Loss of N-linked glycosylation from the hemagglutinin-neuraminidase protein alters virulence of Newcastle disease virus

Abstract

The hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) is an important determinant of its virulence. We investigated the role of each of the four functional N-linked glycosylation sites (G1 to G4) of the HN glycoprotein of NDV on its pathogenicity. The N-linked glycosylation sites G1 to G4 at residues 119, 341, 433, and 481, respectively, of a moderately pathogenic NDV strain Beaudette C (BC) were eliminated individually by site-directed mutagenesis on a full-length cDNA clone of BC. A double mutant (G12) was also created by eliminating the first and second glycosylation sites at residues 119 and 341, respectively. Infectious virus was recovered from each of the cDNA clones of the HN glycoprotein mutants, employing a reverse genetics technique. There was a greater delay in the replication of G4 and G12 mutant viruses than in the parental virus. Loss of glycosylation does not affect the receptor recognition by HN glycoprotein of NDV. The neuraminidase activity of G4 and G12 mutant viruses and the fusogenicity of the G4 mutant virus were significantly lower than those of the parental virus. The fusogenicity of the double mutant virus (G12) was significantly higher than that of the parental virus. Cell surface expression of the G4 virus HN was significantly lower than that of the parental virus. The antigenic reactivities of the mutants to a panel of monoclonal antibodies against the HN protein indicated that removal of glycosylation from the HN protein increased (G1, G3, and G12) or decreased (G2 and G4) the formation of antigenic sites, depending on their location. In standard tests to assess virulence in chickens, all of the glycosylation mutants were less virulent than the parental BC virus, but the G4 and G12 mutants were the least virulent.

FIG. 1.

Schematic representation of the HN glycosylation mutants generated by site-directed mutagenesis. The HN wild-type (wt) protein is shown within the box at the top of the figure. Symbols: open bar, 571-amino-acid polypeptide chain; filled bar, N-terminal transmembrane anchor; Y, functional N-linked glycosylation site. The residue numbers indicate the Asn residues in the HN amino acid sequence. The designations of the carbohydrate chains (G1, G2, G3, and G4) are indicated above each site. HN glycosylation mutants are represented in a similar fashion, with the designated name shown at the left of each diagram. Wild-type HN has four functional Asn-linked glycosylation sites, represented by G1, G2, G3, and G4, present at amino acid residues 119, 341, 433, and 481, respectively. The HN G1 mutant has its glycosylation site G1 at residue 119 eliminated, mutant HN G2 has its G2 glycosylation site at residue 341 eliminated, mutant HN G3 has its G3 glycosylation site at residue 433 eliminated, mutant HN G4 has its G4 glycosylation site at residue 481 eliminated, and the double mutant HN G12 has two of its glycosylation sites at residues 119 and 341 eliminated. All glycosylation sites were eliminated by mutating the asparagine (Asn) residue at each site to glutamine (Glu) by site-directed mutagenesis.

FIG. 2.

Growth kinetics of viruses in tissue culture. Multicycle growth of parental and HN glycosylation mutant viruses in chicken embryo fibroblast (DF1) cells. Cells were infected with the indicated parental or chimeric virus at an MOI of 0.01. Samples were taken at 8-h intervals, and virus titers were determined by plaque assay. Values are averages from the results from three independent experiments.

FIG. 3.

Plaque morphology in DF1 cells by wild-type and HN glycosylation mutant viruses 4 days PI. Recovered viruses were titrated in duplicate in 12-well plates. Supernatant collected from virus-inoculated samples was serially diluted, and 100 μl of each serial dilution was added per well to confluent DF1 cells. After 60 min of adsorption, cells were overlaid with Dulbecco's modified Eagle's medium (containing 2% fetal bovine serum and 0.9% methylcellulose) and incubated at 37°C for 3 to 4 days. The cells were then fixed with ethanol and stained with crystal violet for observation of plaques. Plaque size and morphology are shown for rBC (a), rG1 (b), rG2 (c), rG3 (d), rG4 (e), and rG12 (f) viruses.

FIG. 4.

Immunoprecipitation of parental rBC and HN glycosylation mutants from infected cells and treatment with endo H. DF1 cells were infected with each of the viruses for 4 to 6 h, and proteins were labeled with [³⁵S]methionine-cysteine for 2 h. Equal amounts of cell lysates were immunoprecipitated with a cocktail of NDV-specific polyclonal antibodies and S. aureus protein A and analyzed by SDS-PAGE. The immunoprecipitates were digested (+) or mock-digested (−) with endo H and then analyzed by SDS-PAGE. NP, nucleocapsid protein.

FIG. 5.

Expression of HN protein glycosylation mutants. Equal amounts of proteins from parental and mutant purified viruses were analyzed by SDS-PAGE. The migration and protein stability of the HN protein from each virus were examined after Coomassie blue staining. The HN protein of mutant rG4 virus was found to be degraded. A protein molecular mass marker was also run along with the purified viral samples to assess the viral proteins based on their molecular masses. The molecular mass of the HN protein of NDV is approximately 74 kDa. Three independent experiments with purified viral proteins confirmed the degradation of the HN glycoproteins of the rG4 mutant virus.

FIG. 6.

Detection of cell surface immunofluorescence of parental and mutant HN proteins. Virus-infected DF1 cells (0.1 MOI) were washed with PBS at 48 h PI and fixed with 3% paraformaldehyde in PBS for cell surface fluorescence (b, d, f, h, j, l, and n) or fixed and then permeabilized with 0.1% Triton X-100 in PBS for intracytoplasmic fluorescence (a, c, e, g, i, k, and m). HN proteins were stained with an anti-HN MAb cocktail, followed by affinity-purified fluorescein-labeled goat anti-mouse immunoglobulin (Kirkegaard and Perry Laboratories). (a and b) rBC; (c and d) rG1; (e and f) rG2; (g and h) rG3; (i and j) rG4; (k and l) rG12; (m and n) negative control. Cells were photographed at a magnification of ×200.

FIG.7.

Biological activities of HN glycosylation mutants. The HAd, NA, and fusogenic activities of HN glycosylation mutants were examined. (A) HAd was measured as the percentage of the hemoglobin released compared to the parental rBC virus. The NA and fusion indices (FI) of the mutant viruses were also shown in comparison with those activities of the rBC virus. NA activity of purified viruses was measured by a fluorometric assay. The FI is the ratio of the total number of nuclei to the number of cells in which the nuclei were observed (i.e., the mean number of nuclei per cell). The averages of the results from three experiments are shown. *, P < 0.05. (B) Fusogenicity of mutant viruses in Vero cells in comparison to parental rBC virus. Infected cells were fixed at 48 h p.i. and stained with hematoxylin-eosin. The extent of fusion of the parental rBC (a) and the mutant viruses rG1, rG2, rG3, rG4, and rG12 (b, c, d, e, and f, respectively) in Vero cells was photographed on a Nikon Eclipse TE 300 microscope (magnification, ×168). An uninfected Vero cell monolayer (g) was included as a negative control for comparison.

FIG.7.

Biological activities of HN glycosylation mutants. The HAd, NA, and fusogenic activities of HN glycosylation mutants were examined. (A) HAd was measured as the percentage of the hemoglobin released compared to the parental rBC virus. The NA and fusion indices (FI) of the mutant viruses were also shown in comparison with those activities of the rBC virus. NA activity of purified viruses was measured by a fluorometric assay. The FI is the ratio of the total number of nuclei to the number of cells in which the nuclei were observed (i.e., the mean number of nuclei per cell). The averages of the results from three experiments are shown. *, P < 0.05. (B) Fusogenicity of mutant viruses in Vero cells in comparison to parental rBC virus. Infected cells were fixed at 48 h p.i. and stained with hematoxylin-eosin. The extent of fusion of the parental rBC (a) and the mutant viruses rG1, rG2, rG3, rG4, and rG12 (b, c, d, e, and f, respectively) in Vero cells was photographed on a Nikon Eclipse TE 300 microscope (magnification, ×168). An uninfected Vero cell monolayer (g) was included as a negative control for comparison.