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.

N-Linked Glycosylation Status of Classical Swine Fever Virus Strain Brescia E2 Glycoprotein Influences Virulence in Swine

“…The G4 glycosylation site in the NDV HN protein has been shown to be necessary for correct folding and transport of the protein to the cell surface of infected cells (19,25). Interestingly, G4 virus was considerably attenuated in chickens (25). In the case of CSFV, we have observed previously that other BICv-derived viruses containing recombinant E2 protein (29) showed reduced plaque size as well.…”

“…Similarly, loss of one specific N-linked glycosylation site (G4) from the HN protein of NDV yielded plaques in cultured cells of considerably smaller size (25), while three other single mutants (G1, G2, and G3) produced plaques comparable to the size of the parental virus. The G4 glycosylation site in the NDV HN protein has been shown to be necessary for correct folding and transport of the protein to the cell surface of infected cells (19,25). Interestingly, G4 virus was considerably attenuated in chickens (25).…”

“…In vivo viral glycoproteins have been shown to influence infectivity (2), virulence (13), and host immune response (24,25). Added oligosaccharides confer proper function to viral glycoproteins since alteration of those glycosylation sites has shown dramatic consequences for viruses affecting protein folding (10,17,36) and protein active conformation (20).…”

“…Similarly, degrees of virulence in chickens have been associated with glycosylation patterns of surface proteins hemagglutinin and neuraminidase of the highly pathogenic avian influenza virus H5N1 (13). Pathogenic phenotypes observed upon infection of natural hosts with modified viruses link glycosylation of virus surface proteins with mechanisms such as evasion of the immune system in porcine respiratory and reproductive syndrome virus (2), attenuation in NDV (25), and determinants of virulence in avian influenza (13).…”

“…Recently it has been shown that glycosylation of porcine respiratory and reproductive syndrome virus GP5 affects virus infectivity, antigenicity, and ability to induce neu-tralizing antibodies in swine (2). Loss of N-linked glycosylation from the hemagglutinin-neuraminidase (HN) protein from Newcastle disease virus (NDV), a bird pathogen, results in attenuation of the virus in chickens (25). Similarly, degrees of virulence in chickens have been associated with glycosylation patterns of surface proteins hemagglutinin and neuraminidase of the highly pathogenic avian influenza virus H5N1 (13).…”