Differentially Regulated Interferon Response Determines the Outcome of Newcastle Disease Virus Infection in Normal and Tumor Cell Lines

Newcastle disease virus (NDV) is a negative-strand RNA virus with oncolytic activity against human tumors. Its effectiveness against tumors and safety in normal tissue have been demonstrated in several clinical studies. Here we show that the spread of NDV infection is drastically different in normal cell lines than in tumor cell lines and that the two cell types respond differently to beta interferon (IFN-β) treatment. NDV rapidly replicated and killed HT-1080 human fibrosarcoma cells but spread poorly in CCD-1122Sk human skin fibroblast cells. Pretreatment with endogenous or exogenous IFN-β completely inhibited NDV replication in normal cells but had little or no effect in tumor cells. Thus, the outcome of NDV infection appeared to depend on the response of uninfected cells to IFN-β. To investigate their differences in IFN responsiveness, we analyzed and compared the expression and activation of components of the IFN signal transduction pathway in these two types of cells. The levels of phosphorylated STAT1 and STAT2 and that of the ISGF3 complex were markedly reduced in IFN-β-treated tumor cells. Moreover, cDNA microarray analysis revealed significantly fewer IFN-regulated genes in the HT-1080 cells than in the CDD-1122Sk cells. This finding suggests that tumor cells demonstrate a less-than-optimum antiviral response because of a lesion in their IFN signal transduction pathway. The rapid spread of NDV in HT-1080 cells appears to be caused by their deficient expression of anti-NDV proteins upon exposure to IFN-β

Loss of N-Linked Glycosylation from the Hemagglutinin- Neuraminidase Protein Alters Virulence of Newcastle Disease Virus

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

Biological Significance of the Second Receptor Binding Site of Newcastle Disease Virus Hemagglutinin-Neuraminidase Protein

The paramyxovirus hemagglutinin-neuraminidase (HN) is a multifunctional protein responsible for attachment to receptors containing sialic acid, neuraminidase (NA) activity, and the promotion of membrane fusion, which is induced by the fusion protein. Analysis of the three-dimensional structure of Newcastle disease virus (NDV) HN protein revealed the presence of a large pocket, which mediates both receptor binding and NA activities. Recently, a second sialic acid binding site on HN was revealed by cocrystallization of the HN with a thiosialoside Neu5Ac-2-S-α(2,6)Gal1OMe, suggesting that NDV HN contains an additional sialic acid binding site. To evaluate the role of the second binding site on the life cycle of NDV, we rescued mutant viruses whose HNs were mutated at Arg516, a key residue that is involved in the second binding site. Loss of the second binding site on mutant HNs was confirmed by the hemagglutination inhibition test, which uses an inhibitor designed to block the NA active site. Characterization of the biological activities of HN showed that the mutation at Arg516 had no effect on NA activity. However, the fusion promotion activity of HN was substantially reduced by the mutation. Furthermore, the mutations at Arg516 slowed the growth rate of virus in tissue culture cells. These results suggest that the second binding site facilitates virus infection and growth by enhancing the fusion promotion activity of the HN