Inhibition of endoplasmic reticulum-resident glucosidases impairs severe acute respiratory syndrome coronavirus and human coronavirus NL63 spike protein-mediated entry by altering the glycan processing of angiotensin I-converting enzyme 2

Abstract

Endoplasmic reticulum (ER)-resident glucosidases I and II sequentially trim the three terminal glucose moieties on the N-linked glycans attached to nascent glycoproteins. These reactions are the first steps of N-linked glycan processing and are essential for proper folding and function of many glycoproteins. Because most of the viral envelope glycoproteins contain N-linked glycans, inhibition of ER glucosidases with derivatives of 1-deoxynojirimycin, i.e., iminosugars, efficiently disrupts the morphogenesis of a broad spectrum of enveloped viruses. However, like viral envelope proteins, the cellular receptors of many viruses are also glycoproteins. It is therefore possible that inhibition of ER glucosidases not only compromises virion production but also disrupts expression and function of viral receptors and thus inhibits virus entry into host cells. Indeed, we demonstrate here that iminosugar treatment altered the N-linked glycan structure of angiotensin I-converting enzyme 2 (ACE2), which did not affect its expression on the cell surface or its binding of the severe acute respiratory syndrome coronavirus (SARS-CoV) spike glycoprotein. However, alteration of N-linked glycans of ACE2 impaired its ability to support the transduction of SARS-CoV and human coronavirus NL63 (HCoV-NL63) spike glycoprotein-pseudotyped lentiviral particles by disruption of the viral envelope protein-triggered membrane fusion. Hence, in addition to reducing the production of infectious virions, inhibition of ER glucosidases also impairs the entry of selected viruses via a post-receptor-binding mechanism.

FIG 1

Effects of iminosugars on transduction of a panel of pseudotyped lentiviral particles. Huh7.5 cells seeded in 96-well plates were mock treated (0.5% DMSO) or treated with 50 μM iminosugar, as indicated, for 72 h, followed by inoculation of the pseudotyped lentiviral particles for 3 h. After two washes with DMEM, the cells were cultured with complete DMEM for another 72 h. Luciferase activity in the cell lysates was determined. Relative transduction efficiency represents the luciferase activity normalized to that of mock-treated cells and expressed as the mean ± standard deviation (n = 6). Differences in SARSpp and IAVpp transduction efficiencies between mock-treated cells and cells treated with iminosugar compounds are statistically significant (**, P < 0.001). Representative results from three independent experiments are presented.

FIG 2

IHVR-17028 inhibits pseudotyped lentiviral transduction in a dose- and time-dependent manner. (A) Huh7.5 cells were pretreated with IHVR-17028 at the indicated concentrations for 72 h, followed by infection with the indicated pseudotyped viral particles for 3 h. Luciferase activity in the cell lysates was determined at 72 h postinfection. Relative transduction efficiency represents the luciferase activity normalized to that of mock-treated cells and expressed as the mean ± standard deviation (n = 6). (B) Huh7.5 cells seeded in 96-well plates were pretreated with 50 μM IHVR-17028 for the indicated periods or during infection with pseudotyped lentiviral particles for 2 h. After two washes with DMEM, the cells were cultured with complete DMEM. Luciferase activities in cell lysates were determined at 72 h postinfection. Relative transduction efficiency represents the luciferase activity normalized to that of mock-treated cells and expressed as the mean ± standard deviation (n = 6). Differences in SARS-CoVpp, HCoV-NL63pp, or IAVpp transduction efficiency between cells with mock and IHVR-17028 treatments for 24 h or longer are statistically significant (**, P < 0.001). Representative results from three independent experiments are presented.

FIG 3

IHVR-17028 dose-dependently inhibits the transduction of ACE2-expressing 293 cells by SARS-CoVpp and HCoV-NL63pp but not VSVpp. (A) An FLP/IN T Rex-derived stable cell line that inducibly (via tetracycline) expresses N-terminally myc-tagged ACE2, designated FLP-IN/ACE2, was established as described in Materials and Methods. The cells were cultured in the absence or presence of 1 μg/ml of tetracycline for 24 h. The levels of ACE2 protein expression in cell lysates were determined by a Western blot assay with a monoclonal antibody against the myc tag. β-Actin served as a loading control. (B) FLP-IN/ACE2 cells were cultured in complete DMEM with or without tetracycline for 24 h, followed by infection with the indicated pseudotyped lentiviral particles. Luciferase activity was determined at 72 h postinfection. Luciferase activity is expressed as the mean ± standard deviation (n = 6). (C) FLP-IN/ACE2 cells were cultured in complete DMEM with tetracycline and the indicated concentrations of IHVR-17028 for 48 h and then infected with the indicated pseudotyped lentiviral particles. Luciferase activities in cell lysates were determined at 72 h postinfection. Relative transduction efficiency represents the luciferase activity normalized to that of mock-treated cells and expressed as the mean ± standard deviation (n = 6). (D) FLP-IN/ACE2 cells were cultured with IHVR-17028 at the indicated concentrations in the absence or presence of tetracycline for 48 h. The levels of ACE2 protein expression were determined by a Western blot assay with a monoclonal antibody against the myc tag. β-Actin served as a loading control. The relative amounts of ACE2 were quantified using Scion Image and expressed as percentages of the untreated control level (data are means ± standard deviations from three independent experiments).

FIG 4

IHVR-17028 treatment does not apparently change the total amount of ACE2 but alters its electrophoretic mobility. Huh7.5 cells were treated with IHVR-17028 at the indicated concentrations for 72 h (A) or treated with 50 μM IHVR-17028 for the indicated periods (B). ACE2 in cell lysates was immunoprecipitated with an ACE2 polyclonal antibody and detected by a Western blot assay with an ACE2 polyclonal antibody. (C) 293T cells were transfected with a plasmid expressing ACE2 and treated with 50 μM IHVR-17028 for 48 h. ACE2 in cell lysates was purified by use of protein A/G beads coated with an ACE2 polyclonal antibody. Purified ACE2 was digested by PNGase or endo H and detected by a Western blot assay. Representative results from two independent experiments are presented.

FIG 5

IHVR-17028 treatment alters the glycan structure of ACE2. (A) FLP-IN/ACE2 cells were mock treated or treated with 50 μM IHVR-17028 in the absence or presence of 1 μg/ml tetracycline for 48 h and then lysed with CHAPS buffer. ACE2 protein was purified by use of protein A/G beads conjugated with polyclonal anti-ACE2 antibody and resolved by SDS-PAGE. IP, immunoprecipitation. (B) The ACE2 bands were sliced from the gel and subjected to N-linked glycan analysis. The sialylated N-linked glycan profiles are shown, with the major peaks indicated. The peak labeled A2G2 is a biantennary glycan with terminal galactose residues. The peak labeled FcA2G2 is a core fucosylated biantennary glycan with terminal galactose residues. Sialylated versions of these structures are indicated as S1, containing a single sialic acid, or S2, containing two sialic acid molecules. The profiles also contain large branched glycans that contain various levels of sialylation. All peaks were identified by sequential exoglycosidase digestion. Representative results from two independent experiments are presented.

FIG 6

IHVR-17028 treatment does not alter the cell surface expression of ACE2. FLP-IN/ACE2 cells were mock treated or treated with 50 μM IHVR-17028 in the absence or presence of 1 μg/ml tetracycline for 48 h. (A) Surface expression of ACE2 was examined by flow cytometry using an anti-myc antibody. (B) Binding of a SARS-CoV RBD-Ig fusion protein on the cell surface was assayed by flow cytometry. Mean fluorescence values and standard deviations from three independent experiments, obtained from cells treated under the conditions described above and stained with anti-myc antibody (C) or the SARS-CoV RBD-Ig fusion protein (D), are also presented.

FIG 7

Syncytium formation between SARS-CoV spike protein- and ACE2-expressing cells was inhibited by iminosugar treatment. (A) 293T cells transfected with a plasmid encoding either SARS-CoV S protein or ACE2 were mock treated or treated with 50 μM IHVR-17028 for 48 h. For syncytium formation, the cells were mixed at a 1:1 ratio and cultured for 24 h. The cultures were imaged with a Nikon microscope. (B) 293T cells cotransfected with plasmids encoding either ACE2 and T7 polymerase or SARS-CoV spike protein and T7/luciferase were mock treated or treated with 50 μM IHVR-17028 for 48 h. For syncytium formation, the cells were mixed at a 1:1 ratio and cultured for 24 h. Luciferase activities in the cell lysates were determined, normalized to the mock-treated control level, and then expressed as means ± standard deviations (n = 4). Differences in cell fusion between mock-treated and IHVR-17028-treated cells were analyzed statistically (**, P < 0.001; t test). (C) 293T cells transfected with a plasmid encoding SARS-CoV S protein or ACE2 were treated with 50 μM IHVR-17028 for 48 h and then lysed by use of CHAPS buffer. The ACE2 and SARS-CoV spike proteins were detected by Western blotting. Representative results from two independent experiments are presented.