Gangliosides and N-glycoproteins function as Newcastle disease virus receptors

The interaction of enveloped viruses with cell surface receptors is the first step in the viral cycle and an important determinant of viral host range. Although it is established that the paramyxovirus Newcastle Disease Virus binds to sialic acid-containing glycoconjugates the exact nature of the receptors has not yet been determined. Accordingly, here we attempted to characterize the cellular receptors for Newcastle disease virus. Treatment of cells with tunicamycin, an inhibitor of protein N-glycosylation, blocked fusion and infectivity, while the inhibitor of O-glycosylation benzyl-N-acetyl-alpha-D-galactosamide had no effect. Additionally, the inhibitor of glycolipid biosynthesis 1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol blocked viral fusion and infectivity. These results suggest that N-linked glycoproteins and glycolipids would be involved in viral entry but not O-linked glycoproteins. The ganglioside content of COS-7 cells was analyzed showing that GD1a was the major ganglioside component; the presence of GM1, GM2 and GM3 was also established. In a thin-layer chromatographic binding assay, we analyzed the binding of the virus to different gangliosides, detecting the interaction with monosialogangliosides such as GM3, GM2 and GM1; disialogangliosides such as GD1a and GD1b, and trisialogangliosides such as GT1b. Unlike with other viruses, our results seem to point to the absence of a specific pattern of gangliosides that interact with Newcastle disease virus. In conclusion, our results suggest that Newcastle disease virus requires different sialic acid-containing compounds, gangliosides and glycoproteins for entry into the target cell. We propose that gangliosides would act as primary receptors while N-linked glycoproteins would function as the second receptor critical for viral entry.     

Conformational changes of Newcastle disease virus envelope glycoproteins triggered by gangliosides.

We have investigated the conformational changes of Newcastle disease virus (NDV) glycoproteins in response to receptor binding, using 1,1-bis(4-anilino)naphthalene-5,5-disulfonic acid (bis-ANS) as a hydrophobicity-sensitive probe. Temperature- and pH-dependent conformational changes were detected in the presence of free bovine gangliosides. The fluorescence of bis-ANS was maximal at pH 5. The binding of bis-ANS to NDV was not affected by chemicals that denature the fusion glycoprotein, such as reducing agents, nor by the presence of neuraminidase inhibitors such as N-acetyl neuramicic acid. Gangliosides partially inhibited fusion and hemadsorption, but not neuraminidase hemagglutinin-neuraminidase glycoprotein (HN) activity. A conformational intermediate of HN, triggered by the presence of gangliosides acting as receptor mimics, was detected. Our results indicate that, upon binding to free gangliosides, HN undergoes a certain conformational change that does not affect the fusion glycoprotein.     

Biological consequences of neuraminidase deficiency in Newcastle disease virus.

A second-step revertant (L1) of a temperature-sensitive mutant (C1) of Newcastle disease virus agglutinated erythrocytes normally but had less than 3% of the wild-type (strain AV) levels of neuraminidase activity. Revertant L1 had seven times more virion-associated N-acetylneuraminic acid (NANA) than strain AV. NANA residues on purified virions were specifically labeled with periodate and tritiated borohydride. Analyses of radiolabeled L1 virions on sodium dodecyl sulfate-polyacrylamide gels showed that most of the virion-associated NANA was in a high-molecular-weight component with an electrophoretic mobility different from that of any known viral protein. NANA was also detected in molecules with the electrophoretic mobility of the viral glycoproteins HN and F1. Revertant L1 had a twofold lower rate constant of attachment to HeLa cells than that of the wild-type. Treatment of L1 virions with Vibrio cholerae neuraminidase removed the excess NANA and returned L1 attachment kinetics to normal. Revertant N1, which has 10-fold more neuraminidase activity than L1, penetrated host cells at the same rate as L1. L1 was impaired in elution from erythrocytes. Removal of virion-associated NANA exacerbated this defect. Despite a small disadvantage in attachment and a major defect in elution relative to strain AV, revertant L1 enjoyed a slight advantage over the wild-type during a single reproductive cycle in cultured chicken embryo cells.     

Relationships among the polypeptides of Newcastle disease virus.

We have studied the relationships among the polypeptides of Newcastle disease virus by using both kinetic and tryptic peptide analyses. The results of our tryptic peptide analyses suggest that there are at least six unique viral polypeptides--L, HN, FO(F), NP, M, and a 47,000-dalton polypeptide. The small virion glycopolypeptide F is related to FO, a glycopolypeptide found only in infected cells. In addition, several smaller polypeptides, including a 53,000-dalton polypeptide found both in purified virions and in infected cells, are related to the nucleocaspid protein. Kinetic analysis of each viral polypeptide reveals that all of the major viral polypeptides, with the possible exception of L, are stable after an amino acid chase. A precursor-product relationship between FO and F was not demonstrable by pulse-chase experiments. Also, almost the same relative amount of F, the putative product, was present in infected cultures after either 5 or 30 min of radioisotopic labeling. These results suggest that FO is processed rapidly.     

Fatty acid modification of Newcastle disease virus glycoproteins.

The fatty acid acylation of Newcastle disease virus hemagglutininin-neuraminidase and fusion glycoproteins was assayed. [3H]palmitate label was associated with cytoplasmic fusion proteins (F0 and F1) and virion-associated F1. In contrast, there was no detectable [3H]palmitate label associated with the hemagglutin-neuraminidase protein in Newcastle disease virus-infected Chinese hamster ovary cells or chicken embryo cells or in virions released from these cells. Thus, fatty acid modification may not be important for the maturation of some glycoproteins.     

Three-dimensional organization of Rift Valley fever virus revealed by cryoelectron tomography

Rift Valley fever virus (RVFV) is a member of the Bunyaviridae virus family (genus Phlebovirus) and is considered to be one of the most important pathogens in Africa, causing viral zoonoses in livestock and humans. Here, we report the characterization of the three-dimensional structural organization of RVFV vaccine strain MP-12 by cryoelectron tomography. Vitrified-hydrated virions were found to be spherical, with an average diameter of 100 nm. The virus glycoproteins formed cylindrical hollow spikes that clustered into distinct capsomeres. In contrast to previous assertions that RVFV is pleomorphic, the structure of RVFV MP-12 was found to be highly ordered. The three-dimensional map was resolved to a resolution of 6.1 nm, and capsomeres were observed to be arranged on the virus surface in an icosahedral lattice with clear T=12 quasisymmetry. All icosahedral symmetry axes were visible in self-rotation functions calculated using the Fourier transform of the RVFV MP-12 tomogram. To the best of our knowledge, a triangulation number of 12 had previously been reported only for Uukuniemi virus, a bunyavirus also within the Phlebovirus genus. The results presented in this study demonstrate that RVFV MP-12 possesses T=12 icosahedral symmetry and suggest that other members of the Phlebovirus genus, as well as of the Bunyaviridae family, may adopt icosahedral symmetry. Knowledge of the virus architecture may provide a structural template to develop vaccines and diagnostics, since no effective anti-RVFV treatments are available for human use.     

Noncytopathic mutants of Newcastle disease virus

We have isolated a novel class of mutants of Newcastle disease virus which are less cytopathic than their virulent parent but are still capable of infectious virus production. Unlike wild-type virus, the mutants did not form plaques after 2 days of incubation; they did, however, make hemadsorbing spots. The mutants range in production of infectious virus from 10 to 200% of that of the wild type. They were less cytopathic in a single cycle of infection by light microscopy, loss of protein from the plate, and inhibition of total protein accumulation. All of the mutants exhibited extended mean embryo death times, a correlate of virulence in the adult animal.     

Protection against respiratory syncytial virus by a recombinant Newcastle disease virus vector

Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract disease in infants and the elderly, but no safe and effective RSV vaccine is yet available. For reasons that are not well understood, RSV is only weakly immunogenic, and reinfection occurs throughout life. This has complicated the search for an effective live attenuated viral vaccine, and past trials with inactivated virus preparations have led to enhanced immunopathology following natural infection. We have tested the hypothesis that weak stimulation of innate immunity by RSV correlates with ineffective adaptive responses by asking whether expression of the fusion glycoprotein of RSV by Newcastle disease virus (NDV) would stimulate a more robust immune response to RSV than primary RSV infection. NDV is a potent inducer of both alpha/beta interferon (IFN-alpha/beta) production and dendritic cell maturation, while RSV is not. When a recombinant NDV expressing the RSV fusion glycoprotein was administered to BALB/c mice, they were protected from RSV challenge, and this protection correlated with a robust anti-F CD8+ T-cell response. The effectiveness of this vaccine construct reflects the differential abilities of NDV and RSV to promote dendritic cell maturation and is retained even in the absence of a functional IFN-alpha/beta receptor.