Neutralization map of the hemagglutinin-neuraminidase glycoprotein of Newcastle disease virus: domains recognized by monoclonal antibodies that prevent receptor recognition.

Monoclonal antibodies (MAbs) to the hemagglutinin-neuraminidase (HN) glycoprotein of Newcastle disease virus delineate seven overlapping antigenic sites which form a continuum on the surface of the molecule. Antibodies to five of these sites neutralize viral infectivity principally by preventing attachment of the virion to cellular receptors. Through the identification of single amino acid substitutions in variants which escape neutralization by MAbs to these five antigenic sites, a neutralization map of HN was constructed, identifying several residues that contribute to the epitopes recognized by MAbs which block the attachment function of the molecule. These epitopes are defined, at least in part, by three domains on HN: residues 193 to 201; 345 to 353 (which include the only linear epitope we have identified in HN); and a C-terminal domain composed of residues 494, 513 to 521, and 569. To identify HN residues directly involved in receptor recognition, each of the variants was tested for its ability to agglutinate periodate-modified chicken erythrocytes. One variant with a single amino acid substitution at residue 193 was 2.5- to 3-fold more resistant to periodate treatment of erythrocytes than the wild-type virus, suggesting that this residue influences the binding of virus to a sialic acid-containing receptor(s) on the cell surface.

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.

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.

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.

Quantitative basic residue requirements in the cleavage-activation site of the fusion glycoprotein as a determinant of virulence for Newcastle disease virus.

Newcastle disease virus exhibits a wide range of pathogenicity and virulence which, as with all paramyxoviruses, is directly related to the cleavability of a precursor (F0) of the fusion glycoprotein by cellular proteases. Sequence analyses of the cleavage site of several virulent and avirulent isolates of the Newcastle disease virus serotype reveal a correlation between virulence or pathogenicity and a high content of basic amino acid residues at the cleavage site. A similar correlation has been seen for other paramyxoviruses.

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.

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.

Selection of unique antigenic variants of Newcastle disease virus with neutralizing monoclonal antibodies and anti-immunoglobulin.

Monoclonal antibodies were used to isolate nonneutralizable antigenic variants in the hemagglutinin-neuraminidase glycoprotein of Newcastle disease virus. It had been found that a large percentage of virus retains infectivity despite binding neutralizing antibody. This high persistent fraction of nonneutralized virus precluded the isolation of variants by the standard treatment with antibody alone. Rabbit anti-mouse immunoglobulin was used to reduce the percentage of virus that remains infectious despite the presence of bound antibody. This procedure made possible the isolation of variants of two distinct types: classical variants, not neutralized because they do not bind the antibody used to select them; and unique variants that, although still capable of binding the selecting antibody, are only slightly neutralized. The general applicability of this method for the isolation of antigenic variants in nonneutralizing epitopes is also discussed.

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.

Monoclonal antibodies as functional probes of the HN glycoprotein of Newcastle disease virus: antigenic separation of the hemagglutinating and neuraminidase sites.

A panel of nine monoclonal antibodies has been used to construct a functional inhibition profile of four antibody-binding sites previously delineated on the HN glycoprotein of Newcastle disease virus. Antibodies to all four sites are capable of neutralizing infectivity and inhibiting hemolysis and neuraminidase activity with fetuin. There is a good correlation between the fractions of infectivity and neuraminidase activity which survive antibody treatment. However, the inhibition of hemolysis is in the inverse relative order of the neutralization of infectivity. Antibodies to sites 1 and 2 are capable of inhibiting hemagglutination, whereas only antibodies to site 2 can inhibit neuraminidase activity on the smaller substrate N-acetyl neuraminlactose. This antigenically separates the hemagglutinin and neuraminidase sites on the HN glycoprotein. We used these functional inhibition studies to speculate about the location of the four antigenic sites relative to the hemagglutinin and neuraminidase sites.

Neutralization of Newcastle disease virus by monoclonal antibodies to the hemagglutinin-neuraminidase glycoprotein: requirement for antibodies to four sites for complete neutralization.

The neutralizing characteristics of monoclonal antibodies directed to four antigenic sites on the hemagglutinin-neuraminidase glycoprotein of Newcastle disease virus were determined. Neutralization by each antibody resulted in a persistent fraction of nonneutralized virus which varied from 1 to 17% depending on the hemagglutinin-neuraminidase site recognized, but not on the antibody. The addition of antibodies to all four sites on the hemagglutinin-neuraminidase glycoprotein was required to give a level of neutralization comparable with that obtained with polyclonal mouse antiserum. The high persistent fractions were not due to viral aggregates, a high level of variants in the virus stock, the use of insufficient antibody, low antibody avidity, or an effect peculiar to the use of the chicken cells as host. The addition of rabbit anti-mouse immunoglobulin to the persistent fraction left by any of the antibodies resulted in a further reduction in infectivity, often by as much as two logs. Thus, some viral particles are capable of binding antibody while retaining their infectivity. The implications of these findings to the mechanism of neutralization are discussed.

Mutation in the matrix protein of Newcastle disease virus can result in decreased fusion glycoprotein incorporation into particles and decreased infectivity.

Virus particles produced in eggs by the group D ts mutants of Newcastle disease virus at permissive temperature display low infectious and hemolytic activities (M.E. Peeples and M. A. Bratt , J. Virol. 42:440-446, 1982). These lower activities correlate with a decreased incorporation of F1+2 (fusion glycoprotein) into virus particles, compared with that for wild type. The incorporation of F1+2 into virus particles of the group D mutants is also lower than that for wild type when grown in chicken embryo cells in culture at either permissive or nonpermissive temperature. The infectivity of virions from these mutants correlates with the amounts of F1+2 in the virus particles, below a certain concentration, indicating that the quantity of F1+2 in virus particles is a determining factor in the infectivity of those particles. In addition, one of these mutants, D1, produces an M (matrix protein) which migrates at a faster rate in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three of four revertants of D1 have coreverted to wild-type M electrophoretic mobility, associating M with the ts lesion and the other observed phenotypes. In each of these revertants, as well as in three revertants each from D2 and D3, there has been coreversion from the low specific infectious and hemolytic activities to greater, and often wild-type, activities. There is also a coreversion for F1+2 incorporation into virions. All of the revertants incorporate F1+2 into virions more efficiently than their mutant parents. The coreversions associate those phenotypes with the ts lesion and, in the case of D1, with the M lesion as well.

Monoclonal antibodies to newcastle disease virus: delineation of four epitopes on the HN glycoprotein.

Eighteen independent hybridomas producing monoclonal antibodies to Newcastle disease virus have been prepared by fusion of SP2 cells with spleen lymphocytes from a BALB/c mouse immunized with intact UV-inactivated Newcastle disease virus strain Australia-Victoria. They have been divided into three groups on the basis of radioimmunoprecipitation, infected cell surface and cytoplasmic fluorescence, and isotype. The anti-HN group is made up of nine antibodies which give surface fluorescence on infected cells and immunoprecipitate the HN glycoprotein. These antibodies bind to HN in nitrocellulose transfers of sodium dodecyl sulfate gels, but only if it has been neither reduced nor boiled. To varying degrees, all of these HN antibodies neutralize infectivity. These results suggest that they recognize exposed determinants of a conformational nature on the native HN molecule. They have been used in competition antibody-binding radioimmunoassays and additive neutralization assays, and on the basis of these studies the epitopes they recognize have been subdivided into four domains, two of which are overlapping, on the HN glycoprotein. The relatively weaker neutralizing activity observed with some of these antibodies cannot be explained by lower avidities for their epitopes because there is not an inverse correlation between estimated binding constant and neutralizing activity. The four antibodies in the second group all give a predominantly cytoplasmic fluorescence pattern, immunoprecipitate the nucleocapsid protein, and bind to nucleocapsid protein in nitrocellulose transfers of reduced and nonreduced sodium dodecyl sulfate-polyacrylamide gels. All five of the antibodies in the third group are of the immunoglobulin M class, unlike the others which are all immunoglobulin G antibodies. Members of this group show variable fluorescence patterns, but none is able to immunoprecipitate or bind to a specific viral antigen transferred to nitrocellulose paper from sodium dodecyl sulfate-polyacrylamide gels.

Thermostabilities of virion activities of Newcastle disease virus: evidence that the temperature-sensitive mutants in complementation groups B, BC, and C have altered HN proteins.

Four virion activities of Newcastle disease virus (hemagglutinating, neuraminidase, hemolytic, and infectious activities) were examined before and after heat stress in low-salt buffer and physiological salt buffer (phosphate-buffered saline). The hemagglutinating and neuraminidase activities of the Australia-Victoria wild-type (AV-WT) strain were thermostable at both salt concentrations tested, whereas the thermostabilities of the hemolytic and infectious activities were salt dependent (thermostable in phosphate-buffered saline but not in low-salt buffer). Virions of RNA(+) temperature-sensitive (ts) mutants of AV-WT were tested for the stabilities of the four activities. Some mutants in groups B, BC, and C were as stable as AV-WT in all functions, but others were much less stable in all functions. The unstable mutants in groups B, BC, and C affirmed the assignment of the ts lesions of these mutants to the hemagglutinin/neuraminidase (HN) protein gene because HN function(s) are required for all four activities. The instability of these ts mutants was not related to their decreased virion HN protein content and was not due to physical loss of the HN protein from the virions. Three of four ts(+) plaque-forming revertants of the least stable mutant, BC2, coreverted for stability, confirming that the unstable phenotype is indeed the result of the mutation responsible for the ts phenotype. Group D mutants were approximately as stable as AV-WT in hemagglutinating, neuraminidase, and hemolytic activities; this is consistent with this group representing a lesion in a gene other than the HN protein gene. However, the infectivities of two of the three group D mutants were less stable than the infectivity of AV-WT in low-salt buffer.

RNA synthesis by Newcastle disease virus temperature-sensitive mutants in two RNA-negative complementation groups.

The temperature-sensitive RNA-negative mutants of Newcastle disease virus comprise two complementation groups, group A (seven members) and group E (one member). The RNA-synthesizing activities of four representative members of group A and the single member of group E were compared with the activity of the wild type. These mutants were defective to varying extents in primary transcription at the nonpermissive temperature, ranging from mutant A1, which had no activity, to mutant E1, which lost only 50% of its activity. All of the mutants were also defective in a postprimary transcriptive process since after preincubation at the permissive temperature in the presence of cycloheximide, there was no subsequent RNA synthesis at the nonpermissive temperature upon removal of the cycloheximide. Similarly, in experiments in which cycloheximide was not used, shifts from the permissive temperature to the nonpermissive temperature before 3 h postinfection did not result in RNA synthesis. However, later shifts to the nonpermissive temperature did allow RNA synthesis. With the exception of mutant A1, all of the mutants maintained this RNA-synthetic ability for at least 3 h, suggesting that RNA synthesis from progeny genomes was not the major postprimary transcriptive defect in these mutants. In contrast, the RNA-synthetic ability of mutant A1 rapidly decayed at the nonpermissive temperature, suggesting that the A gene product is involved in RNA synthesis from progeny genomes. The postprimary transcriptive defect(s) of the other mutants may be in the processing or stability of a protein, in the processing of mRNA, or in replication. Plaque-forming revertants (ts+) of all of the mutants coreverted for RNA synthesis. This finding strengthens the relationship between temperature sensitivity for plaquing and both the primary and postprimary RNA-negative phenotypes.

Virion functions of RNA+ temperature-sensitive mutants of Newcastle disease virus.

Virions from Newcastle disease virus mutants in four temperature-sensitive RNA+ groups were grown in embryonated hen eggs at the permissive temperature, purified, and then analyzed for biological properties at both the permissive and nonpermissive temperatures. At the permissive temperature, virions of mutants in groups B, C, and BC (11 mutants) were all lower in specific (per milligram of protein) hemagglutination, neuraminidase, and hemolysis activities compared with the wild type. These deficiencies were related to decreased amounts of hemagglutinin-neuraminidase glycoprotein in the virions. Activities of these mutant virions at both the permissive and nonpermissive temperatures were similar, indicating that hemagglutinin-neuraminidase synthesized at the permissive temperature was not temperature sensitive in function. The three group D mutants displayed a different pattern. At the permissive temperature, they had wild-type hemagglutination and neuraminidase activities but were deficient compared with the wild type in hemolysis. Again, functions were similar at both temperatures. Most of the B, C, and BC mutants had specific infectivities similar to that of the wild type despite lower hemagglutination, neuraminidase, and hemolysis functions. However, the D mutants were all less infectious. This evidence is consistent with a shared hemagglutinin-neuraminidase defect in the B, C, and BC mutants and a defect in either the F glycoprotein or the M protein in the D mutants.

UV irradiation analysis of complementation between, and replication of, RNA-negative temperature-sensitive mutants of Newcastle disease virus.

Random UV irradiation-induced lesions destroy the infectivity of Newcastle disease virus (NDV) by blocking downstream transcription from the single viral promoter. The nucleocapsid-associated polypeptides most likely to be involved in RNA synthesis are located at the extreme ends of the genome: NP and P are promoter proximal genes, and L is the most distal gene. We attempted to order the two temperature-sensitive (ts) RNA-negative (RNA-) mutant groups of NDV by determining the UV target sizes for the complementing abilities of mutants A1 and E1. After UV irradiation, E1 was unable to complement A1, a result compatible with the A mutation lying in the L gene. In contrast, after UV irradiation, A1 was able to complement E1 for both virus production and viral protein synthesis, with a target size most consistent with the E mutation lying in the P gene. UV-irradiated virus was unable to replicate as indicated by its absence in the yields of multiply infected cells, either as infectious virus or as particles with complementing activity. After irradiation, ts mutant B1 delta P, with a non-ts mutation affecting the electrophoretic mobility of the P protein, complemented E1 in a manner similar to A1, but it did not amplify the expression of delta P in infected cells. This too is consistent with irradiated virus being unable to replicate despite the presence of the components needed for replication of E1. At high UV doses, A1 was able to complement E1 in a different, UV-resistant manner, probably by direct donation of input polypeptides. Multiplicity reactivation has previously been observed at high-multiplicity infection by UV-irradiation paramyxoviruses. In this case, virions which are noninfectious because they lack a protein component may be activated by a protein from irradiation virions.

Relationships among virus spread, cytopathogenicity, and virulence as revealed by the noncytopathic mutants of Newcastle disease virus.

We have studied protein synthesis in cultured cells infected with the six noncytopathic (nc) mutants of the Australia-Victoria strain (AV-WT) of Newcastle disease virus and their plaque-forming revertants. Virus-specific polypeptides accumulated at 30 to 63% of wild-type levels in nc mutant-infected cells and between 66 and 175% of wild-type levels in revertant-infected cells. An exception was the L polypeptide, which accumulated in nc mutant-infected cells at only 5 to 20% of the levels found in wild-type infection. The reduced accumulation of the L polypeptide did not appear to be due to increased degradation of that polypeptide. A new polypeptide (X) accumulated instead of polypeptide P in cells infected with mutants nc4 or nc16 and in virions released from them. Peptide mapping identified X as an altered form of P. A revertant of mutant nc4 (nc4S1), which forms larger hemadsorbing spots, but still does not form plaques, accumulated P instead of the X polypeptide. Thus, a lesion in P can affect virus spread without affecting cytopathogenicity. Virions of mutant nc7 and two naturally occurring avirulent strains of Newcastle disease virus (NJ LaSota and B1-Hitchner) contained polypeptides (F(7) and F(A), respectively) related to, but migrating more rapidly than, F(0) in sodium dodecyl sulfate-polyacrylamide gels. As previously reported for avirulent strains, a brief treatment of nc7 virions with trypsin converted F(7) to F and increased infectivity. Similarly, culturing nc7-infected cells in the presence of trypsin facilitated fusion from within and viral spread from cell to cell. A plaque-forming revertant of nc7 still accumulated F(7) in virions, indicating that the lesions responsible for the F(7) and noncytopathic phenotypes are genetically separable. The virulent parental strain, AV-WT, exhibited a mean embryo death time of 42 h. Both the larger-spot-forming revertant of nc4 (nc4S1) and the small-plaque-forming revertant of nc7 exhibited a decrease in mean embryo death time (increase in virulence) from 74 to 63 h. A second-step, plaque-forming revertant derived from nc4S1 (nc4S1R1) exhibited a further decrease in mean embryo death time from 63 to 44 h. The results suggest that the F(A)-F(7) and X lesions affect the ability of virus to spread from cell to cell. In addition, these lesions appear to be genetically separable from those responsible for the noncytopathic phenotype. However, both types of lesions cause an extension of mean embryo death time and, thus, may be relevant to virulence in vivo.

Noncytopathic mutants of Newcastle disease virus are defective in virus-specific RNA synthesis.

We have studied virus-specific RNA synthesis in cells infected by six noncytopathic (nc) mutants of the Australia-Victoria wild-type strain (AV-WT) of Newcastle disease virus (NDV) (19). The rates of NDV-specific RNA synthesis in mutant infection were three to sevenfold lower than those observed in wild-type infection. Velocity sedimentation of this NDV-specific RNA revealed that the lower rates of synthesis in mutant infection correlated with reduced accumulation of 18S and 35S mRNA. Electrophoresis in polyacrylamide-urea gels showed that accumulation of all of the 18S mRNA species was reduced and no new species could be detected. Primary transcription appeared unaltered in mutant infection. Cells infected with two naturally occurring avirulent strains of NDV also showed less accumulation of 18S mRNA. Electrophoresis of this RNA resulted in patterns which differed from those obtained with RNA from either AV-WT or nc mutant infection. Complementation for RNA accumulation between the nc mutants and RNA- temperature-sensitive mutants of AV-WT (32) suggested a common defect in the nc mutants. Analysis of plaque-forming revertants of five of the nc mutants revealed that viral RNA synthetic capacity, cell killing, and plaque-forming ability correlated absolutely. These results suggest that viral RNA synthesis and cytopathogenicity may be causally related. In addition, several of the plaque-forming (and cell-killing) revertants were found to be unable to induce fusion from within in infected cell cultures. This result, coupled with the finding that several of the nc mutants are capable of wild-type levels of fusion from within, suggests that the ability to cause such fusion does not correlate with the ability to kill cells.

Noncytopathic mutants of Newcastle disease virus.

We have isolated a novel class of mutants of Newcastle disease virus which are less cytopathic than their virulent parent but are still capable of infectious virus production. Unlike wild-type virus, the mutants did not form plaques after 2 days of incubation; they did, however, make hemadsorbing spots. The mutants range in production of infectious virus from 10 to 200% of that of the wild type. They were less cytopathic in a single cycle of infection by light microscopy, loss of protein from the plate, and inhibition of total protein accumulation. All of the mutants exhibited extended mean embryo death times, a correlate of virulence in the adult animal.