A single asparagine-linked glycosylation site of the severe acute respiratory syndrome coronavirus spike glycoprotein facilitates inhibition by mannose-binding lectin through multiple mechanisms

Abstract

Mannose-binding lectin (MBL) is a serum protein that plays an important role in host defenses as an opsonin and through activation of the complement system. The objective of this study was to assess the interactions between MBL and severe acute respiratory syndrome-coronavirus (SARS-CoV) spike (S) glycoprotein (SARS-S). MBL was found to selectively bind to retroviral particles pseudotyped with SARS-S. Unlike several other viral envelopes to which MBL can bind, both recombinant and plasma-derived human MBL directly inhibited SARS-S-mediated viral infection. Moreover, the interaction between MBL and SARS-S blocked viral binding to the C-type lectin, DC-SIGN. Mutagenesis indicated that a single N-linked glycosylation site, N330, was critical for the specific interactions between MBL and SARS-S. Despite the proximity of N330 to the receptor-binding motif of SARS-S, MBL did not affect interactions with the ACE2 receptor or cathepsin L-mediated activation of SARS-S-driven membrane fusion. Thus, binding of MBL to SARS-S may interfere with other early pre- or postreceptor-binding events necessary for efficient viral entry.

FIG. 1.

Binding of pseudotyped viruses to mannose-binding lectin. Pseudovirions were bound to plates coated with various concentrations of pdMBL (A) or 10 μg ml⁻¹ of rMBL (B). The binding of the pseudotyped virus to MBL was assessed by lysing the virus with detergent and measuring released p24 core protein by ELISA. The data are presented as percentage of recovered p24 antigen bound ± standard deviations. A single experiment carried out in triplicate is presented. Similar results were obtained in three independent experiments.

FIG. 2.

Inhibition of the infectivity of SARS-S pseudoviruses and live SARS-CoV by mannose-binding lectin. Pseudoviruses preincubated with pdMBL or rMBL were added to 293T-ACE2 cells (A), and pseudoviruses preincubated with pdMBL were added to 293T-ACE2 and Huh7.5 cells for SARS-S pseudoviruses, 293T cells for VSV and Ebola virus pseudoviruses, and Huh7.5 cells for HCV pseudoviruses (B). The percentage of infection of no-MBL controls ± standard deviations is presented for a single experiment carried out in triplicate. Similar results were obtained in three independent experiments. (C) SARS-S pseudoviruses were incubated with pdMBL (3 μg ml⁻¹) in the presence of either Ca²⁺, EDTA, or anti-human MBL antibody (1.5 or 6 μg ml⁻¹), and infectivity was detected in 293T-ACE2 cells. (D) pdMBL and rMBL inhibition assays were carried out with live SARS-CoV strain Frankfurt-1 using a plaque reduction assay in Vero E6 cells. Following 48 h of incubation at 37°C, cells were fixed, and plaques were visualized by staining with crystal violet. Numbers in parentheses indicate the concentrations of pdMBL and rMBL (in μg/ml). The percentage of infection of no-MBL controls ± standard deviations is presented for a single experiment carried out in triplicate. Similar results were obtained in a further experiment. Ab, antibody.

FIG. 3.

MBL blocks binding of SARS-CoV with DC-SIGN. (A) Virus pseudotyped with SARS-S or particles with no envelope protein (no Env) were preincubated with pdMBL (0 to 10 μg ml⁻¹) before incubation with DC-SIGN⁺ or parental B-THP cells. Cells were washed and lysed, and cell-bound virus was measured by p24 ELISA. Binding of pseudotyped viruses to DC-SIGN ± standard deviations was calculated as follows: p24 bound to DC-SIGN⁺ B-THP cells minus p24 bound to DC-SIGN⁻ parental B-THP cells. A single experiment carried out in triplicate is shown. Similar results were obtained in three independent experiments. (B) Transmission of the bound SARS-CoV pseudotypes to target 293T-ACE2 cells. Numbers in parentheses indicate the concentrations of MBL and mannan (in μg/ml). Differences with or without 0.1 μg ml⁻¹ purified pdMBL were analyzed by pairwise t tests (*, P < 0.02). Ab, antibody.

FIG. 4.

Complement-mediated neutralization of pseudotyped viruses. Pseudotyped viruses were incubated with 1:2 or 1:4 dilutions of pooled sera from either NHPS, MDPS, or heat-inactivated pooled sera of these two sources (HINHPS and HIMDPS, respectively). Treated viruses (100 μl) were then incubated with 293T-ACE2 cells. After 40 h, luciferase activity in cells was measured, and the percentage of neutralization ± standard deviations is presented for a single experiment carried out in triplicate. Raw luciferase values for no-serum controls are also presented. Similar results were obtained in three independent experiments. (A) Comparison of VSV-G luciferase activity in cells. A comparison of the percent neutralization of VSV-G (B) and SARS-S (C) pseudotyped viruses by NHPS and MDPS is also shown. (D) SARS-S pseudotyped viruses were incubated alone (0) or with a 1:4 dilution of MDPS and with or without 0.05 μg ml⁻¹ purified pdMBL. Differences were analyzed by pairwise t tests (P < 0.02).

FIG. 5.

Specific glycosylation sites are critical for MBL and SARS-CoV interaction. (A) A schematic diagram of SARS-CoV S glycoprotein. Functional S1 and S2 domains, receptor-binding domain (RBD) and receptor-binding motif (RBM), heptad repeat regions HR1 and HR2, transmembrane domain TM, three clusters of potential N-linked glycosylation sites (I, II, and III), and four glycosylation sites used to make site mutations are indicated. (B) Western blot analysis of wild-type (WT) or mutant S glycoprotein expression performed under reducing and denaturing conditions and detected using rabbit anti-SARS-S antiserum. (C) Effects of glycosylation site mutations of SARS-S on binding of pseudotyped viruses to rMBL-coated plates (10 μg ml⁻¹) (main panel) or rabbit anti-SARS S antiserum-coated plates (inset). The data are presented as the percentage of recovered p24 antigen bound ± standard deviations. (D) Effects of glycosylation site mutations on ACE2-mediated SARS-S pseudovirus infectivity (inset) or MBL-mediated inhibition (main panel). The percentage of infection ± standard deviations for a single experiment carried out in triplicate is presented. Similar results were obtained in three independent experiments.

FIG. 6.

The effect of mannosidase I inhibitor, DMJ, on the interaction of MBL with SARS-CoV. (A) Binding of wild-type (WT) and N330Q mutant SARS-S pseudoviruses, produced in the presence or absence of 1 mM DMJ, to rMBL-coated plates (10 μg ml⁻¹). (B) MBL-mediated neutralization of wild-type and N330Q mutant pseudoviruses, produced in the presence or absence of 1 mM DMJ. The percentage of infection ± standard deviations is presented for a single experiment carried out in triplicate. Similar results were obtained in three independent experiments.

FIG. 7.

Effect of MBL on virus interaction with ACE2 receptor and cathepsin L-mediated activation of SARS-S intervirion fusion. (A) Viruses pseudotyped with SARS-CoV wild-type or mutant S glycoproteins were preincubated with pdMBL (0, 1.0, and 10 μg ml⁻¹) or 80R antibody (5 μg ml⁻¹) in VBS-Ca buffer before incubation with recombinant human ACE2 (2 μg ml⁻¹)- or BSA-coated plates. Wells were washed, and bound viruses were lysed and assayed for p24. A single experiment was carried out in triplicate. Similar results were obtained in three independent experiments. (B) Purified S1-Ig binding to ACE2-coated plates. S1-Ig was preincubated with pdMBL, and binding to ACE2-coated plates was detected using a goat anti-rabbit Ig antibody conjugated to alkaline phosphatase (AP) followed by a chemiluminescent substrate for AP activity. Ebola virus envelope glycoprotein with Ig conjugate (Ebola GP-Ig) was used as a negative control. (C) A diagram of intervirion assays with MBL, with the three steps indicated as S1, S2, and S3. (D) Intervirion assays with pdMBL. HIV-luc(ACE2) and HIV-gfp(SARS S/ASLV-A) particles were mixed and kept at 4°C for 30 min to allow binding. Samples were raised to 37°C for 15 min to allow for conformational rearrangements. Trypsin or CTSL was then added. The mixed viruses were used to infect HeLa/Tva cells pretreated with leupeptin. pdMBL (50 or 100 μg ml−¹) was added to the mixture at three different steps: S1, concurrent with initial virion mixing; S2, concurrent with protease addition; and S3, after proteolysis. The percentage of infection achieved for mixed particles on untreated cells ± standard deviations is presented for a single experiment carried out in triplicate. Similar results were obtained in three independent experiments.