Michael A. Bratt

Functional and neutralization profile of seven overlapping antigenic sites on the HN glycoprotein of Newcastle disease virus: monoclonal antibodies to some sites prevent viral attachment

We have previously identified five antigenic sites on the hemagglutinin-neuraminidase (HN) glycoprotein of the Australia-Victoria isolate of Newcastle disease virus (Iorio and Bratt, J. Virol. 48, 440-450; Iorio et al., J. Gen. Virol. 67, 1393-1403). Two additional sites (designated 12 and 23) are now described, bringing to a total of seven the number of antigenic sites defined by our panel of neutralizing anti-HN antibodies. Competition antibody binding and additive neutralization assays reveal that each of these newly-identified sites overlaps two previously-defined ones. The seven HN antigenic sites thus form a continuum in the three-dimensional conformation of the molecule. Studies on the inhibition of hemagglutination (HA), neuraminidase (NA) and the attachment of virus to chick cell monolayers have been used to construct a functional profile of each antigenic site. Monoclonal antibodies (mAbs) to three overlapping sites (12, 2 and 23) inhibit HA and NA and prevent viral attachment to chick cell monolayers. These findings are consistent with the domains recognized by these mAbs being close to the NA and receptor-binding sites. MAbs to two other overlapping sites, 14 and 1 (which in turn, overlap site 12), inhibit HA quite effectively, and attachment to a lesser extent. Sites 14 and 1 probably identify a second domain involved in receptor recognition. MAbs to the two remaining sites (3 and 4), though neutralizing, are negative in all three assays, thus recognizing domains not involved in HA or NA or attachment to chick cells.

Genetic variation within a neutralizing domain on the haemagglutinin-neuraminidase glycoprotein of Newcastle disease virus

Previously, a panel of monoclonal antibodies recognizing epitopes in four antigenic sites on the haemagglutinin-neuraminidase (HN) glycoprotein of the Australia-Victoria strain of Newcastle disease virus were used in strain comparisons. Epitopes in three sites were found to be conserved while the epitope recognized by the single antibody to site 3 was not. A new panel of antibodies is described, two of which bind to epitopes in site 3 and six of which bind to a site (site 1,4) that overlaps with sites 1 and 4 as determined by analyses of variants, temperature-sensitive mutants, and strains by assays of neutralization of infectivity and binding in a radioimmunoassay. Neutralization of heterologous strains with the panel of antibodies revealed that both new site 3 epitopes are also highly divergent, while three additional epitopes outside site 3 (those in site 1,4) are highly conserved. The new site 3 antibodies can bind to virions of several heterologous strains without neutralizing infectivity. Thus, of the 10 epitopes we have now examined, all of three in site 3 are specific with respect to neutralization of infectivity for the homologous strain, while all of seven in other sites are conserved in heterologous strains. This suggests that the strain specificity originally described for a single site 3 epitope is, instead, a property of a much more extensive, poorly conserved domain on the HN molecule.

Reducing agent-sensitive dimerization of the hemagglutinin-neuraminidase glycoprotein of newcastle disease virus correlates with the presence of cysteine at residue 123

Viruses within the Newcastle disease virus (NDV) serotype induce a wide array of disease manifestations ranging from an almost apathogenic pattern to the high mortality caused by avirulent or virulent isolates, respectively. A disulfide-linked dimer form of the NDV hemagglutinin-neuraminidase (HN) glycoprotein can be demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions for only some of these isolates. For others, indeed the majority of those we have studied, no such reducing agent-sensitive dimeric form of HN is demonstrable. Apparently, there is no causal relationship between disulfide-linked dimeric HN and virulence. Using the deduced amino acid sequence of the dimeric HN of isolate AV as a basis for selection of oligonucleotide primers, we sequenced three additional reducing agent-sensitive dimeric HN glycoproteins and eight for which a disulfide-linked dimer has not been identified, using primer extension and dideoxy sequencing. The deduced amino acid sequences reveal a strict correlation between the presence of cysteine at residue 123 and reducing agent-sensitive dimerization of HN.

Identification of amino acid residues important to the neuraminidase activity of the HN glycoprotein of Newcastle disease virus

Monoclonal antibodies (MAbs) to three overlapping antigenic sites (designated 12, 2, and 23) on the hemagglutinin-neuraminidase glycoprotein (HN) of Newcastle disease virus (NDV) were previously shown to inhibit neuraminidase activity (NA) on neuraminlactose (R. M. Iorio and M. A. Bratt, 1984a, J. Immunol. 133, 2215-2219; R. M. Iorio et al., 1989, Virus Res. 13, 245-262). However, a competitive inhibitor of NA blocks the binding of only MAbs to site 23, suggesting that the domain they recognize may be closely related to the NA site. Antigenic variants selected with site 23 MAbs have single amino acid substitutions at HN residues 192, 193, or 200. Virions of variants, which have a substitution at residue 193 or 200, have alterations in NA which are not attributable to a commensurate change in HN content. A revertant of a temperature-sensitive mutant, which has markedly diminished NA relative to the wild type, has an amino acid substitution at residue 175. A second step revertant having partially restored NA has an additional substitution at residue 192 identical to that in one of the site 23 variants, which, in turn, also makes the revertant resistant to neutralization by site 23 MAbs. Thus, an amino acid substitution at residue 175, 193, or 200 of the HN of NDV can have marked effects on the NA of the protein. The amino acids in the region around residue 175 are highly conserved between the HNs of NDV and other paramyxoviruses, suggesting that this domain is important to the integrity of the NA site in this group of viruses.

Genetics and Paragenetic Phenomena of Paramyxoviruses

This is the companion chapter to Chapter 10, “Genetics of Orthomyxoviruses.” These two groups of viruses, paramyxoviruses and orthomyxoviruses, known since the turn of the century, possess many similarities of structure and biological properties but molecular biologies which differ greatly (see Choppin and Compans, 1975; Compans and Choppin, 1975). The orthomyxoviruses undergo extensive recombination or chromosomal reassortment, and numerous reviews of their genetics have been written. For paramyxoviruses, there is no evidence for recombination, and their genetic properties have never been reviewed. Finally, although we have worked on paramyxoviruses for 15 years, we have never worked on any aspect of orthomyxoviruses.

Monoclonal antibodies identify a strain-specific epitope on the HN glycoprotein of Newcastle disease virus strain Australia-Victoria

A panel of monoclonal antibodies raised against the hemagglutinin-neuraminidase glycoprotein (HN) of the Australia-Victoria strain of Newcastle disease virus has been used to compare that strain and eight other strains of the virus. The ability of the antibodies to neutralize infectivity, inhibit hemagglutination and neuraminidase, and bind to purified virions in solid-phase radioimmunoassays was determined for each strain. Of the four antigenic sites delineated by these antibodies on the HN of the homologous strain, site 1 (that with the greatest neutralizing susceptibility), is apparently conserved in all the strains tested as revealed by neutralization assays. The least neutralizing site, number 4, is also conserved in most of the strains tested. Site 2, which lies at or near the neuraminidase site, appears to be conserved in the avirulent strains but not in the virulent strains. An antibody to site 3 is unable to bind to a significant extent to any of the heterologous strains tested, and thus recognizes a strain-specific epitope. Inhibition of hemagglutination and neuraminidase by antibodies to each site were also examined and the results suggest that antibodies to sites 1 and 2 may distinguish virulent and avirulent strains at least with respect to these functions.

Temperature-sensitive mutants of Newcastle disease virus altered in HN glycoprotein size, stability, or antigenic maturity

It has been suggested that the 11 group B, C, and BC temperature-sensitive (ts) mutants of Newcastle disease virus (NDV), strain Australia-Victoria (AV-WT), have lesions in the gene for the hemagglutinin/neuraminidase glycoprotein (HN), and that complementation between groups B and C is intracistronic. Virions produced by these mutants even at permissive temperature contain greatly reduced amounts of HN, and the accompanying hemagglutinating and neuraminidase functions. To explore the basis for decreased HN incorporation into virions and the temperature sensitivity of these mutants, infected chick embryo cells were examined for changes in HN characteristics. The HN of two of the mutants was clearly altered in electrophoretic migration rates in both virions and infected cells. The migrational differences were not due to differences in glycosylation because altered migration rates were also observed in the presence of tunicamycin. In all cases, cells infected by these mutants produced as much HN as did AV-WT-infected cells, but the HN of six of these mutants was metabolically unstable. All of the mutants, including those with metabolically stable HN, exhibited greatly restricted ability to convert HN to an antigenically reactive form, indicating an early block in processing. For most of these mutants, the neuraminidase activities of infected cells were somewhat temperature sensitive, but the production of hemadsorbing activities on cell surfaces was not temperature sensitive. In contrast, the hemadsorbing and neuraminidase activities of cells infected by one mutant, BC2, were temperature sensitive, probably a reflection of the previously described extreme thermolability of the HN of this mutant. The relationship between these mutant characteristics, their temperature sensitivity and the virion phenotypes, is discussed. The data presented here confirm the assignment of these 11 group B, C, and BC mutants to defects in HN and begin to separate them into groups with different characteristics.

Genetics of Orthomyxoviruses

The influenza viruses of man and animals comprise the orthomyxovirus group (Melnick, 1973). Originally, these viruses were grouped with the viruses which now constitute the paramyxovirus group under the designation “myxoviruses” (Andrewes et al., 1955). Members of both groups possess RNA genomes and lipid-containing envelopes (ether lability), and exhibit strong affinities for various mucoid substances. Despite these common properties, the elucidation of the genome strategy of these viruses has revealed dramatic differences between paramyxoviruses and influenza viruses. Most of these differences stem from the physical nature of the genome: continuous single-stranded RNA in paramyxoviruses and a segmented single-stranded RNA genome in orthomyxoviruses. Orthomyxoviruses also have a nuclear phase in their reproduction which is absent in paramyxoviruses. Long before molecular studies revealed the disparate nature of these groups, clear differences in their genetic interactions were apparent. Over a quarter century ago, Burnet and Lind (1951) observed that recombinant viruses were produced with unexpectedly high frequency after mixed infection with two different strains of influenza virus. “High-frequency recombination,” as it was usually described, can now be more aptly termed “genetic reassortment,” in light of our current knowledge of the segmented genome. Both terms are used in the contemporary literature and we will use them interchangeably here.

The 50S and 35S RNAs from Newcastle Disease Virus-Infected Cells

Summary Electrophoretic analyses of the 50S and 35S Newcastle disease virus-specific RNAs from infected cells before and after heat denaturation make it possible to demonstrate that these regions each contain single-stranded RNAs with corresponding S values as well as partially base-paired structures. The partially base-paired structures which sediment at 50S (40 to 60S) have a distribution in gels similar to that of the in vitro transcriptive intermediates, and they remain when 50S RNA synthesis (replication) is blocked by cycloheximide. The partially base-paired 35S RNA is more homogeneous and is neither labelled in the in vitro transcription reaction nor when infected cells are treated with cycloheximide. These base-paired structures may, therefore, be involved in transcription and replication, respectively.

A GENETIC APPROACH TO CYTOPATHOGENICITY, VIRUS SPREAD, AND VIRULENCE OF NEWCASTLE DISEASE VIRUS (NDV)

ABSTRACT The noncytopathic (nc) mutants of the virulent AV strain of NDV, selected to form hemadsorbing spots but not plaques on chicken embryo cells (1,2) are restricted in viral RNA synthesis, accumulate less L protein intracellularly, and complement with the Group E ts RNA - mutant, but not the Group A ts RNA - mutants of NDV. Plaque-forming revertants of 5 of these mutants show at least partial co-reversion of each of these properties, suggesting that RNA synthesizing capacity and cytopathogenicity are causally related. At least 3 of the nc mutants (those chosen originally as small hemadsorbing spot-formers) have a second mutation resulting in either the accumulation of an F protein precursor (nc7) or an altered polypeptide (X) related to viral polypeptide p55 (nc4 and nc16). A small plaque-forming revertant of the F protein mutant shows no reversion of the F alteration but reversion for RNA synthesis. A large spot-forming revertant of the X protein mutant shows no increased RNA synthesis, but loss of the X protein. A second step plaque-forming revertant of this spot-size revertant shows increased RNA synthesis. The independent reversion of properties relevant to cytopathogenicity and spread suggest that these are separate properties. Incremental effects of each of these mutations on mean embryo death times suggest that each contribute to virulence.