Fusogenic activity of reconstituted newcastle disease virus envelopes: a role for the hemagglutinin-neuraminidase protein in the fusion process.

Enveloped viruses, such as newcastle disease virus (NDV), make their entry into the host cell by membrane fusion. In the case of NDV, the fusion step requires both transmembrane hemagglutinin-neuraminidase (HN) and fusion (F) viral envelope glycoproteins. The HN protein should show fusion promotion activity. To date, the nature of HN-F interactions is a controversial issue. In this work, we aim to clarify the role of the HN glycoprotein in the membrane fusion step. Four types of reconstituted detergent-free NDV envelopes were used, on differing in their envelope protein contents. Fusion of the different virosomes and erythrocyte ghosts was monitored using the octadecyl rhodamine B chloride assay. Only the reconstituted envelopes having the F protein, even in the absence of HN protein, displayed residual fusion activity. Treatment of such virosomes with denaturing agents affecting the F protein abolished fusion, indicating that the fusion detected was viral protein-dependent. Interestingly, the rate of fusion in the reconstituted systems was similar to that of intact viruses in the presence of the inhibitor of HN sialidase activity 2,3-dehydro-2-deoxy-N-acetylneuraminic acid. The results show that the residual fusion activity detected in the reconstituted systems was exclusively due to F protein activity, with no contribution from the fusion promotion activity of HN protein.

Recombinant Newcastle disease virus as a vaccine vector.

A complete cDNA clone of the Newcastle disease virus (NDV) vaccine strain Hitchner B1 was constructed, and infectious recombinant virus expressing an influenza virus hemagglutinin was generated by reverse genetics. The rescued virus induces a strong humoral antibody response against influenza virus and provides complete protection against a lethal dose of influenza virus challenge in mice, demonstrating the potential of recombinant NDV as a vaccine vector.

Membrane glycoproteins of Newcastle disease virus: nucleotide sequence of the hemagglutinin-neuraminidase cloned gene and structure/function relationship of predicted amino acid sequence.

The nucleotide sequence of the glycoprotein hemagglutinin-neuraminidase (HN) gene of the Newcastle disease virus (NDV) strain Clone-30 has been determined. The open reading frame of the HN gene contains 1731 nucleotides and encodes a protein of 577 amino acids. Three highly conserved patterns among all paramyxovirus HN glycoproteins, and one additional conserved species-specific region are present. The protein contains five potential N-glycosylation sites, all but one located in the C-terminal external domain. The secondary structure prediction shows that the C-terminal external domain is mostly arranged in beta-sheets, while alpha-helices are predominantly located in the N-terminal domain. The nucleotide sequence data of the HN gene reported in this paper has been deposited in the GenBank database, under accession number AF098289.

Study of the influence of salt concentration on Newcastle disease virus matrix protein aggregation.

The aggregation process of Newcastle disease virus matrix protein (M protein) has been studied using light scattering. We observed that the aggregation of M protein is inversely correlated with ionic strength and that this process can be reversed by high salt concentrations. It was found that the oligomeric structure of NDV matrix protein is different from those described earlier for other matrix proteins of enveloped viruses.

A differential scanning calorimetric study of Newcastle disease virus: identification of proteins involved in thermal transitions.

The irreversible thermal denaturation of Newcastle disease virus was investigated using different techniques including high-sensitivity differential scanning calorimetry, thermal gel analysis intrinsic fluorescence, and neuraminidase activity assays. Application of a successive annealing procedure to the scanning calorimetric endotherm of Newcastle disease virus furnished four elementary thermal transitions below the overall endotherm; these were further identified as coming from the denaturation of each viral protein. The shape of these transitions, as well as their scanrate dependence, was explained by assuming that thermal denaturation takes place according to the kinetic scheme N-->(k)D, where k is a first-order kinetic constant that changes with temperature, as given by the Arrhenius equation; N is the native state; and D is the denatured state. On the basis of this model, activation energy values were calculated. The data obtained with the other methods used in this work support the proposed two-state kinetic model.

Modulation of the neuraminidase activity of HN protein from Newcastle disease virus by substrate binding and conformational change: kinetic and thermal denaturation studies.

A statistical study of the enzyme kinetics of the soluble fraction of Haemagglutinin-Neuraminidase (HN) protein from Newcastle Disease virus reveals that a high substrate concentrations its neuraminidase activity follows substrate-inhibition kinetics, in which the binding of a second molecule of substrate to the protein inhibits its enzymatic activity. Results show that the enzymatic activity is modulated by the second substrate to a different extent when the protein is in different environments. Taken together with results obtained by thermal denaturation studies of HN under varying conditions the data show that conformational changes leading to a loss of rigidity of HN are concomitant with the loss of catalytic activity. Also, electrophoretic and sucrose gradient analyses show that the soluble domain of HN behaves as a monomer.