Differential downregulation of ACE2 by the spike proteins of severe acute respiratory syndrome coronavirus and human coronavirus NL63

Abstract

The human coronaviruses (CoVs) severe acute respiratory syndrome (SARS)-CoV and NL63 employ angiotensin-converting enzyme 2 (ACE2) for cell entry. It was shown that recombinant SARS-CoV spike protein (SARS-S) downregulates ACE2 expression and thereby promotes lung injury. Whether NL63-S exerts a similar activity is yet unknown. We found that recombinant SARS-S bound to ACE2 and induced ACE2 shedding with higher efficiency than NL63-S. Shedding most likely accounted for the previously observed ACE2 downregulation but was dispensable for viral replication. Finally, SARS-CoV but not NL63 replicated efficiently in ACE2-positive Vero cells and reduced ACE2 expression, indicating robust receptor interference in the context of SARS-CoV but not NL63 infection.

FIG. 1.

SARS-S binds to ACE2 with higher efficiency than NL63-S. (A) For production of recombinant SARS-S and NL63-S proteins, the S1 unit of the respective proteins was fused to the Fc portion of human immunoglobulin, and the soluble S proteins and the Fc control protein were transiently expressed in 293T cells. The S proteins and the Fc control protein were purified from the culture supernatants by protein A chromatography, and protein content was analyzed by Western blot analysis using an anti-human Fc antibody. Lane 1, Fc control; lane 2, SARS-S; lane 3, NL63-S. (B) 293T cells stably expressing ACE2 were incubated with SARS-S, NL63-S, and Fc control protein at a concentration of 22.5 μg/ml, and protein binding was analyzed by a fluorescence-activated cell sorter (FACS). Results from a representative experiment are shown in the left panel, and the geometric mean channel fluorescence measured upon analysis of duplicate samples is shown in the right panel. (C) Binding of SARS-S and NL63-S to ACE2 was assessed by employing a sandwich ELISA. The ELISA plate was coated with anti-human Fc antibody to capture the soluble spike Fc proteins or a Fc control protein (2 μg/ml), followed by addition of different concentrations of recombinant ACE2 labeled with a FLAG tag, which was detected using an anti-FLAG antibody-horseradish peroxidase (HRP) conjugate. (D) An anti-human Fc antibody was immobilized onto a BIAcore chip surface, and SARS-S Fc protein was added. Subsequently, unbound protein was removed, and binding of different concentrations of recombinant ACE2 was assessed. The binding of the receptor was measured by a decreased angle of the surface plasma resonance (SPR), which is illustrated in response units (RU) over time. (E) 293T cells overexpressing ACE2 were incubated with the indicated concentrations of recombinant SARS-S (left) or NL63-S (right) for 1 h at 37°C and infected with lentiviruses pseudotyped with SARS-S, NL63-S, or vesicular stomatitis virus G protein (VSV-G). At 72 h postinfection, the luciferase activity in the cell lysates was determined using a commercially available kit. The results of a representative experiment are shown and are graphed as relative infection efficiency. Upon infection in the absence of recombinant spike protein, the following luciferase counts per second (c.p.s.) were measured: VSV-G, 3,612,752 ± 2,523,541 c.p.s.; SARS-S, 2,290,442 ± 347,006 c.p.s.; NL63-S, 168,575 ± 20,581 c.p.s.

FIG. 2.

SARS-S but not NL63-S induces efficient ACE2 shedding. (A) Vero E6 cells were incubated for 0, 2, 8, and 24 h with VLPs harboring SARS-S, NL63-S, or control particles containing no viral glycoprotein. As a positive control for ACE2 shedding, cells were treated with the indicated concentrations of PMA for 1 h. Thereafter, the supernatants were harvested, OVA was added, and the samples were precipitated with TCA. In parallel, the cells were lysed, pellets were obtained upon TCA precipitation, and cell lysates were analyzed for ACE2 expression by Western blot analysis. As precipitation and loading control, respectively, OVA content (supernatants) and β-actin expression (cell lysates) were also determined by Western blot analysis. (B) The signals obtained for ACE2 in the culture supernatants examined in panel A (left) or in the supernatants of cells treated with Fc control protein, SARS-S Fc, or NL63-S Fc protein (right) were quantified employing ImageJ software. The average of two independent experiments is shown in the left panel. The right panel depicts the average of three independent experiments. (C) To confirm the importance of TACE for ACE2 shedding, Vero E6 cells were incubated with 0.1 μM PMA in the presence of the indicated concentrations of TAPI-0, a TACE inhibitor. ACE2 shedding was analyzed as described for panel A. (D) Vero E6 cells were incubated with SARS-S for 3 h at 4°C or 37°C in the absence (top) and presence (middle) of TAPI-0. Alternatively, cells were incubated with NL63-S protein for 3 h at 4°C or 37°C (bottom). Thereafter, unbound protein was removed by washing, and protein binding was determined by FACS analysis. (E) 293T cells engineered to express high levels of ACE2 were transiently transfected with 3 μg of plasmids encoding SARS-S, NL63-S, or the indicated SARS-CoV proteins, and ACE2 surface expression was determined by FACS analysis. (F) The experiment was carried out as described in panel E. However, the cells were harvested at 48 h posttransfection, and ACE2 expression in cell lysates was analyzed by Western blot analysis. Expression of β-actin was determined as a loading control.

FIG. 3.

Shedding of ACE2 is dispensable for SARS-CoV and NL63 spread. (A) In order to determine the importance of TACE activity for SARS-S- and NL63-S-driven infectious entry, ACE2-transfected 293T cells were incubated with the indicated concentrations of TAPI-0 and infected with infectivity-normalized lentiviral pseudotypes bearing the indicated glycoproteins. At 72 h postinfection, the cells were lysed and the luciferase activities in cell lysates were determined by employing a commercially available kit. (B) To assess the importance of ACE2 shedding to SARS-CoV and NL63 spread, Vero E6 cells were pretreated with the indicated concentrations of TAPI-0 or pretreated with dimethyl sulfoxide (DMSO) as a control and then infected with SARS-CoV (Frankfurt strain) or NL63 at an MOI of 0.001. Supernatants of the infected and noninfected cells were taken at the indicated time points postinfection, and the number of viral genome copies was determined by real-time reverse transcription-PCR (RT-PCR). The following primers and probes were used for detection of the SARS-CoV genome: BNITMSARS1 (5′-TTATCACCCGCGAAGAAGCT-3′) (forward primer), BNITMSARAs2 (5′-CTCTAGTTGCATGACAGCCCTC-3′) (reverse primer), BNITMSARP (5′-FAM-TCGTGCGTGGATTGGCTTTGATGT-TAMRA-3′) (probe). For detection of the NL63 genome, the following primers and probes were used: 63RF2 (5′-CTTCTGGTGACGCTAGTACAGCTTAT-3′) (forward primer), 63RR2 (5′-AGACGTCGTTGTAGATCCCTAACAT-3′) (reverse primer), and 63RP (5′-FAM-CAGGTTGCTTAGTGTCCCATCAGATTCAT-3′-TAMRA) (probe). The SARS-CoV and NL63-specific primers both recognize ORF1B sequences. The result of a representative experiment carried out in duplicates is shown.

FIG. 4.

Downmodulation of ACE2 expression in SARS-CoV-infected cells. (A) Vero E6 cells were infected with SARS-CoV (Frankfurt strain) or NL63 at an MOI of 0.001 in duplicates. At the indicated times postinfection, cells were washed with phosphate-buffered saline (PBS), lysed with sodium dodecyl sulfate (SDS) lysis buffer, and analyzed for expression of ACE2 or viral proteins by Western blot analysis. For detection of viral proteins, an antibody directed against SARS-CoV Nsp8 and a serum reactive against NL63-infected cells were used. (B) The signals for ACE2 in the cellular lysates shown in panel A were quantified using ImageJ software and are shown relative to the signals measured at 2 h postinfection. (C) The supernatants of the infected cells described above were analyzed for viral genome copies by real-time RT-PCR.