Identification of N-linked glycosylation sites in the spike protein and their functional impact on the replication and infectivity of coronavirus infectious bronchitis virus in cell culture

Abstract

Spike (S) glycoprotein on the viral envelope is the main determinant of infectivity. The S protein of coronavirus infectious bronchitis virus (IBV) contains 29 putative asparagine(N)-linked glycosylation sites. These post-translational modifications may assist in protein folding and play important roles in the functionality of S protein. In this study, we used bioinformatics tools to predict N-linked glycosylation sites and to analyze their distribution in IBV strains and variants. Among these sites, 8 sites were confirmed in the S protein extracted from partially purified virus particles by proteomics approaches. N-D and N-Q substitutions at 13 predicted sites were introduced into an infectious clone system. The impact on S protein-mediated cell-cell fusion, viral recovery and infectivity was assessed, leading to the identification of sites essential for the functions of IBV S protein. Further characterization of these and other uncharacterized sites may reveal novel aspects of N-linked glycosylation in coronavirus replication and pathogenesis.

Keywords: Cell-cell fusion; Clone; Coronavirus; Infectious bronchitis virus; Infectious cDNA; N-linked glycosylation; Spike protein; Virus infectivity.

Fig. 1

a Diagram showing the IBV spike protein with different functional domains indicated. Signal sequence(SS), amino acids 1–18; S1, amino acids 19–537; S2, amino acids 538–1162; Heptad Repeat 1 (HP1), amino acids 790–911; Heptad Repeat 2 (HP2), amino acids 1056–1089; Trans-membrane domain (TM), amino acids 1097–1118. Also indicated are the putative N-linked glycosylation sites in three clusters, and amino acid positions of the N-linked glycosylation sites in Cluster I. The relative importance of these N-linked glycosylation sites in Cluster I is indicated with colored triangles, with red indicating less importance and yellow indicating critical importance. b The 29 putative glycosylation sites on the IBV spike protein as predicted by NetNGlyc 1.0 software. The threshold and glycosylation potential are shown.

Fig. 2

a Enzymatic reaction mechanism of PNGase F-mediated Asn to Asp conversion in H₂O¹⁸ solvent. b Western blot analysis of IBV S protein from partially purified IBV particles before and after PNGase F digestion. Polypeptides were separated on a 10% SDS-PAGE gel and analyzed by Western blot with anti-IBV S antiserum. The full-length glycosylated S (S*), full-length unglycosylated S (S), glycosylated S1/S2 (S1/S2*) and de-glycosylated S1/S2 (S1/S2) are indicated. Numbers on the left indicate molecular masses in kilodalton.

Fig. 3

a Glycosylation peptide (ALAYFVN*GTAQDVILCDGSPR) of IBV S protein following PNGase F treatment of purified IBV. N* represents N-glycosylation site. Identities of the b-ions and y-ions in the spectrum shown in (b). b LC-MS/MS spectrum of the glycosylation peptide in (a) with a [M+2 H]²⁺ precursor ion at 1136.08 m/z. c Another glycosylation peptide (NFIFHN*ETGANPNPSGVQNIQTY) of IBV S protein following PNGase F treatment of purified IBV. N* represents N-glycosylation site. Identities of the b-ions and y-ions in the spectrum shown in (d). d LC-MS/MS spectrum of the glycosylation peptide in (c) with a [M+2 H]²⁺ precursor ion at 1283.61 m/z.

Fig. 4

a Detection of membrane fusion in cells overexpressing wild type and N-D mutant S proteins at the predicted N-linked glycosylation sites in Cluster I by indirect immunofluorescence. Vero cells were infected with the vaccinia/T7 recombinant virus and transfected with the indicated constructs. At 18 h post-transfection, cells were fixed with 4% paraformaldehyde and stained with rabbit anti-IBV S polyclonal antibodies and anti-rabbit IgG conjugated to FITC. b Western Blot analysis of cells expressing wild type and N-D mutant S proteins at the predicted N-linked glycosylation sites in Cluster I. Vero cells were infected with the vaccinia/T7 recombinant virus and transfected with the indicated constructs. At 18 h post-transfection, cells were harvested and lysates prepared. Polypeptides were separated on a 10% SDS-PAGE gel and analyzed by Western blot with anti-IBV S antiserum. The full-length glycosylated S (S*), full-length unglycosylated S (S) and glycosylated S1/S2 (S1/S2*) are indicated. Numbers on the left indicate molecular masses in kilodalton. c Western Blot analysis of cells expressing wild type and double N-D mutant S proteins at selected N-linked glycosylation sites in Cluster I. Vero cells were infected with the vaccinia/T7 recombinant virus and transfected with the indicated constructs. At 18 h post-transfection, cells were harvested and lysates prepared. Polypeptides were separated on a 10% SDS-PAGE gel and analyzed by Western blot with anti-IBV S and anti-actin antibodies, respectively. The full-length glycosylated S (S*), full-length unglycosylated S (S) and glycosylated S1/S2 (S1/S2*) are indicated. Numbers on the left indicate molecular masses in kilodalton.

Fig. 5

a Cytopathic effect of Vero cells transfected with wild type and selected N-D mutant full-length transcripts. Cells were transfected with RNA and syncytium formation was observed by microscopy at 24–36 h post-transfection. b Western blot analysis of cells infected with wild type and a set of recombinant IBV with N-D mutation(S) in N-linked glycosylation sites. Vero cells were infected with virus, harvested at 24 h post-infection and lysates prepared. Polypeptides were separated on a 10% SDS-PAGE gel and analyzed by Western blot with anti-IBV S and anti-actin antibodies, respectively. The full-length glycosylated S (S*), full-length unglycosylated S (S) and glycosylated S1/S2 (S1/S2*) are indicated. Numbers on the left indicate molecular masses in kilodalton. c Growth kinetics of wild type and N51D, N77D and N103D recombinant IBV. Confluent Vero cells were infected with IBV, harvested at 0, 4, 8, 12, 18, 24 and 36 h post-infection, respectively. Viral stocks were prepared by freezing/thawing of the cells three times and TCID₅₀ was determined. d Growth kinetics of wild type and N144D, N163D and N51-77D recombinant IBV. Confluent Vero cells were infected with IBV, harvested at 0, 4, 8, 12, 18, 24 and 36 h post-infection, respectively. Viral stocks were prepared by freezing/thawing of the cells three times and TCID₅₀ was determined.

Fig. 6

a Detection of membrane fusion in cells overexpressing wild type and N-D/Q mutant S proteins at three predicted N-linked glycosylation sites in Cluster I by indirect immunofluorescence. Vero cells were infected with the vaccinia/T7 recombinant virus and transfected with the indicated constructs. At 18 h post-transfection, cells were fixed with 4% paraformaldehyde and stained with rabbit anti-IBV S polyclonal antibodies and anti-rabbit IgG conjugated to FITC. b Western Blot analysis of cells expressing wild type and N-D/Q mutant S proteins at three predicted N-linked glycosylation sites in Cluster I. Vero cells were infected with the vaccinia/T7 recombinant virus and transfected with the indicated constructs. At 18 h post-transfection, cells were harvested and lysates prepared. Polypeptides were separated on a 10% SDS-PAGE gel and analyzed by Western blot with anti-IBV S antiserum. The full-length glycosylated S (S*), full-length unglycosylated S (S) and glycosylated S1/S2 (S1/S2*) are indicated. Numbers on the left indicate molecular masses in kilodalton. c Growth kinetics of wild type and N283Q recombinant IBV. Confluent Vero cells were infected with IBV, harvested at 0, 4, 8, 12, 18, 24 and 36 h post-infection, respectively. Viral stocks were prepared by freezing/thawing of the cells three times and TCID₅₀ was determined.