Analysis of pathotype-specific structural features and cleavage activation of Newcastle disease virus membrane...

Peptides were synthesized, that correspond to cleaved and trimmed carboxyl termini of the F2 polypeptide regions of fusion (F) protein precursors (F0 proteins) in four different strains of Newcastle disease virus (NDV). These peptides differed only within the four carboxyl-terminal residues and represent F2 polypeptides of virulent (AV), low-virulence (EG) and avirulent (V4 and WA) pathotypes of NDV. Polyclonal rabbit antisera against each peptide reacted with their corresponding monomeric F2 polypeptides and F protein oligomers as analysed by immunoblotting of egg-propagated virions. Bidirectional cross-reactivity was observed between V4 and EG antisera and F2 polypeptides which differ only by a single variation of lysine and arginine at position 3 from their carboxyl termini. The other two antisera (AV and WA) were specific for their corresponding F2 polypeptides. All of these antisera were shown to react in a strain-specific manner with intact egg-propagated virions in an ELISA. A previously described antiserum, designed to target the haemagglutinin-neuraminidase (HN) protein precursor (HN0 protein) of avirulent strains of NDV, has been shown to be specific for residual HN0 protein of avirulent virions propagated in embryonated chicken eggs. Whereas the antiserum targeted at the carboxyl terminus of the V4 F2 polypeptide did not react with F0 proteins of cell culture-propagated strains in immunoblotting, antipeptide antibodies targeted at another region of the F2 polypeptide and a segment of the F1 polypeptide did react with the F0 protein from infected cells. These data are consistent with inclusion of the terminal carboxylate of the F2 polypeptides in the recognition determinants of the antibodies targeted at the carboxyl terminus of the V4 F2 polypeptide. The antisera described herein are ideally suited to rapid immunochemical pathotyping of NDV isolates and immunochemical characterization of the sites of intracellular cleavage activation of F0 and HN0 proteins and may be useful for defining interactions involved in F protein folding.