124
J. J. GORMAN et al.
nant C-terminal ion series, consisting principally ofy- and z-type ions, and comparatively less intense ions from the N-terminal series (Fig. 8.3D; Table 8.1).
Whereas the C-terminal ions were approximately IDa heavier than the comparable ions of the unmodified asparaginyl peptide up to and including the z3/y3
pair, the z2/y2 ion pair was equivalent to the corresponding pair of the unmodified sequence. No ions were observed at the position postulated above to correspond with the probable N~-butyl-Asn immonium ion.
3.2
Characterization of the Sites of Proteolytic Activation
of Fusion Protein Precursors of Newcastle Disease Virus Isolates
Newcastle disease virus (NDV) is a serious avian pathogen that exists in a variety
of forms with virulence ranging from highly lethal to innocuous (Waterson et al.
1967). NDV is a member of the paramyxoviridae family of viruses that infect cells
by fusion of the viral lipid bilayer membrane with the plasma cell membrane.
Fusion is mediated by a fusion (F) protein that consists of two disulfide-linked
polypeptide (Fl and F2) chains. The F-protein is produced as an inactive single
chain (FO) bioysynthetic precursor that is activated by a combination of endoproteolytic and exoproteolytic cleavages (Gorman et al; 1988, 1990a; Homma et al.
1975; Scheid et al. 1978). Variability of the virulence of NDV strains is dependent
upon the susceptibilities of their various FO-proteins to proteolytic activation
(Nagai et al. 1976). Susceptibility to proteolysis is dictated by the nature of the
amino acids preceding the susceptible peptide bond (Glickman et al. 1988; Toyoda
et al. 1987). Highly virulent strains have a cleavage signalling motif of two pairs of
basic amino acids, separated by a single glutamine, preceding the susceptible peptide bond. The positions equivalent to that occupied by the first basic amino acids
of these pairs are occupied by another type of amino acid, usually glycine, in avirulent and low-virulence strains. Antibodies capable of detecting these differences
were produced using synthetic peptides as a means of rapid pathotyping of NDV
isolates (Hodder et al. 1993). However, isolates have been encountered that do not
react with the first generation of antibodies due to unusual variability in the cleavage signalling motifs such as the presence of amino acids other than glycine or
basic amino acids (Hodder et al. 1994; Scanlon et al. 1999). When such isolates are
encountered it is necessary to define the structural features of the cleavage sites
for production of antibodies to accommodate the new sequences. The order of the
polypeptide chains in the FO-protein prior to proteolysis is NH2-F2-F1-COOH.
Thus the cleavage signalling motif is at the C-terminus of the F2-polypeptide of
the mature F-protein. Consequently, characterization of cleavage signalling motifs
using isolated mature F-proteins corresponds to characterization of the C-termini
of their F2-polypeptides. A factor that must be borne in mind is that basic amino
acids are exposed at the C-termini as a consequence of primary endoproteolytic
cleavage of the FO-protein and these newly exposed basic amino acids are subsequently removed by a carboxypeptidaseB-like enzyme (Gorman et al. 1990a). A
single basic amino acid is removed from the C-termini of the F2-polypeptides of
avirulent and low-virulence strains and two basic amino acids are removed from
F2-polypeptides of virulent strains.
J. J. GORMAN et al.
nant C-terminal ion series, consisting principally ofy- and z-type ions, and comparatively less intense ions from the N-terminal series (Fig. 8.3D; Table 8.1).
Whereas the C-terminal ions were approximately IDa heavier than the comparable ions of the unmodified asparaginyl peptide up to and including the z3/y3
pair, the z2/y2 ion pair was equivalent to the corresponding pair of the unmodified sequence. No ions were observed at the position postulated above to correspond with the probable N~-butyl-Asn immonium ion.
3.2
Characterization of the Sites of Proteolytic Activation
of Fusion Protein Precursors of Newcastle Disease Virus Isolates
Newcastle disease virus (NDV) is a serious avian pathogen that exists in a variety
of forms with virulence ranging from highly lethal to innocuous (Waterson et al.
1967). NDV is a member of the paramyxoviridae family of viruses that infect cells
by fusion of the viral lipid bilayer membrane with the plasma cell membrane.
Fusion is mediated by a fusion (F) protein that consists of two disulfide-linked
polypeptide (Fl and F2) chains. The F-protein is produced as an inactive single
chain (FO) bioysynthetic precursor that is activated by a combination of endoproteolytic and exoproteolytic cleavages (Gorman et al; 1988, 1990a; Homma et al.
1975; Scheid et al. 1978). Variability of the virulence of NDV strains is dependent
upon the susceptibilities of their various FO-proteins to proteolytic activation
(Nagai et al. 1976). Susceptibility to proteolysis is dictated by the nature of the
amino acids preceding the susceptible peptide bond (Glickman et al. 1988; Toyoda
et al. 1987). Highly virulent strains have a cleavage signalling motif of two pairs of
basic amino acids, separated by a single glutamine, preceding the susceptible peptide bond. The positions equivalent to that occupied by the first basic amino acids
of these pairs are occupied by another type of amino acid, usually glycine, in avirulent and low-virulence strains. Antibodies capable of detecting these differences
were produced using synthetic peptides as a means of rapid pathotyping of NDV
isolates (Hodder et al. 1993). However, isolates have been encountered that do not
react with the first generation of antibodies due to unusual variability in the cleavage signalling motifs such as the presence of amino acids other than glycine or
basic amino acids (Hodder et al. 1994; Scanlon et al. 1999). When such isolates are
encountered it is necessary to define the structural features of the cleavage sites
for production of antibodies to accommodate the new sequences. The order of the
polypeptide chains in the FO-protein prior to proteolysis is NH2-F2-F1-COOH.
Thus the cleavage signalling motif is at the C-terminus of the F2-polypeptide of
the mature F-protein. Consequently, characterization of cleavage signalling motifs
using isolated mature F-proteins corresponds to characterization of the C-termini
of their F2-polypeptides. A factor that must be borne in mind is that basic amino
acids are exposed at the C-termini as a consequence of primary endoproteolytic
cleavage of the FO-protein and these newly exposed basic amino acids are subsequently removed by a carboxypeptidaseB-like enzyme (Gorman et al. 1990a). A
single basic amino acid is removed from the C-termini of the F2-polypeptides of
avirulent and low-virulence strains and two basic amino acids are removed from
F2-polypeptides of virulent strains.