to an Äkta FPLC system (Äkta, Sweden) and preequilibrated with binding buffer. Proteins were eluted with 500 mM imidazole in IMAC binding buffer. Collected peak fractions were pooled and fractionated again to remove imidazole and excess NaCl. For this purpose, a 26/10 desalting column (GE Healthcare, Poland) (preequilibrated with 20 mM Na₂HPO₄-NaH₂PO₄ [pH 7.7], 250 mM NaCl, and 5% glycerol) was used. Purified 6×His-M_153–226 protein was analyzed by SDS-PAGE and Western blotting.

LLC-Mk2 cells were seeded into a 96-well plate 48 h prior to the assay and incubated at 37°C under 5% CO₂. Purified 6×His-M_153–226 protein (initial concentration of 0.15 mg/ml) was 2-fold serially diluted with PBS and transferred to a plate with confluent LLC-Mk2 cells. As a control, 6×His-N HKU1 was prepared in the same manner and added to the cells at the same molar ratio. After 2 h of incubation at 32°C under 5% CO₂, the culture plate was inverted to remove unbound material, and cells were washed 4 times with PBS. Next, unspecific binding sites on the plate were blocked with 5% bovine serum albumin in PBS for 1 h, and subsequently, samples were incubated for 90 min with anti-His antibody conjugated with HRP (1:10,000; Sigma, Poland). The signal was visualized with the 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (OptiEIA; Becton, Dickinson, USA), and the reaction was stopped with 1 M HCl. The absorbance was measured at a wavelength (λ) of 450 nm using a Tecan Infinite 200 Pro microplate reader. All measurements were performed in triplicate, and the background signal (from control wells) was subtracted. Another in situ ELISA was performed to evaluate 6×His-M_153–226 protein binding to LLC-Mk2 cells in the presence or absence of soluble HS. Briefly, 6×His-M_153–226 protein was preincubated for 30 min at room temperature with HS (1 mg/ml in PBS) and transferred to a plate with confluent LLC-Mk2 cells. As a control, 6×His-M_153–226 (diluted in the same manner) was added to cells. After 2 h of incubation at 32°C under 5% CO₂, the culture plate was processed as described above.

A cellulose membrane displaying 54 immobilized peptides covering the whole membrane protein (15 amino acids each, with a sliding window with a 4-aa step) was designed and purchased from JPT Peptides GmbH (Germany). The cellulose membrane was activated in methanol for 5 min, washed three times with TBS-T, and blocked for 2 h with 2.5% skimmed milk (in TBS-T). Next, the membrane was incubated for 3 h with 10 ml of heparin conjugated with biotin (10 μg/ml in PBS; Sigma-Aldrich, Poland), washed 4 times in TBS-T, and incubated for 2 h with 5 ml of streptavidin-HRP (2 μg/ml in PBS; Sigma-Aldrich, Poland). Following extensive washing (4 times) with TBS-T, the signal was developed using the Immobilon Western chemiluminescent HRP substrate (Millipore, Poland) and visualized by exposing the membrane to an X-ray film (Thermo Fisher Scientific, Poland).

The TMHMM server, v. 2.0, for prediction of transmembrane helices in proteins (68) was used to evaluate M protein topology.

To verify if anti-M antibody inhibits binding to target cells, purified MEN and MENS VLPs and iodixanol-concentrated HCoV-NL63 were incubated for 1 h at room temperature with an equal volume of rabbit polyclonal anti-M serum at 1:10 or PBS. Next, samples were added to LLC-Mk2 cells seeded on glass coverslips 48 h earlier and grown to 80% confluence and washed with PBS directly before the experiment. After 4 h of incubation at 4°C, cells were washed three times with PBS, fixed, and stained with anti-N antibody for confocal microscopy analysis, as described above. As a control, an identical experiment was conducted with preimmune rabbit serum. Additionally, the effect of anti-M serum on HCoV-OC43 adhesion to HRT-18G cells was examined in a corresponding experiment.

The number of particles and number of cells were quantified using the ImageJ Fiji 3D Objects Counter tool.

All data were tested for normality using a Shapiro-Wilk test and a Mann-Whitney test (for pseudoneutralization assays), a Kruskal-Wallis test with Dunn’s multiple-comparison test (for the anti-ACE2 antibody effect on VLP adhesion to cells), or one-way analysis of variance (ANOVA) with a Dunnett multiple-comparison test (for the HS effect on VLPs adhesion to cells). P values of <0.05 were considered significant. All graphs were prepared using GraphPad Prism 7.

The nucleotide sequences of the N, M, E, and S genes appear in the GenBank database under accession numbers NC_005831.2, MH050812, MH050811, and MH050813, respectively.