Entry of Human Coronavirus NL63 into the Cell

KEYWORDS: HCoV-NL63, clathrin, Coronaviridae, coronavirus, endocytosis, entry, infection, internalization

HCoV-NL63 enters the cell via endocytosis.

We first determined whether entry of HCoV-NL63 requires endocytosis and acidification of endosomes. For this, we studied the effect of ammonium chloride (NH₄Cl) and bafilomycin A, lysosomotropic agents that inhibit acidification of endosomes (21–23), using two models of HCoV-NL63 infection: permissive LLC-Mk2 cells and HAE cultures. Cells were preincubated with NH₄Cl (50 mM), bafilomycin A (100 nM), or control dimethyl sulfoxide (DMSO) for 1 h at 37°C and subsequently incubated with the virus at a 50% tissue culture infective dose (TCID₅₀) of 100/ml for LLC-Mk2 cells or 400/ml for HAE for 2 h at 32°C in the presence of the inhibitor. Subsequently, supernatants were removed, and cells were washed thrice with acidic buffer to inhibit the fusogenic activity of the virions retained on the surface (24). Next, LLC-Mk2 cells were washed with 1× phosphate-buffered saline (PBS) (pH 7.4), overlaid with culture medium, and incubated at 32°C for 4 days. Supernatant samples were collected for virus replication analysis. Simultaneously, HAE cultures were washed with 1× PBS (pH 7.4) and further maintained at an air-liquid interphase at 32°C for 5 days. During this time, HAE cultures were washed every 24 h with 1× PBS supplemented with a given inhibitor for 10 min at 32°C, and apical washes were collected for virus replication analysis. Subsequently, viral RNA was isolated and reverse transcribed (RT), and the HCoV-NL63 yield was determined using a quantitative real-time PCR (qPCR).

Bafilomycin A and NH₄Cl inhibited HCoV-NL63 infection in LLC-Mk2 cells, proving that acidification is a requirement for the virus infection in vitro. No inhibition was observed in HAE cultures (Fig. 1A). No cytotoxic effect was observed in the presence of these inhibitors (Fig. 1B).

Next, we analyzed HCoV-NL63 colocalization with early endosome antigen 1 (EEA1), a hydrophilic protein localizing exclusively to early endosomes (25). LLC-Mk2 cells were fixed after 10, 20, 30, or 40 min postinoculation (p.i.) with gradient-purified virus, stained with antibodies specific to HCoV-NL63 N protein and EEA1, and analyzed under a confocal microscope. Measured colocalization, expressed as Manders' coefficient, increases with time and reaches 0.68 at 40 min p.i. (n = 6 cells) (Fig. 1C).

We validated the obtained results using the HAE model. Briefly, HAE cultures were inoculated with gradient-purified HCoV-NL63 and incubated at 32°C for 2 h. For this culture model, a longer incubation was required to observe virus attachment and entry, most likely due to the requirement to cross the mucus layer. Subsequently, cells were fixed and labeled with specific antibodies against HCoV-NL63 N protein and EEA1. Colocalization of HCoV-NL63 virus particles with EEA1 protein was analyzed using a confocal microscope. Colocalization of virus and EEA1 was observed in inoculated cells (Fig. 1D).

Endocytosis of virus particles is induced by binding to the entry receptor.

HCoV-NL63 virus employs the ACE2 protein for cellular entry, while heparan sulfate proteoglycans serve as attachment receptors (19). Here, we analyzed the consequence of interaction between the virus particle and ACE2. First, we inoculated naturally permissive LLC-Mk2 cells with HCoV-NL63 and incubated them for 40 min at 4°C to enable virus adhesion to a cell surface. Subsequently cells were fixed, the virus was labeled with specific antibodies, and its colocalization with ACE2 and clathrin was studied. As shown in Fig. 2A, HCoV-NL63 particles attach efficiently to the cell surface. However, only a proportion of virions colocalize with the ACE2 (Manders' coefficient = 0.573; n = 5), suggesting that binding to the heparan sulfate precedes interaction with the entry receptor. At that point, there is no colocalization of virus particles and clathrin-coated pits (Manders' coefficient = 0.140; n = 5) (Fig. 2B). Next, we tested whether the virus binding to the adhesion or entry receptor triggers recruitment of common cellular proteins responsible for pit formation by incubating cells for 5 min at 32°C. Immunostaining showed that the virus particles bound to ACE2 start to colocalize with clathrin (Manders' coefficient = 0.849; n = 6) (Fig. 2C), while there is no colocalization between non-ACE2-bound virions and clathrin (Manders' coefficient = 0.189; n = 6).

HCoV-NL63 colocalizes with clathrin during entry.

To determine whether colocalization with clathrin following the ACE2 binding is relevant and the virus indeed enters the cell by use of clathrin-coated pits, we analyzed colocalization of intracellular virions with clathrin. Briefly, LLC-Mk2 cells were incubated at 32°C for 5 to 20 min with gradient-purified HCoV-NL63, fixed, immunostained, and analyzed by confocal microscopy. The results showed colocalization of virions entering the cell with clathrin (Manders' coefficient = 0.584; n = 7) (Fig. 3A), whereas no colocalization with caveolin 1 was observed (Manders' coefficient = 0.053; n = 5) (Fig. 3B). HCoV-NL63 colocalization with clathrin and caveolin was also studied in the HAE model. For this, cultures were incubated with gradient-purified HCoV-NL63 at 32°C for 2 h; the virus and the cellular proteins were immunostained and analyzed by confocal microscopy. HCoV-NL63 virions also colocalized with clathrin in this model, whereas no colocalization was observed for caveolin 1 (Fig. 3).

Clathrin and dynamin are important for HCoV-NL63 entry.

As we already knew that HCoV-NL63 virions migrate to clathrin-coated pits, in the subsequent step we aimed to determine whether the clathrin-mediated endocytosis is indeed important for the virus entry. For this reason, we blocked the pathway using Pitstop 2 {N-[5-(4-bromobenzylidene)-4-oxo-4,5-dihydro-1,3-thiazol-2-yl]naphthalene-1-sulfonamide}, a selective clathrin inhibitor targeting its amino-terminal domain, and tetradecyltrimethylammonium bromide (MiTMAB), a dynamin I and II GTPase inhibitor. Activity of these compounds was verified with the positive control, fluorescently labeled transferrin (26, 27). LLC-Mk2 cells were treated with Pitstop 2, MiTMAB, or control DMSO for 30 min at 37°C following transferrin uptake for 45 min at 37°C. Confocal images showed that both inhibitors blocked transferrin endocytosis, as the protein was present only on the cell surface (Fig. 4A to D).

Subsequently, LLC-Mk2 cells were incubated with one of the inhibitors at 37°C for 30 min and inoculated with gradient-purified HCoV-NL63 at 32°C for 5 min. Following immunostaining of the HCoV-NL63 N protein and actin, virus endocytosis was analyzed using confocal microscopy. The results showed that virus internalization was hampered in cells pretreated with clathrin and dynamin inhibitors compared to the DMSO-treated cells (Fig. 4D to G). Simultaneously, a cytotoxicity test of the entry inhibitors was performed, which showed no toxic effect of the tested compounds on LLC-Mk2 cells (Fig. 5). In order to ensure that our observations were not biased, statistical analysis of virus entry was performed. For this, an algorithm was prepared for image analysis, a 3D representation of the cell was prepared, and the virus position in the cell was determined (Fig. 6).

A similar experiment was conducted using HAE cultures. For this, cultures were incubated for 1 h at 37°C with the inhibitors described above following incubation with gradient-purified HCoV-NL63 at 32°C for 2 h. A strong inhibition of virus internalization in cultures preincubated with clathrin or dynamin inhibitors compared to control cells was observed (Fig. 7). No cytotoxicity to HAE was observed for the tested inhibitors after 3 h of incubation at 37°C (Fig. 8).

Clathrin-mediated endocytosis is the main entry route for HCoV-NL63.

Even though certain cargo is usually internalized by a single route, frequently other pathways may be used as alternatives. We therefore aimed to test whether inhibition of clathrin-mediated entry with chemical inhibitors results in inhibition of virus replication. To address this, we incubated LLC-Mk2 cells with a given inhibitor at 37°C for 1 h and infected them with HCoV-NL63 (TCID₅₀ = 400 per ml) for 2 h at 32°C. Subsequently, media were removed and cells were washed thrice with acidic buffer following washing with 1× PBS (pH 7.4). Next, cells were overlaid with culture medium containing a given inhibitor and incubated at 32°C for 4 days. Cells were fixed and immunostained for HCoV-NL63 N protein to assess the number of infected cells. To assess the nonspecific effect of entry inhibitors, control cells were also treated with these at 4 h p.i. Clearly, in the presence of clathrin-mediated endocytosis inhibitors (Pitstop 2 and MiTMAB), the number of HCoV-NL63-infected cells was much lower than in the control. However, MiTMAB also inhibited virus replication at later stages of the infection (Fig. 9). To ensure that entry inhibitors affected HCoV-NL63 infection in LLC-Mk2 cells, we analyzed by RT-qPCR virus replication at 120 h p.i. in the presence of the tested compounds. The analysis showed an ∼2-log decrease in virus progeny production in the presence of Pitstop 2 and MiTMAB compared to that in DMSO-treated cells and a slight increase of RNA copy levels in the presence of nystatin (Fig. 10A). Importantly, no cytotoxic effect was observed for the tested inhibitors applied to LLC-Mk2 cells for 4 days at 32°C (Fig. 10B). The influence of the tested inhibitors on HCoV-NL63 infection was also analyzed in HAE cultures. For this, cultures were preincubated with a given inhibitor (Pitstop 2, MiTMAB, nystatin, or control DMSO) for 1 h at 37°C and infected with HCoV-NL63 at a TCID₅₀ of 400 per ml for 2 h at 32°C. Subsequently, noninternalized virions were inactivated by acid washing, and cultures were washed with 1× PBS and incubated with a given inhibitor for 10 min. After that time, supernatants were discarded and cultures were incubated for 5 days at 32°C. During this period, cultures were incubated with a given inhibitor for 10 min at 32°C every 24 h. Viral RNA from these samples was quantified by RT-qPCR. Virus replication in HAE was not affected by any of the tested inhibitors (Fig. 10A).

TMPRSS2 is important during early stages of the infection.

It was previously suggested that coronaviruses may bypass the endocytic entry route employing transmembrane protease serine 2 (TMPRSS2), which primes the fusion protein and enables fusion of viral and cellular membranes on the cell surface (28, 29). We have tested whether inhibition of TMPRSS2 with camostat affects the HCoV-NL63 infection. We observed that inhibition of TMPRSS2 hampers virus infection in HAE cultures, while it has no effect on virus replication in LLC-MK2 cells (Fig. 11A). No inhibition of virus entry was observed in any of the models, as tracked with confocal microscopy visualizing the nucleoprotein (Fig. 11B). As only single entry events per view were observed, several images for camostat-treated and control cells are presented. In total, 500 entry events into HAE cells were tracked, and no difference between the camostat-treated sample and the control sample was noted.

HCoV-NL63 entry requires actin remodeling.

We studied trafficking of HCoV-NL63 inside the cell. As entry by endocytosis would probably require remodeling of the cytoskeleton, we evaluated virus internalization in the presence of cytochalasin D, jasplakinolide, or nocodazole. The first chemical inhibits actin polymerization, whereas the second binds F-actin and stabilizes actin filaments (30, 31). The last compound interferes with microtubule formation. The analysis showed that actin inhibitors prevented virus particles from penetrating the cell, with visible viral particle accumulation on actin cortex or unstructured actin deposits. The microtubule inhibitor did not affect virus entry (Fig. 12). No cytotoxicity was observed for the tested inhibitors (Fig. 13).

Cell culture.

LLC-Mk2 cells (ATCC CCL-7; Macaca mulatta kidney epithelial) were maintained in minimal essential medium (MEM) (two parts Hanks' MEM and one part Earle's MEM; Thermo Fisher Scientific, Poland) supplemented with 3% heat-inactivated fetal bovine serum (Thermo Fisher Scientific, Poland), penicillin (100 U/ml), streptomycin (100 μg/ml), and ciprofloxacin (5 μg/ml). Cells were cultured at 37°C under 5% CO₂.

Ethics statement.

Human tracheobronchial epithelial cells were obtained from airway specimens resected from adult patients undergoing surgery under Silesian Center for Heart Diseases-approved protocols. This study was approved by the Bioethical Committee of the Medical University of Silesia in Katowice, Poland (approval no. KNW/0022/KB1/17/10 dated 16 February 2010). Participants provided their written informed consent to participate in the study, as approved by the Bioethical Committee.

HAE cultures.

Primary human tracheobronchial epithelial cells were expanded on plastic to generate passage 1 cells and plated on permeable Transwell insert (6.5-mm-diameter) supports. Human airway epithelium (HAE) cultures were generated by provision of an air-liquid interface for 6 to 8 weeks to form well-differentiated, polarized cultures that resemble in vivo pseudostratified mucociliary epithelium. Cultures were prepared and maintained as previously described (24).

Cell viability assay.

LLC-Mk2 cells were cultured on 96-well plates, and HAE cultures were prepared as described above. Cell viability assay was performed by using the 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide salt (XTT) cell viability assay (Biological Industries, Israel) according to the manufacturer's instructions. Briefly, on the day of the assay, 100 μl of the culture medium (for LLC-Mk2) or 1× PBS (for HAE) with 30 μl of the activated XTT solution was added to each well/culture insert. Following 2 h of incubation at 37°C, the solution was transferred onto a 96-well plate and the signal was measured at λ = 490 nm using a colorimeter (Spectra Max 250; Molecular Devices). The results obtained were further normalized to the control sample, for which cell viability was set to 100%.

Virus preparation and titration.

The HCoV-NL63 stock (isolate Amsterdam 1) was generated by infecting monolayers of LLC-Mk2 cells. The virus-containing liquid was aliquoted and stored at −80°C. A control LLC-Mk2 cell lysate from mock-infected cells was prepared in the same manner. The virus yield was assessed by titration on fully confluent LLC-Mk2 cells in 96-well plates according to the method described by Reed and Muench (37).

Purification of HCoV-NL63.

The virus stock was concentrated 25-fold using centrifugal protein concentrators (Amicon Ultra, 10-kDa cutoff; Merck, Poland) and subsequently overlaid on 15% iodixanol solution in 1× PBS (OptiPrep medium; Sigma-Aldrich, Poland). Following virus concentration using an iodixanol cushion (centrifugation at 175,000 × g for 3 h at 4°C), it was overlaid on a 10 to 20% iodixanol gradient in 1× PBS and centrifuged at 175,000 × g for 18 h at 4°C. Fractions (1 ml) collected from the gradient were analyzed by Western blotting using anti-HCoV-NL63 N IgGs (0.25 μg/ml; Ingenansa, Spain) and a secondary antibody coupled with horseradish peroxidase (65 ng/ml; Dako, Denmark). The virus-containing fractions were aliquoted and stored at −80°C. The control cell lysate (mock) was concentrated and prepared in the same manner as the virus stock.

Inhibition of virus entry.

LLC-Mk2 cells were seeded on coverslips in six-well plates (TPP, Switzerland) and cultured for 2 days at 37°C. Subsequently, cells were incubated with a given inhibitor for 30 min at 37°C and later with 50 μl of purified HCoV-NL63 or mock sample for 1 h at 32°C. For the ex vivo experiment, HAE cultures were exposed to the tested inhibitor or control PBS for 1 h at 37°C following inoculation with iodixanol-concentrated HCoV-NL63 or mock sample. Following 2 h of incubation at 32°C, unbound virions were removed by washing with 1× PBS. Cells were then washed with 1× PBS and fixed with 4% paraformaldehyde (PFA).

Transferrin and albumin were used as positive controls, as they were previously described to serve as a cargo in clathrin- and caveolin-dependent endocytosis, respectively (38, 39). LLC-Mk2 cells were seeded on coverslips in six-well plates (TPP, Switzerland) and cultured for 2 days at 37°C. Subsequently, cells were incubated with a given inhibitor for 30 min at 37°C following incubation with Alexa Fluor 488-labeled transferrin (100 μg/ml; Molecular Probes), fluorescein isothiocyanate (FITC)-labeled albumin (500 μg/ml; Sigma-Aldrich, Poland), or control PBS for 45 min at 32°C. Cells were then washed with 1× PBS and fixed in 4% PFA.

Immunostaining and confocal imaging.

Fixed cells were permeabilized with 0.1% Triton X-100 in 1× PBS and incubated overnight at 4°C in 1× PBS supplemented with 5% bovine serum albumin (BSA) and 0.5% Tween 20. To visualize HCoV-NL63 particles, cells were incubated for 2 h at room temperature with mouse anti-HCoV-NL63 N IgGs (0.25 μg/ml; Ingenansa, Spain), followed by 1 h of incubation with Alexa Fluor 488-labeled goat anti-mouse IgG (2.5 μg/ml; Thermo Fisher Scientific, Poland). The following antibodies were used for endosomal markers: polyclonal goat anti-human clathrin HC coupled with tetramethylrhodamine (10 μg/ml; Santa Cruz Biotechnology), polyclonal rabbit anti-human early endosome antigen 1 (2 μg/ml; Santa Cruz Biotechnology), polyclonal rabbit anti-human caveolin 1 (2 μg/ml; Sigma-Aldrich, Poland), and Alexa Fluor 633-labeled goat anti-rabbit (2.5 μg/ml; Thermo Fisher Scientific, Poland). Actin filaments was stained using phalloidin coupled with Alexa Fluor 633 (0.2 U/ml; Thermo Fisher Scientific, Poland). Nuclear DNA was stained with DAPI (4′,6′-diamidino-2-phenylindole) (0.1 μg/ml; Sigma-Aldrich, Poland). Immunostained cultures were mounted on glass slides in ProLong Gold antifade medium (Thermo Fisher Scientific, Poland). Fluorescent images were acquired under a Leica TCS SP5 II confocal microscope (Leica Microsystems GmbH, Mannheim, Germany) and a Zeiss LSM 710 confocal microscope (Carl Zeiss Microscopy GmbH). Images were acquired using Leica Application Suite Advanced Fluorescence LAS AF v. 2.2.1 (Leica Microsystems CMS GmbH) or ZEN 2012 SP1 software (Carl Zeiss Microscopy GmbH) deconvolved with Huygens Essential package version 4.4 (Scientific Volume Imaging B.V., The Netherlands) and processed using ImageJ 1.47v (National Institutes of Health, Bethesda, MD, USA).

Flow cytometry.

LLC-Mk2 cells were seeded on 6-well plates (TPP) and cultured for 2 days at 37°C with 5% CO₂. Cells in monolayer were incubated with each entry inhibitor for 1 h at 37°C following infection with HCoV-NL63 at a TCID₅₀ of 100/ml or inoculation of the mock sample. On day 4 p.i., cells were washed with sterile PBS, fixed with 3% PFA, permeabilized with 0.1% Triton X-100 in 1× PBS, and incubated for 1 h with 3% BSA in 1× PBS with 0.1% Tween 20. To quantify HCoV-NL63 infection, fixed cells were scraped from the plastic and incubated for 2 h at room temperature with mouse anti-HCoV-NL63 N IgG antibodies (1 μg/ml; Ingenansa), followed by 1 h of incubation with Alexa Fluor 488-labeled goat anti-mouse antibody (2.5 μg/ml; Molecular Probes). Cells were then washed, resuspended in 1× PBS, and analyzed with a FACSCalibur instrument (Becton Dickinson) using Cell Quest software.

Isolation of nucleic acids and reverse transcription.

Viral nucleic acids were isolated from cell culture supernatants (LLC-Mk2 cells) or apical washes (HAE cultures) using the viral RNA/DNA isolation kit (A&A Biotechnology, Poland) according to the manufacturer's instructions. Reverse transcription was carried out with a high-capacity cDNA reverse transcription kit (Thermo Fisher Scientific, Poland), according to the manufacturer's instructions.

RT-qPCR.

The HCoV-NL63 yield was determined by RT-qPCR (7500 Fast Real-Time PCR machine; Life Technologies, Poland). Viral cDNA (2.5 μl per sample) was amplified in a 10-μl reaction mixture containing 1× master mix (RT Mix Probe; A&A Biotechnology, Poland), a specific probe labeled with 6-carboxyfluorescein (FAM) and 6-carboxytetramethylrhodamine (TAMRA) (100 nM) (5′-ATG TTA TTC AGT GCT TTG GTC CTC GTG AT-3′), and primers (450 nM each) (sense, 5′-CTG TGG AAA ACC TTT GGC ATC-3′; antisense, 5′-CTG TGG AAA ACC TTT GGC ATC-3′). Rox was used as the reference dye. The reaction conditions were as follows: 2 min at 50°C and 10 min at 92°C, followed by 40 cycles of 15 s at 92°C and 1 min at 60°C. In order to assess the copy number for the N gene, DNA standards were prepared. Briefly, the N gene of HCoV-NL63 was amplified and cloned into pTZ57R/T (Thermo Fisher Scientific, Poland) plasmid using the InsTAclone PCR cloning kit (Thermo Fisher Scientific, Poland). Subsequently, DNA vectors were amplified and linearized with EcoRI restriction enzyme. Linear nucleic acids were further purified with the GeneJET PCR purification kit (Thermo Fisher Scientific, Poland) according to the manufacturer's instructions, and its concentration was assessed using a spectrophotometer. The number of DNA copies per milliliter was assessed using Avogadro's constant and the molecular mass of RNA molecules. Samples were serially diluted and used as an input for real-time PCR.

In this article, the data from quantitative PCR are presented as log removal values (LRVs) in order to enable comparison of results obtained from different assays. The LRV was calculated according to the formula LRV = −log (c_i/c₀), where c_i is the number of viral RNA copies per milliliter in the sample from the culture treated with a given polymer and c₀ is the number of viral RNA copies per milliliter in the control sample (untreated cells).

Image analysis.

To evaluate the infection inhibition in the presence of various endocytosis inhibitors, image analysis was performed on 2-mm by 2-mm tile scan images. On each image, the number of nuclei (expressed as a number of cells) and the mean pixel intensity for the virus were calculated. For that, histograms of all images were adjusted to the minimum/maximum value, excluding signal from the virus derived from images with no infected cells. Results are presented as mean intensity of fluorescence per cell.

Colocalization analyses were performed under ImageJ using the JACoP plugin (41), where Manders' coefficient was calculated for 3D images of more than 5 cells.

Quantitative analysis of virus internalization in the presence of inhibitors was performed with the algorithm previously described by Berniak et al. with modifications (40). The cell surface was estimated on each image slice manually using the polygon selection tool in ImageJ, and based on this information, the 3D cell surface was modeled. Coordinates of virus particles were determined using the 3D Object Counter ImageJ plugin. The relative localization and distance between the virus particle and cell surface were calculated. Results are presented as a ratio between virus particles inside a cell and the particles on the surface (up to 1.5 μm above).

Statistical analysis.

All the experiments were performed in triplicate, and the results are presented as mean ± standard deviation (SD). To determine the significance of the obtained results, a comparison between groups was conducted using the Student t test. P values of <0.05 were considered significant.

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