Role of mass spectrometry in mapping strain variation and post-translational modifications of viral proteins.

Enzymatically derived fragments of the nucleocapsid protein from one strain (V4) of the paramyxovirus, New castle disease virus (NDV), have been aligned with the sequence deduced for a related strain (D26) by gene sequence analysis. This process involved extensive use of fast atom bombardment (FAB) mass spectrometry of unfractionated tryptic digests and fragments separated from tryptic or AspN protease digests by high-performance liquid chromatography (HPLC). Amino acid analysis and stepwise Edman degradation sequence analysis were used to complement FAB mass spectral data or as alternatives where no ions were produced by FAB. The nature of biosynthetic processing and blockage (acetylation) at the N-terminus of the protein were confirmed using collision-induced dissociation. Data obtained by direct analysis of the V4 nucleocapsid protein facilitated mapping of sequence variations within the nucleocapsid protein of the antigenically distinct WA2116 strain of NDV. Most of the WA2116 protein was mapped by FAB mass spectrometric analysis of HPLC fractions, thus amino acid analysis or stepwise sequence analysis were only required where FAB mass spectral data were inconclusive or indicated amino acid variations. This approach to comparison of NDV nucleocapsid proteins is proposed as a general strategy for mapping strain variation and post-translational modifications of viral proteins.

Mature, cell-associated HN protein of Newcastle disease virus exists in two forms differentiated by posttranslational modifications.

Characterization of the posttranslational modifications of the mature, cell-associated hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) revealed that the HN protein exists in two forms differentiated by disulfide bonds and glycosylation. One form, HNa, contains intermolecular disulfide bonds and is endoglycosidase H partially resistant. The other form, HNb, is not linked by disulfide bonds and is endoglycosidase H sensitive. Both forms of the protein are modified with fucose indicating transport to the Golgi membranes. Both forms are detected at the cell surface by monoclonal antibody. Furthermore, both forms are transported to the cell surface with identical kinetics. HNa is incorporated into virions. HNb is not incorporated into virions and is presumably degraded. The cDNA derived from the HN gene was expressed from a retrovirus vector. The majority of the protein expressed was in the nonvirion-associated form b. Evidence is presented that the level of gene expression determines the ratio of the two forms of HN protein. At high levels of expression, the virion-associated form is favored while at low levels of expression the nonvirion-associated form is favored. The results presented have implications for persistent infections as well as expression of viral genes from different vectors.

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.

Detection of polycistronic transcripts in Newcastle disease virus infected cells and identification of their sequence content.

The synthesis of six to seven polycistronic transcripts of Newcastle disease virus (NDV) in BHK cells was detected by Northern hybridization using cDNA clones generated by reverse transcription of five NDV mRNAs. Within the molecular weight range resolved by the gel electrophoresis system employed, four of the transcripts were suggested to be distronic, containing sequences of two genes, NP-P, P-M, M-F0 and F0-HN, respectively. In addition, tricistronic molecules of M-F0-HN and possibly of NP-P-M as well as P-M-F0 appeared to develop, although they were very low in amount. These data suggest a gene order of NP-P-M-F0-HN on the NDV genome. The polycistronic as well as monocistronic transcripts were generated with an almost constant proportion in amount throughout the virus replication. Further, at least several of them were also generated under the conditions where only the primary transcription was allowed by inhibiting de novo protein synthesis. Therefore, it appears likely that there is no distinct temporal control in NDV genome expression.

Expression at the cell surface of native fusion protein of the Newcastle disease virus (NDV) strain Italien from cloned cDNA.

A cDNA library was constructed with poly(A)+-mRNAs from NDV-Italien infected BHK-21 cells. A clone, that hybridized to the F gene mRNA, was sequenced. A long open reading frame encodes for a protein of 553 amino acids, with a calculated molecular weight of 59,153, consisting of twelve cysteine residues and six potential glycosylation sites. The protein sequence contains a hydrophobic region at the N-terminus of F1 and a presumptive long transmembrane fragment near the C-terminus. Comparison of the F proteins from NDV strains Italien and Australia-Victoria shows that the sequences are very similar, with conservation of most cysteine residues and of the potential glycosylation sites. The F coding sequence was inserted into the genome of vaccinia virus under the control of vaccinia P7.5 transcriptional regulatory sequences. Expression of F protein was demonstrated by indirect immunofluorescence with five anti-F monoclonal antibodies known to react with conformational epitopes.

Pathogenicity of antigenic variants of Newcastle disease virus Italian strain selected with monoclonal antibodies.

Antigenic variants of the Italian strain of NDV were selected using monoclonal antibodies directed against the HN and F proteins of Italian virus. Antigenic mapping of the HN and F proteins using variant viruses in cross neutralization tests revealed the presence of at least two different epitopes on HN and four epitopes on F protein. Immunoselected variant viruses were demonstrated to have different intravenous pathogenicity index than the parental Italian virus.

Nucleotide sequence of the gene encoding the fusion glycoprotein of Newcastle disease virus.

The nucleotide sequence of the gene encoding the fusion (F) glycoprotein of the Beaudette C strain of Newcastle disease virus (NDV) has been determined from cDNA clones obtained from virion RNA. The gene is 1792 nucleotides long, including mRNA start and polyadenylation signals typical of paramyxoviruses. The single open reading frame encodes a polypeptide of 553 amino acids, with a predicted molecular weight of 59042. The F polypeptide has three regions of high hydrophobicity: an N-terminal signal peptide, the N terminus of F1 (known from protein sequencing) and a C-terminal membrane-spanning region by which the F glycoprotein is anchored to the membrane. The cleavage site of F0 is located in a highly basic region of the F polypeptide. Five potential asparagine-linked glycosylation sites are present in the amino acid sequence, of which one is in F2 and the others in F1. Comparison of the NDV F amino acid sequence to those from other paramyxoviruses reveals homology to Sendai virus, simian virus 5 and human respiratory syncytial virus. There is also limited homology between the N terminus of F1 of NDV and the N termini of HA2 of influenza viruses. Post-translational modifications of the NDV F polypeptide are discussed in the light of information provided by the amino acid sequence.

Differentiation of Newcastle disease virus strains by one-dimensional peptide mapping.

One-dimensional peptide mapping was used for the differentiation of Newcastle disease virus (NDV) strains. Virions were purified in one step, and digested with Staphylococcus aureus V8 protease or chymotrypsin without prior separation of their proteins. Peptides were separated by polyacrylamide gel electrophoresis and stained with Coomassie blue. This method proved to be a simple, economic and reproducible means of differentiating NDV strains.

Structure of the major oligosaccharides in the fusion glycoprotein of Newcastle disease virus.

The fusion glycoprotein (F0) was isolated from Newcastle disease virus (NDV) particles metabolically labelled with [2-3H]mannose; it was successively digested with protease and with endo-beta-N-acetylglucosaminidase from Streptomyces griseus. In this manner, the majority of the oligosaccharides in NDV F0 could be liberated. After reduction with NaBH4, they were separated by high-performance liquid chromatography, and were subjected to structural analysis. Using micromethylation/capillary gas chromatography/mass fragmentography, alpha-mannosidase digestion, and acetolysis, it was found that the enzymatically released NDV F0 oligosaccharides are common oligomannosidic glycoprotein glycans of size classes (Man)8GlcNAc, Man)7GlcNAc, (Man)6GlcNAc, (Man)9GlcNAc, and (Man)5GlcNAc (in order of prevalence). The major structural isomers present in the NDV F0 (Man)8GlcNAc to (Man)5GlcNAc fractions were shown to lack mannose residues D2, D1D2 or D2D3, D1D2D3, and CD1D2D3, respectively, of (Man)9GlcNAc.

The carboxyterminus of the hemagglutinin-neuraminidase of Newcastle disease virus is exposed at the surface of the viral envelope.

The amino-terminal and the carboxy-terminal amino acids of the hemagglutinin-neuraminidase glycoprotein of the Ulster strain of Newcastle disease virus have been analyzed before and after proteolytic activation of the precursor HNo (Mr approximately 82K). The amino termini of HNo and of the large cleavage fragment HN (approximately 74K) obtained by in vivo and in vitro proteolysis could not be sequenced by Edman degradation. This indicates that in both instances the amino termini are blocked. The carboxy termini of HNo and HN are different as demonstrated by end-point digestion with carboxypeptidase A. Furthermore, a small cleavage fragment (approximately 9K) of HNo that was removed from the virion after trypsin treatment could be purified by HPLC. In contrast to HN, this fragment displays a free amino terminus susceptible to Edman degradation. These data indicate that conversion of HNo involves removal of a 9K glycopeptide from the carboxy-terminal end. Thus, it has to be concluded that, unlike most other viral glycoproteins, the hemagglutinin-neuraminidase is inserted in the envelope with its carboxy terminus exposed at the surface of the virus particle.

Biological functions of monospecific antibodies to envelope glycoproteins of Newcastle disease virus.

Monospecific antisera to HN and F glycoproteins of Newcastle disease virus were prepared, and their effects on the biological activities of the virus were investigated. Anti-HN serum inhibited hemagglutinating and neuraminidase activity, as well as hemolysis. Anti-F serum had no effect on hemagglutination or neuraminidase but inhibited hemolysis and virus-induced cell fusion. Anti-HN serum was highly neutralizing, while neutralization by anti-F serum was very inefficient in conventional plaque reduction tests, although both sera were estimated to contain comparable amounts of antibody reacting with the virus as indicated by complement fixation and immuno-diffusion tests. The neutralizing activity of anti-F serum was greatly enhanced by the addition of anti-IgG serum or fresh guinea pig serum, whereas that of anti-HN serum was little enhanced. Anti-HN serum incorporated in the agar overlay suppressed the development of plaques to some degree, while anti-F serum had little effect. The combination of anti-HN and anti-F sera resulted in a marked decrease in the number and size of plaques, demonstrating the synergistic effect of the two species of antibody in the containment of the spread of viral infection.

A temperature-sensitive mutant of Newcastle disease virus which is affected in both haemagglutinin-neuraminidase and matrix proteins.

Virions prepared from a non-revertible temperature-sensitive (ts) mutant (ts53) of Newcastle disease virus (NDV) grown in ovo at the permissive temperature (34 degrees C) possessed thermolabile haemagglutination and neuraminidase activities compared with parental (ts+) virions. Purified haemagglutinin-neuraminidase (HN) protein from ts53 virions was also more thermolabile than ts+ HN protein. SDS-PAGE analysis of [3H]leucine pulse- and pulse/chase-labelled NDV proteins synthesized in chick embryo fibroblasts following infection with ts+ and ts53 virus revealed that ts53 matrix (M) protein was unstable and disappeared during chase incubations only at the non-permissive temperature (42 degrees C). The non-revertibility of the ts53 mutant may indicate that it is a double mutant affected in both HN and M genes; alternatively this mutant may only be affected in the HN gene, the close physical association of the thermolabile HN with the M protein during virus maturation resulting in the lack of protection of the M protein from the action of cellular proteases at the non-permissive temperature.

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.

Separation and sugar component analysis of the oligosaccharides in the surface glycoproteins of Newcastle disease virus.

The precursor glycoproteins HN0 and F0 in the surface spikes of Newcastle Disease Virus strain Ulster as produced by MDBK cells, were found to contain 10.4 and 11.9 weight per cent, respectively, of the sugars typical for N-glycosidically linked glycoprotein glycans. A molar ratio of D-mannose:D-galactose: L-fucose:N-acetyl-D-glucosamine approaching 1.0:1.1:0.5:1.0 was found for HN0, and of 1.0:0.7:0.3:0.6 for F0. By a sequence of degradation (with pronase, with endo-beta-N-acetylglucosaminidase H [endo H], and by hydrazinolysis) and separation procedures (Concanavalin A-affinity and Biogel P-4 chromatography), the radiolabelled carbohydrate moieties of NDV HN0 and F0 (as oligosaccharitols) were separated into (at least) ten and eight fractions, respectively. Separate in vivo labelling with tritiated derivatives of the four sugars showed that both glycoproteins contain oligosaccharides of the oligomannosidic ("high mannose"), of the N-acetyllactosaminic ("complex"), as well as of the "mixed" type. The majority of the oligosaccharides in F0, but not of those in HN0, was found to be endo H-sensitive.

Topological location and biological significance of phospholipids in the membrane of Newcastle disease virus. Hydrolysis of phospholipids in intact virion with pure phospholipases A2, C, and D.

The composition, topological distribution and biological significance of phospholipids in the membrane of Newcastle disease virus (NDV) grown in embryonated chicken eggs were investigated. Phosphatidylethanolamine and sphingomyelin were the predominant phospholipids in NDV membrane. The location of phospholipids in the lipid bilayer of the membrane was studied by assessing their reactivities with highly purified phospholipase A2 (Agkistrodon halys blomhoffi) and phospholipase D (Streptomyces chromofuscus), and the biological role of membrane phospholipids was also investigated by using pure phospholipases A2, C (Bacillus cereus) and D. Choline-containing phospholipids were found predominantly in the outer layer of the membrane. The inner layer was composed mainly of aminoglycerophospholipids, though a fair amount of them also appeared to be located in the outer half of the bilayer. When intact virion was treated with phospholipase C, marked decreases in hemolytic activity and infectivity mediated by viral fusion (F) glycoprotein were observed, but hemagglutinating and neuraminidase activities did not change significantly. Apparently complete hydrolysis of phospholipids in the outer half of the lipid bilayer with phospholipase D caused about 22% decrease in the original hemolytic activity. On the other hand, when all phosphatidylcholine and aminoglycerophospholipids in the outer half of the viral membrane were hydrolyzed with purified phospholipase A2, no significant change in viral hemolytic activity or morphology was detected. No marked change of hemagglutinating and neuraminidase activities was detected on treatment of NDV with phospholipases A2 and D. The above results suggest that the integrity of fatty acid ester of glycerophospholipids in NDV membrane is not essential for the manifestation of viral activities, though polar groups of the phospholipids in the outer half of the membrane may be involved in the function of fusion (F) glycoprotein, but not in that of hemagglutinating and neuraminidase (HN) glycoprotein of NDV.

Evidence for an interaction between the membrane protein of a paramyxovirus and actin.

Evidence for an interaction of the membrane (M) protein of Newcastle disease and Sendai viruses with cellular actin was obtained by three different techniques. M protein linked to Sepharose 4B was found to bind actin, but not myoglobin or bovine serum albumin, and to selectively remove actin from a mixture of these three proteins. Sedimentation of a mixture of M protein and F-actin through a sucrose gradient resulted in sedimentation of M protein with actin. Control proteins, bovine serum albumin and cytochrome c, did not sediment with actin. In circular dichroism studies, M protein added to actin in a 1:1 complex resulted in a significant increase in negative ellipticity at 220 nm, which corresponds to an increase in alpha-helix and a decrease in beta-structure and random coil. This is indicative of an interaction between M protein and actin. It is possible that the frequent identification of cellular actin in a number of enveloped viruses may be attributed to the interaction of actin and M protein or its equivalent.

Differentiation of exotic strains of Newcastle disease virus by oligonucleotide fingerprinting.

The oligonucleotide fingerprints of five of six strains of velogenic-viscerotropic Newcastle disease (VVND) virus were readily distinguishable, even though the strains could not be differentiated by a series of in vivo and in vitro procedures. Two of the VVND virus strains tested (10333-0 and 8231412) showed marked similarity to the prototype strain (MIL-0). Two of the remaining three strains (80-27489 and 80-28634) had identical oligonucleotide fingerprints, even though they had a different isolation history and exhibited small differences in their virulence for chickens. Fingerprints of the sixth VVND strain, 80-12927, demonstrated some similarities when it was compared with the prototype virus fingerprint, but it was quite different from strains 80-27489, 10333-0 and 8231412.

Non-structural proteins in Newcastle disease virus-infected cells.

Examination of pulse-labelled Newcastle disease virus (NDV)-infected chick embryo fibroblasts (CEF) by two-dimensional polyacrylamide gel electrophoresis revealed the presence of two-virus-coded non-structural polypeptides of mol. wt. 36K and 33K. Longer pulses and pulse-chase incubations revealed the production of an additional, glycosylated, non-structural polypeptide of mol. wt. 40K (gp40). Kinetic arguments suggest that 36K and 33 K are primary translation products but that gp40 is not. 36K was stable in chase incubations, but 33K was not. Partial digest peptide analysis showed that gp40 and an additional glycosylated polypeptide gp62, which is sometimes present (Chambers & Samson, 1980), are related to the HN polypeptides. Partial digest peptide analysis of the 36K polypeptide generated only a few peptides, which were not sufficient to conclude whether 36K was related to the major virus polypeptides, and since polypeptides 33K was metabolically unstable, insufficient radioactivity was incorporated for peptide studies. Extensive strain-dependent variation in the isoelectric points and mol. wt. of all the NDV polypeptides which are soluble in the isoelectric focusing gels, including 36K and 33K, is reported. This variation, and the insensitivity of the synthesis of 36K and 33K to actinomycin D, show that both non-structural polypeptides are virus-coded.