Isolation and Characterization of Monoclonal Antibody-resistant Mutants of Newcastle Disease Virus

Abstract

SUMMARYNeutralizing monoclonal antibodies incorporated into plaque assay overlay medium were used to select antibody-resistant (Ab ~) mutants of both the Herts (using antifusion protein monoclonal 481) and Beaudette C (using anti-haemagglutininneuraminidase protein monoclonal 445) strains of Newcastle disease virus at the permissive temperature of 34 °C. Certain of the Herts, but none of the Beaudette C, Ab R mutants were also temperature-sensitive (ts-) and failed to form plaques at the non-permissive temperature of 41.5 °C. pS]Methionine-labelled proteins from chick embryo fibroblasts infected with wild-type, ts ÷ Ab R and ts-Ab R virus when separated by two-dimensional polyacrylamide gel electrophoresis revealed a variety of changes in the isoelectric point of the fusion protein F (using monoclonal 481) and the haemagglutinin-neuraminidase protein HN (using monoclonal 445). The ts ÷ 'revertants' of ts-Ab R mutants remained Ab R and also showed changed isoelectric points in the F protein.Monoclonal antibodies raised against viruses are of value in identifying virus antigens in virus-infected cells, in aiding purification of virus proteins by affinity chromatography (Varsanyi et al., 1984), and in investigating site(s) on virus surface antigens that are important in virus neutralization (Wiley et aL, 1981; Emini et al., 1983;Dimmock, 1984), This study investigates the utility of neutralizing monoclonal antibodies directed against the fusion (F) and haemagglutinin-neuraminidase (HN) surface glycoproteins of Newcastle disease virus (NDV) to select temperature-sensitive (is-) antibody-resistant (Ab R) mutants. Such mutants would be expected to have lesion(s) within one or the other glycoprotein and ts ÷ 'revertants' of these mutants should shed light on interactions of the antibody-binding sites with other sites in the same or indeed other virus proteins.The monoclonal antibodies 481 (Abe81, anti-F) and 445 (Ab~5, anti-HN-2) used in this study, and isolated and characterized earlier by one of us (Russell et al., 1983), were raised against the Ulster strain of NDV. This strain requires the presence of trypsin in the overlay medium to allow plaque formation; therefore, for convenience the Herts (sensitive to Ab4sl) and Beaudette C (sensitive to Ab44s) strains of NDV were used in this study, as these strains do not require the addition of trypsin in the plaque overlay medium.Serial dilutions of chorioallantoic fluids from eggs infected with plaque-purified Herts and Beaudette C virus were plated on chick embryo fibroblast (CEF) secondary monolayers in 50 mm diam. Petri dishes (Flow Laboratories) and overlaid with 5 ml HEPES-buffered Medium 199 containing 5~ calf serum and t ~ agar to which was added unheated ascites fluid (5 ~tI per monolayer) prepared from the monoclonal antibody-producing cells. Selection and control plates were incubated at 34 + 0-1 °C in sealable plastic boxes which were completely immersed in a water-bath for 4 days. Plaques formed at 34 °C in the presence of monoclonal antibody were picked usin...

Anomalous Behaviour of Newcastle Disease Virus Haemagglutinin-Neuraminidase Protein in Western Blotting Analysis of Monoclonal Antibody Binding Sites

“…The first explanation is very unlikely because of the way in which MAbs are selected and recloned several times. In addition, immunoprecipitation assays using radioactive proteins made in chick embryo fibroblasts following infection of the Ulster or Beaudette C strains of N D V and precipitated with either rabbit anti-mouse Ig or Staphyloccocus aureus cells respectively, precipitate only HN (74K) (Russell et al, 1983b;Samson et al, 1985). Finally, M A b 445 fails to bind H N from M A b 445-resistant mutants of Beaudette C when assayed by immunoprecipitation and these mutants show an altered isoelectric point only in H N (Samson et al, 1985).…”

“…(74K) haemagglutinin-neuraminidase protein (HN) from NDV strains (Russell et al, 1983b) and have been used to select independent mutants of the Beaudette C strain resistant to neutralization by MAb 445 and MAb 14. All of these mutants have an altered HN (revealed by isoelectric focusing gel electrophoresis) (Samson et al, 1985; and unpublished data). In an attempt to use the technique of Western blotting to investigate further the properties of a temperature-sensitive (but still MAb 445-and MAb 14-sensitive) Beaudette C mutant ts53 [which had been shown earlier to possess a thermolabile HN (Harper et at., 1983)], it was found that three proteins from wild-type virions reacted with either MAi~ 445 or 14 but only the expected HN reacted from the ts53 virions.…”

Location of a Neutralizing Epitope for the Haemagglutinin-Neuraminidase Glycoprotein of Newcastle Disease Virus

“…This clone was constructed by simultaneous ligation to pUC19 cut within the multiple cloning site with SphI and SstI of three fragments from clones described by Chambers et al (1986) MAbs could possibly be located by synthesis of portions of the HN protein in Escherichia coli followed by Western blot analysis of bacterial lysates. MAb-resistant mutants have been isolated from the Beaudette C strain of NDV (Samson et al, 1985). Mutants raised against any of the antibodies that recognized reduced denatured HN were cross-resistant to the other antibodies that recognized reduced denatured HN (Samson et al, 1988).…”

Identification of Haemagglutinin-Neuraminidase Antibody Binding Sites by Western Blot Analysis of Antibody-resistant Mutants and Partial Digest Fragments of Newcastle Disease Virus

“…Monoclonal antibodies (MAbs) against either glycoprotein can neutralize the virus and be used to select MAb-resistant mutants Iorio & Bratt, 1985;Samson et al, 1985;Meulemans et al, 1986).…”

“…The procedure involved plating dilutions of plaquepurified wild-type virus and overlaying with HEPES-buffered Medium 199 containing 5 ~o calf serum and 1 ~o agar to which was added unheated ascites fluid at 1/500 or 1/1000 dilution as previously described (Samson et al, 1985). Wild-type plaque-purified antibody-resistant mutants were grown in ovo; chorioallantoic fluid was collected, clarified by centrifugation at 3000 g for 5 min and prepared for SDS-PAGE and Western blotting as previously described (Samson, 1986).…”