ACE2 Receptor Expression and Severe Acute Respiratory Syndrome Coronavirus Infection Depend on Differentiation of Human Airway Epithelia

Epithelial cell culture.Primary cultures of human airway epithelia were prepared from trachea or bronchi cells and grown at the air-liquid interface (ALI) on collagen-coated porous filters as described previously (17). In select experiments, the apical surface of primary cells was submerged under 500 μl of cell culture medium for 7 days to induce dedifferentiation or grown in nonpolarized fashion on tissue culture plastic. This study was approved by the Institutional Review Board at the University of Iowa. A549 cells (ATCC CCL-185) were maintained in Dulbecco's modified Eagle's medium/F12 (Gibco) containing 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 mg/ml).

Immunoblot analysis.Cells or tissues were lysed in 0.1% Triton X-100 in phosphate-buffered saline (PBS), and total protein was separated on a sodium dodecyl sulfate-7.5% polyacrylamide gel electrophoresis gel and transferred to polyvinylidene difluoride membrane. Goat anti-ACE2 polyclonal primary antibody (catalog no. AF933, R & D Systems, Minneapolis, MN), horseradish peroxidase-conjugated donkey anti-goat secondary antibody, mouse anti-foxj1 monoclonal primary antibody (gift of S. Brody, Washington University), and a horseradish peroxidase-conjugated goat anti-mouse secondary antibody were used. Primary antibody binding was visualized using SuperSignal chemiluminescent substrate (Pierce, Rockford, IL).

Immunofluorescence staining.Airway epithelia were fixed in 4% paraformaldehyde for 5 min at room temperature and washed with PBS. Five percent bovine serum albumin in PBS was used to block nonspecific antibody binding. An anti-ACE2 monoclonal antibody (MAB933; R & D Systems, Minneapolis, MN) was applied to cells (both apical and basolateral surfaces for air-liquid interface-cultured cells). Epithelia were then incubated at 37°C for 1 h and again washed with PBS. Rabbit anti-mouse fluorescein isothiocyanate-conjugated secondary antibody (F-4143; Sigma, St. Louis, MO) was added to cells and incubated at 4°C overnight. Cells were then washed with PBS, and a Cy3-labeled mouse anti-β-tubulin IV monoclonal antibody (C-4585; Sigma) was added. Cells were incubated at 37°C for 1 h, followed by PBS washes, and then mounted with DAPI (4′,6′-diamidino-2-phenylindole) VectaShield (Vector Labs, Burlingame, CA). Nuclei were stained with To-pro-3 (T3605; Sigma, St. Louis, MO). The SARS-CoV nsp1 protein was localized in cells fixed with 100% methanol using a rabbit polyclonal anti-nsp1 primary antibody (gift of M. Denison) (33) and a mouse anti-rabbit fluorescein isothiocyanate-conjugated secondary antibody (catalog no. A11088, Molecular Probes, Eugene, OR).

Surface biotinylation.The apical or basolateral surfaces of airway epithelia were treated with 1 mg/ml N-hydroxysulfosuccinimidobiotin (Pierce, Rockford, IL) in PBS for 30 min at 25°C. The epithelial surface was washed and then incubated with 100 mM glycine in PBS for 20 min at 25°C to quench unreacted biotin. Epithelial protein lysates were prepared by sonication in lysis buffer, and biotinylated proteins were precipitated using neutravidin covalently linked to immobilized diaminodipropylamine (Pierce). Biotinylated proteins were released in 8% sodium dodecyl sulfate-containing loading buffer, boiled, and analyzed by immunoblotting for ACE2. ErB2 was detected as a control basolateral membrane marker (43) using rabbit polyclonal anti-c-erbB2 antibody (Dako Corporation, Carpinteria, CA).

Scanning electron microscopy (SEM).Epithelia were fixed in 2.5% glutaraldehyde in Na cacodylate buffer for 30 min and rinsed with 0.1 M Na cacodylate buffer three times. Samples were postfixed in 1% OsO₄ for 1 h and sequentially rinsed with 0.1 M Na cacodylate buffer and H₂O. Samples were then serially dehydrated using 25% to 100% ethanol. After critical-point drying, samples were mounted on stubs, sputter coated, and examined using a Hitachi F-4000 electron microscope.

Real-time reverse transcription-PCR mRNA analysis.Total cellular RNA was isolated using TRI-Reagent (MRC, Cincinnati, OH) or a commercial spin column isolation kit (Stratagene, La Jolla, CA), and 1 μg was reverse transcribed using a reverse transcription (RT)-PCR kit (Ambion, Austin, TX). An aliquot of cDNA was subjected to PCR using an iCycler iQ fluorescence thermocycler (Bio-Rad, Hercules, CA) with SYBR green I DNA dye (Molecular Probes, Eugene, OR) and Platinum Taq DNA polymerase (iQ SYBR green Supermix kit, Bio-Rad). PCR conditions included denaturation at 95°C for 3 min and then for 35 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s, followed by 5 min at 72°C for elongation. The following primers were used: (i) human ACE2 forward 5′-GGACCCAGGAAATGTTCAGA-3′ and reverse 5′-GGCTGCAGAAAGTGACATGA-3′, (ii) SARS-CoV N gene forward/leader 5′-ATATTAGGTTTTTACCCAGG-3′and reverse 5′-CTTGCCCCATTGCGTCCTCC-3′, (iii) SARS-CoV S gene forward/leader 5′-ATATTAGGTTTTTACCCAGG-3′ and reverse 5′-CTCCTGAGGGAACAACCCTG-3′, and (iv) human hypoxanthine phosphoribosyltransferase (HPRT) forward 5′-CCTCATGGACTGATTATGGAC-3′and reverse 5′-CAGATTCAACTTGCGCTCATC-3′. Fluorescence data was captured during the 10-s dwell to ensure that primer dimers were not contributing to the fluorescence signal, and specificity of the amplification was confirmed using melting curve analysis. Data was collected and recorded by iCycler iQ software (Bio-Rad) and initially determined as a function of threshold cycle (C_T). Levels of mRNA were expressed relative to control HPRT levels, which were calculated as 2^ΔCT. In some samples, PCR products were visualized on 2% agarose gels with ethidium bromide.

Adenoviral vectors.E1-deleted replication incompetent adenoviral vectors expressing human ACE2, Escherichia coli β-galactosidase, and foxj1 under the control of the cytomegalovirus promoter were produced as previously described (1).

Preparation of SARS S protein-pseudotyped lentivirus and infection of airway epithelia.The SARS-CoV S protein cDNA (Urbani strain “S-H2,” as described reference 21) was used to pseudotype feline immunodeficiency virus (FIV) expressing a nuclear-targeted β-galactosidase by using previously described methods (45). The virus was concentrated 250-fold by centrifugation, and titers were determined on HT1080 cells, obtaining titers of ∼2× 10⁵ to 4 × 10⁶transducing units/ml. Well-differentiated human airway epithelia were transduced with the pseudotyped FIV by applying 100 μl of solution to the apical surface of airway epithelia. After 1 h of incubation at 37°C, virus was removed and cells were incubated at 37°C for 2 days. To infect epithelia from the basolateral side, the Millicell insert was turned over, and virus was applied to the basolateral surface for 1 h in 100 μl of medium. Following the 1-h infection, virus was removed and the insert was turned upright and incubated at 37°C in 5% CO₂ for 2 days.

β-Galactosidase activity assays.The Galacto-Light chemiluminescent reporter assay (Tropix, Bedford, MA) was used to quantify β-galactosidase activity following the manufacturer's protocol. Relative light units were quantified using a luminometer (Monolight 3010; Pharmingen, San Diego, CA) and standardized to total protein in the sample.

Preparation of wild-type SARS-CoV and infection of airway epithelia.SARS-CoV (Urbani strain) was produced in Vero E6 cells under BSL3 containment conditions. Virus titers were determined on Vero E6 cells with typical yields of ∼4 × 10⁶ PFU/ml. Epithelia derived from three donors were infected in duplicate with SARS-CoV from the apical surface (multiplicity of infection [MOI], 0.8). Following a 30-min incubation at 37°C, virus was removed and 10 washes with PBS were performed. A sample was collected by adding and removing 500 μl of medium from the apical side of one epithelium from each donor. A total of 500 μl of medium was added to the basolateral side of the remaining epithelia, and 24 h later, samples were collected for titer determinations.

ACE2 expression in human airway epithelia.ACE2 serves as the SARS-CoV receptor, at least for tissue culture cell entry (21). To determine if ACE2 is also the SARS-CoV receptor in the respiratory tract, we first looked for evidence of ACE2 protein expression in human lung tissue by Western blotting. An immunoreactive band consistent with the glycosylated form of ACE2 was identified in lysates from human airway and distal lung tissue (Fig. 1A). However, the cell type expressing ACE2 could not be identified in this experiment, as both endothelial and epithelial cells can express ACE2. Accordingly, we next evaluated ACE2 protein expression in well-differentiated primary cultures of airway epithelia using immunohistochemistry and observed airway epithelial cell expression, with the most abundant signal for ACE2 on the apical rather than the basolateral surface (Fig. 1B). Furthermore, the signal intensity was strongest on ciliated cells, as demonstrated by colocalization with the β-tubulin IV marker of cilia (23), suggesting that ciliated cells express ACE2 abundantly. To confirm a polar distribution of ACE2 in differentiated epithelia, selective apical or basolateral surface biotinylation with subsequent precipitation was performed (Fig. 1C). Immunoblot analysis of precipitated proteins confirmed that ACE2 is expressed in greater abundance on the apical surface of conducting airway epithelia, although in some experiments a weak ACE2 signal was also detected basolaterally. In contrast, ErbB2, the receptor for heregulin-alpha, was more abundant on the basolateral surface as previously reported (43), confirming selective biotinylation.

• Open in new tab
• Download powerpoint

FIG. 1.

ACE2 is expressed on human airway epithelia. (A) ACE2 protein levels were determined using immunoblot analysis of extracts from human airway and alveolar tissue. The control is recombinant ACE2 released into the supernatant from A549 cells infected with an adenoviral vector expressing human ACE2. The positions of ACE2 and α-tubulin are indicated by arrows. MW, molecular weight in thousands. (B) ACE2 protein location in polarized human airway epithelia was determined using immunofluorescence staining for ACE2 (green),β -tubulin IV (red), and nuclear DAPI (blue). Confocal fluorescence photomicroscopic images are presented en face (top) and from vertical sections (bottom). Colocalization is shown by yellow in the merged images. Bar, 10 μm. (C) ACE2 protein location in polarized human airway epithelia was determined by selective apical or basolateral biotinylation, immunoprecipitation of biotinylated surface proteins, and immunoblot analysis for ACE2 or control basolateral erbB2. MW, molecular weight in thousands. The positions of ACE2 and erbB2 are indicated by arrows.

Results from polarized epithelia suggested that ACE2 expression might depend on the state of cellular differentiation. To validate a model to test this hypothesis, we used SEM to compare the apical surface morphology of well-differentiated epithelia with that of well-differentiated cells subsequently grown with medium present on their apical surfaces for 7 days. Of note, submersion of the apical surfaces of primary cells caused loss of cilia (Fig. 2A). Loss of this important marker of cell differentiation with resubmersion was associated with markedly diminished expression of ACE2 mRNA and protein (Fig. 2B and C). In contrast to results in polarized epithelia, poorly differentiated primary human tracheobronchial epithelia (hTBE) or A549 cells grown on tissue culture plastic expressed little ACE2 mRNA or protein. Interestingly, foxj1, a transcription factor required for a well-differentiated ciliated epithelia phenotype (3) was also coordinately expressed with ACE2, indicating that ACE2 positively correlates with the state of epithelial differentiation. We also asked whether foxj1 might directly regulate ACE2 expression in airway epithelia. Primary tracheobronchial cells grown in submersion culture were transduced with an adenoviral vector expressing ACE2, a negative-controlβ -galactosidase, or foxj1. Only transduction with the ACE2 vector conferred ACE2 expression, suggesting that foxj1 expression alone does not regulate ACE2 (Fig. 2D and data not shown).

• Open in new tab
• Download powerpoint

FIG. 2.

ACE2 expression is associated with airway epithelial cell differentiation. (A) Ciliated epithelial cell differentiation in cultures of primary airway epithelial cells under air-liquid interface or resubmerged conditions was verified by SEM of the apical epithelial surface. Bar, 10 μm. (B) ACE2 mRNA levels were determined using real-time RT-PCR analysis of samples from A549 cells or primary hTBE cells cultured under undifferentiating submerged (Sub), differentiating ALI, or resubmerged (Resub) conditions. Values are expressed as mean mRNA levels relative to control HPRT mRNA levels plus or minus standard deviations (SD) (n = 3), and the asterisk indicates a significant difference in mRNA levels between air-liquid interface and resubmerged conditions. (C) ACE2 protein levels were determined using immunoblot analysis of extracts from A549 or primary hTBE cells. The positions of ACE2, foxj1 (to verify epithelial cell differentiation status), and β-actin are indicated by arrows. MW, molecular weight in thousands. (D) ACE2 protein levels were determined using immunoblot analysis of extracts from hTBE cells under submerged conditions that were infected with a recombinant adenoviral vector that expressed ACE2, control transgene (β-galactosidase), or foxj1. MW, molecular weight in thousands.

SARS-CoV infection of human airway epithelia.To evaluate the polarity of entry of the SARS-CoV in airway epithelia, we prepared FIV virions pseudotyped with SARS S protein. The ACE2 dependence of transduction with SARS S protein-pseudotyped FIV virions was first evaluated on 293 cells with or without cotransfection with human ACE2 cDNA. We observed that 293 cell transduction with this vector was almost completely ACE2-dependent (Fig. 3A). To establish the ACE2 dependence of human airway epithelia for SARS-CoV, we transduced poorly differentiated A549 cells and submerged primary hTBE cells that do not express constitutive ACE2 with increasing amounts of an adenoviral vector expressing human ACE2. After 48 h, SARS-CoV S protein-pseudotyped-FIV was applied to the apical surface at an MOI of 0.1 (based on HT1080 titers). There was an inoculum-dependent increase in the transduction of cells that expressed ACE2 (Fig. 3B). S protein-pseudotyped FIV was then used to contrast the efficiency of entry in A549 cells, poorly differentiated (submerged) human airway epithelia, and well-differentiated (ALI) epithelia. Only well-differentiated epithelial cells showed significantβ -galactosidase expression following transduction (Fig. 3C). We next applied the pseudotyped virus to the apical or basolateral surfaces of well-differentiated primary cultures of human airway epithelia to investigate whether the virus preferentially entered from one cell surface. After 2 days, the cells were harvested and entry was evaluated by β-galactosidase activity. Transduction of human airway epithelia by the S protein-pseudotyped virions occurred more efficiently when applied from the apical rather than the basolateral surface (Fig. 3D). This pattern of entry correlates with ACE2 expression on polarized cells (Fig. 1). In contrast, FIV pseudotyped with the vesicular stomatitis virus-G envelope entered polarized cells better from the basolateral surface.

• Open in new tab
• Download powerpoint

FIG. 3.

SARS-CoV S protein-pseudotyped FIV infects differentiated airway epithelia best from the apical surface. (A) β-galactosidase levels were determined by enzyme activity in 293 cells transfected with a plasmid expressing control (Ctl) transgene or human ACE2 and then infected with SARS-S protein-pseudotyped FIV expressing β-galactosidase. ND, not detected. (B) β-galactosidase levels were determined in A549 cells (black bars) or primary hTBE cultured under submerged conditions (white bars) that were first infected with an adenoviral vector expressing ACE2 at the indicated MOI and then infected with SARS-S protein-pseudotyped FIV expressing β-galactosidase. (C) β-Galactosidase levels determined in extracts from A549 cells or primary hTBE cultured under submerged or ALI conditions that were infected from the apical surface with SARS-S protein-pseudotyped FIV expressing β-galactosidase. (D) β-galactosidase levels determined in primary hTBE cultured under ALI conditions that were infected from the apical or basolateral surface with vesicular stomatitis virus-G or SARS-S protein-pseudotyped FIV. In panels A through D, values are expressed as means plus or minus SD (n = 4 to 6) and a significant difference from levels on uninfected cells (A and B), hTBE cells cultured under submerged conditions (C), or cells infected from the apical surface (D) is indicated by an asterisk. RLU, relative light units.

We went on to perform select experiments using wild-type SARS-CoV (Urbani strain). Using protocols similar to those outlined for Fig. 3, we evaluated the ability of SARS-CoV to infect multiple human airway epithelial cell culture models. Under BSL3 containment, we applied the virus to A549 cells, poorly differentiated (submerged) primary cultures of airway epithelia, or well-differentiated (ALI) human airway epithelia at an MOI of 0.8. A549 and hTBE cells cultured under submerged conditions expressed little detectable SARS-CoV N or S gene mRNA after infection (Fig. 4A). In contrast, in well-differentiated cells infected with SARS-CoV from the apical surface, the N and S gene mRNAs were detected at high levels. We confirmed that the gene products detected in real-time RT-PCR assays were generated from new SARS-CoV mRNA templates rather than viral genome by verifying the appropriate size of the amplified products (Fig. 4B). The primers used included a forward primer in the conserved SARS CoV 5′ leader sequence and gene-specific reverse primers. These results suggest that SARS-CoV infects undifferentiated human airway epithelial cells poorly or not at all, while well-differentiated airway epithelia are susceptible.

• Open in new tab
• Download powerpoint

FIG. 4.

Infection of differentiated airway epithelia by SARS-CoV. (A) SARS-CoV N and S gene mRNA levels were determined using real-time RT-PCR analysis of A549 cells or primary hTBE cultured under submerged or ALI conditions and infected with SARS-CoV from the apical surface at an MOI of 0.8 for 24 h. Values are expressed as mean mRNA levels relative to control HPRT mRNA levels plus or minus SD (n = 2). ND, not detected. An asterisk indicates a significant difference in mRNA levels between submerged and ALI conditions. (B) PCR products in panel A for hTBE cells cultured under ALI conditions were visualized by ethidium bromide. bp, base pairs. (C) SARS-CoV nsp1 replicase protein location in polarized human airway epithelia that were left uninfected or infected from the apical or basolateral side with SARS-CoV. Twenty-four hours following infection with SARS-CoV viral replication, complexes were localized using immunofluorescence staining for nsp1 (green) and nuclear To-pro-3 (red). Bar, 50 μm. (D) Colocalization of SARS-CoV nsp1 protein and cilia in polarized human airway epithelia. Airway epithelia were infected as described in the legend for Fig. 4C and then fixed and immunostained for nsp1 (green) or β-tubulin IV (red) as a marker for ciliated cells. The merged image shows colocalization of nsp1 and β-tubulin, indicating that the predominant infected cell types were ciliated epithelia. Bar, 10μ m.

To document that SARS-CoV productively infects human airway epithelia, we applied the virus to the apical surface of well-differentiated human airway epithelia at an MOI of ∼0.8 for 30 min and then measured the release of virus 24 h later. As shown in Table 1, these results indicate that, following apical application of SARS-CoV, a productive infection occurred and virus was preferentially released apically. We confirmed SARS-CoV infection of polarized epithelia by immunostaining cells for the SARS-CoV nsp1 protein 24 h following infection. Confocal microscopy revealed viral nsp1 protein in the cytoplasm, consistent with replication complexes, following apical application of the virus (Fig. 4C). Control, noninfected cells showed no staining. The infection of permissive Vero E6 cells showed intense cytoplasmic staining only in the presence of SARS-CoV infection (data not shown). To better define the cell types infected by SARS-CoV in this model, we colocalized the nsp1 and β-tubulin IV proteins (Fig. 4D). Nsp1 staining was observed in perinuclear regions and distributed throughout the cytoplasm. In addition, we observed some areas of colocalization of nsp1 and β-tubulin IV, suggesting that replication complexes may assemble in the cytoplasm near or within cilia. These findings indicate that ciliated cells are the predominant cell type infected by SARS-CoV in well-differentiated airway epithelia.