Pathotyping isolates of Newcastle disease virus using antipeptide antibodies to pathotype-specific regions of their fusion and hemagglutinin-neuraminidase proteins.

Antipeptide antibodies have been evaluated for their abilities to predict the characteristics of the cleavage motifs of the fusion protein precursors (F0) of 25 isolates of Newcastle disease virus (NDV) with a range of virulences, grouped into 12 sets according to their monoclonal antibody reactivities. A Western blot format was used to show that antisera to synthetic peptides representing sequences at the C-termini of the F2-polypeptides of defined pathotypes of NDV usually distinguish between pathotypes on the basis of their Fo cleavage sequences. However, exceptions were found with three groups of virulent isolates. Protein sequencing and mass spectral analysis of the F2-polypeptide of isolate Texas GB from one of these groups, identified an anomalous cleavage/activation process which removed the amino acids required for recognition by the antisera. This probably also explained the lack of reactivity of the Roakin isolate and low reactivity of the Komarov isolate from this group. The other exceptions involved isolates in groups with cleavage region variations from the usual motif of virulent isolates or isolates with undefined cleavage motifs. Antipeptide antisera were also raised to sections of the 45 residue C-terminal extension the hemagglutinin-neuraminidase precursor (HN0) encoded by the genes of some avirulent isolates. Western blot analysis showed that positive reactions with antibodies to peptides based on sequences between residues 577 and 613 of the HN0 was evidence for the presence of an avirulent isolate but did not exclude the presence of other pathotypes. Antisera designed to target residues 569-577 detected HN0 extensions of 6 residues on isolates known to encode such extensions. These antisera also enabled differentiation of isolates with HN0 extensions of 6 residues from those with no extension, however, it was not possible to determine the virulence of isolates based on reaction with these antisera.

The analysis of Merino wool cuticle and cortical cells by Fourier transform Raman spectroscopy.

Wool fibers are comprised of proteins known as alpha-keratins and have a complex morphological structure. The major components of this structure, the cuticle and cortical cells, differ in the conformations of their chains as well as their amino acid compositions. High quality Fourier transform Raman spectra of cortical and cuticle cells isolated from fine Merino wool fibers have been obtained. Raman spectroscopy has been shown to be sensitive to the differences in both secondary structure and amino acid composition. The cortical cells were found to be higher in alpha-helical content as compared to the cuticle cells, which had an increased disordered content. Specific information, consistent with amino acid analysis results, regarding cystine, tyrosine, tryptophan, and phenylalnine residues, were obtained for both the cortical and cuticle cells. In addition, the Raman spectra provided information about free thiol groups, amino acids residues with amide group side chains, and residues with protonated carboxyl group side chains. Middle ir transmission spectra of these isolated cells were also obtained. In comparison to the Raman data, the middle ir spectra were found to be not as rich in information.

Characterization of field isolates of Newcastle disease virus using antipeptide antibodies.

A recent Australian field isolate of Newcastle disease virus (NDV) was analyzed with antipeptide antibodies capable of differentiating between the sequences at the cleavage activation sites of fusion proteins of different NDV pathotypes. The isolate was found to have the same fusion protein cleavage activation signal as the V4 isolate of the Queensland strain of NDV. However, the isolate failed to react with an antibody specific for the carboxyl-terminal extension on the hemagglutinin/neuraminidase (HN)-protein precursor (HNo-protein) of the V4 isolate and other similar strains (e.g., Ulster and D26). Identity of the fusion protein cleavage activation motif of the isolate was confirmed by chemical analysis of purified fusion protein subunits of the isolate. The combination of a V4-like fusion protein cleavage activation motif but lack of an HNo-protein has not been described before, and the findings indicate that the isolate is a distinct strain of NDV. Analysis of a range of isolates from the state of Victoria, Australia, indicated that similar strains have been present in Australia since at least 1976. Other isolates examined appeared to have fusion protein cleavage activation motifs that could not be defined with the fusion-protein-targeted antibodies. These isolates also appeared to lack the HNo-protein characteristic of the Queensland strain.

Comparison of the positions and efficiency of cleavage activation of fusion protein precursors of virulent and avirulent strains of Newcastle disease virus: insights into the specificities of activating proteases.

The F1- and F2-polypeptide components of in ovo activated fusion proteins of one virulent (AV or Australia-Victoria) strain, one low-virulence (EG or Eaves-Grimes) strain, and two avirulent (V4 or Queensland and WA2116) strains of Newcastle disease virus (NDV) were isolated and subjected to structural analysis. This included complementary application of amino acid analysis, fast atom bombardment-mass spectrometry, and N-terminal sequence analysis to fragments isolated from AspN protease digests of the F2-polypeptides using HPLC. As a result, the complete sequences of the F2-polypeptides were determined, including documentation of glycosylation of asparagine 54. The sequence of the cleavage-activation site of the WA2116 F0-protein was found to be distinctly different from this site in any other NDV F0-protein. Cleavage activation at the C termini of the F2-polypeptide regions was found to have occurred to approximately equivalent extents at arginines 82 and 85 of the AV and EG strains, but was restricted largely to arginine 85 of the V4 strain and completely to arginine 85 of the WA2116 strain. In each case cleavage activation was apparently succeeded by trimming of the basic residues from the newly formed C termini. Immunochemical analysis with antipeptide antisera showed that the extent of cleavage was influenced by amino acids adjacent to these arginines. These data provide insight into the substrate specificities of the enzymes involved in cleavage activation of the fusion protein precursors.

Characterization of the sites of proteolytic activation of Newcastle disease virus membrane glycoprotein precursors.

The F1- and F2-polypeptide components of the fusion proteins and the hemagglutinin/neuraminidase proteins of the avirulent Queensland (V4) and virulent Australia-Victoria (AuV) strains of Newcastle disease virus have been isolated and subjected to extensive primary structural analysis including amino-terminal sequence analysis and fast atom bombardment-mass spectrometry mapping. Nucleotide sequence analysis was performed on the gene which encodes the V4 hemagglutinin/neuraminidase protein. Signal peptidase cleavage was found to have occurred at the Ser31-Leu32 peptide bond of the primary translation products of the fusion protein genes. Activation cleavage of the V4 fusion protein precursor generated a sequence of -Gly-Lys-Gln-Gly84 at the carboxyl terminus of the F2-polypeptide and an amino-terminal sequence of the F1-polypeptide commencing with 86Leu-Ile-Gly-. The V4 hemagglutinin/neuraminidase protein gene was found to encode a primary translation product 45 amino acids longer at the carboxyl terminus than obtainable from the corresponding gene of the AuV strain (McGinnes, L. W., and Morrison, T. G. (1986) Virus Res. 5, 343-356). However, post-translational proteolytic processing, exclusive to the primary translation product of the V4 hemagglutinin/neuraminidase protein gene, was found to have removed the last 42 residues of this carboxyl-terminal appendage.

Fluorescent labeling of cysteinyl residues. Application to extensive primary structure analysis of proteins on a microscale.

The specificity and efficiency of fluorescent labeling of proteins by reduction and subsequent alkylation with 5-N-[(iodoacetamidoethyl)amino]naphthalene-1-sulfonic acid (5-I-AEDANS) [J.J. Gorman, (1987) Anal. Biochem. 160, 376-387] has been investigated. Proteins studied include porcine insulin, chicken ovalbumin and bovine serum albumin. Amino acid analysis of the B-chain derivative of insulin revealed quantitative recovery of cysteine in its S-carboxymethyl form and no other carboxymethylated amino acid derivatives. Fast-atom-bombardment mass spectrometric (FAB-MS) analysis of this derivative also indicated specific labeling of cysteine residues and automated stepwise protein sequence analysis of the derivative was performed to completion with initial and average repetitive yields of 73% and 96%, respectively. Tryptic peptides produced from the ovalbumin and serum albumin derivatives were fractionated by HPLC and subsequently analysed by amino acid analysis, FAB-MS and automated stepwise protein sequence analysis. These analyses have revealed that the labeling procedure exhibits a high degree of efficiency and is specifically directed towards S-alkylation of cysteine residues. The high level of fluorescence intensity of the label enabled specific detection of trace quantities of cysteine-containing peptides derived from contaminating protein(s). It is apparent that in addition to facilitating isolation of small quantities of proteins the labeling procedure is compatible with standard protein chemistry techniques involved in obtaining extensive structural data on isolated proteins.