Temperature-sensitive Mutant of Newcastle Disease Virus which has an Altered Nucleocapsid-associated Protein

Samson, A. C. R.; Chambers, P. L.; Lee, Catherine M.; Simon, Edward H.

doi:10.1099/0022-1317-54-1-197

Cited by 11 publications

(10 citation statements)

References 6 publications

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“…Virus assembly and budding processes are other possible sites of action. One way to determine the role of non-structural polypeptides in replication and assembly is to identify mutants with a lesion in a particular polypeptide showing an electrophoretic shift, and to correlate this physical variable with a biological function (King & Newman, 1980;Samson et al, 1981). A second approach is to examine the kinetics of nucleocapsid and virus assembly, and the polypeptides associated with nucleocapsids and cellular fractions (Portner & Kingsbury, 1976;Nagai et aL, 1976 b;Smith & Hightower, 1981 b).…”

Section: Discussionmentioning

confidence: 99%

“…NP40 was used in all IEF procedures, although the virus nucleocapsid protein is less soluble than in gels containing Tween 80 . The overall results were more reproducible, and the anomalous, slow migration of F in IEF gels containing Tween 80 was avoided (Samson et al, 1981). The general procedure of O'Farrell (1975) was followed, with slight modifications, depending on the isoelectric point (pI) of the polypeptides under study.…”

Section: R Adioiabelled Monolayersmentioning

confidence: 99%

“…(ii) To resolve neutral and acidic polypeptides, such as HN, F 0, F l, NAP and 36K, IEF gels contained 1% ampholines pH range 3.5 to 10 and 1% ampholines pH range 5 to 7. These were loaded at the anode and electrofocused for 4000 V.h (Samson et al, 1981). Measurement of pH gradients, equilibration of the IEF gels and running the second dimension were as described by O'Farrell (1975).…”

Section: R Adioiabelled Monolayersmentioning

confidence: 99%

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Non-structural Proteins in Newcastle Disease Virus-infected Cells

Chambers

Samson

1982

Journal of General Virology

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SUMMARYExamination of pulse-labeUed 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 ofmol, 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 33K 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 , are related to the HN polypeptide. 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 polypeptide 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.

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Section: Discussionmentioning

confidence: 99%

Section: R Adioiabelled Monolayersmentioning

confidence: 99%

Section: R Adioiabelled Monolayersmentioning

confidence: 99%

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Non-structural Proteins in Newcastle Disease Virus-infected Cells

Chambers

Samson

1982

Journal of General Virology

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“…Radioactively labelled proteins from virus-infected CEF secondary monolayers incubated at 34 °C for all Herts analyses and at 37 °C for all Beaudette C analyses were analysed by isoelectric focusing : SDS-PAGE (2D-PAGE) as described previously (Samson et al, 1981) except that focusing was for 4800 Vh and the radioactive amino acid used was [35S]methionine (Amersham) at 4 ~tCi/dish. Ab4sl R mutants were cloned from the Herts wild-type stock and approximately half of these were also ts-.…”

confidence: 99%

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

Samson

Russell

Hallam

1985

Journal of General Virology

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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...

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“…To determine whether the cysteine-rich protein detected in NDV-infected CEFs was a novel NDV protein and whether it comigrated with the anti-P MAb-reactive 35K to 40K protein, a high resolution two-dimensional isoelectric focusing/SDS-PAGE (IEF/SDS-PAGE) (O'FarreU, 1975) system was used as described previously (Samson et al, 1981).…”

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confidence: 99%