1084
BIOCHEMICAL SOCIETY TRANSACTIONS
Activation of Haemolytic Activity in Chick-Embryo Fibroblast Cells Infected with Newcastle-Disease Virus Strain N J. HELEN DICKINSON* and A. C. R. SAMSON Department of Genetics, University of Newcastle upon Tyne, Newcastle upon Tyne N E l 7RU, U.K.
The glycoprotein F of N D virus is synthesized in infected cells in a precursor form (Samson & Fox, 1973) now designated F,,. In virulent strains of N D virus this precursor protein is cleaved by proteinases within the host cell to give the F glycoprotein in the form Fl,z. In avirulent strains this sequence of events does not occur; the precursor protein Fo is incorporated uncleaved into the plasma membrane of the host cell (Nagai et al., 1976). In this position it can be cleaved by incubating infected cells with trypsin. Nagai et al. (1976) have also shown that virions purified from chick-embryo fibroblasts infected with N D virus strain La Sota do not possess haemolytic activity. This property is restored when virions are treated with trypsin. The activation of haemolytic activity in chick-embryo fibroblasts infected with the avirulent strain N of N D virus cultured in the presence of trypsin has been examined in this present work. Chick-embryo fibroblasts infected with the avirulent N D virus strain N or the mesovirulent strain Baudette (Bd) were cultured in 199 medium with or without trypsin at 37°C for 7 h before assay for haemolytic activity. Where trypsin was added to cultures this was at the start of infection and was at a concentration of O.Spg/rnl. For the haemolysis assay monolayers of cells were washed with phosphate-buffered saline, pH7, and were disrupted with a glass spreader. The cell suspension (in 1ml of phosphate-buffered saline) was frozen at -2O"C, thawed, sonicated and mixed with 1ml of erythrocytes ( l x 10' cells/ml in phosphate-buffered saline, pH7). Incubation Abbreviation used: ND virus. Newcastle-disease virus * Present address: Department of Biology, University of York, Heslington,York YO1 5DD, U.K.
Table 1. Comparison of haemolysis and haemadsorption assays in cells infected with Newcastle-disease virus strain N o r Bd in thepresence or absence 0j"trypsin Readings are absorbance at 415nm. Where readings are > 2 the sample was diluted in phosphate-buffered saline, pH 7, for the haemolysis assay and with distilled water for haemadsorption assays. This value was then multiplied by the appropriate dilution factor. Mock-infected controls gave readings of 0.027 absorbance units for haemolysis and 0.00 for haemadsorption activity. Further details of the experimental procedure are in the text. N D virus strain Bd
N D virus strain N
, Assay Haemolysis
\
,
No tryspin 0.59 0.55 0.62
With trypsin 2.43 2.73 2.31
No trypsin 6.04 6.08 5.04
With trypsin 4.72 4.64 3.62
Mean Haemadsorpt ion
..
0.60 2.8 2.8 3.4
2.49 3.0 3.44 3.44
5.12 3.36 3.54 3.66
4.33 3.04 3.45 3.64
Mean
..
3.0
3.29
3.52
3.38 1979
583rd MEETING, CAMBRIDGE
1085
was for 1h at 37°C. Subsequently cell debris was removed by centrifugation and the absorbance of the supernatant was monitored at 415 nm. Haemadsorption activity in infected cultures was examined to test whether trypsin would affect this viral glycoprotein activity. To assay haemadsorption monolayers of cells were washed with phosphate-buffered saline, pH7, covered with 1ml of erythrocytes ( 5 x lo7 cells/ml in phosphate-buffered saline) and placed at 4°C for 30 min for adsorption to occur. Monolayers were washed to remove unabsorbed erythrocytes until mock-infected monolayers were clear as viewed by microscopy. Adsorbed erythrocytes were then lysed by adding 1 ml of distilled water. The lysate was removed and the cells were then washed with a further 1 ml of distilled water. The two washings were combined and centrifuged to remove cell debris, and the absorbance of the lysate was monitored at 415nm. Results are shown in Table 1. Addition of trypsin to the culture medium does not affect the haemadsorption activity of cultured cells infected with either strain of virus. Also, the addition of trypsin does not stimulate the haemolytic activity of ND-virusstrain-Bd-infected cells. The small decrease in haemolytic activity (5.72 down to 4.33) is because some cells begin to detach from the monolayer before the assay as a result of the action of the trypsin. Incubation of infected cells with trypsin did, however, induce a stimulation of haemolytic activity for N D virus strain N (Table 1). In another approach to the problem chick-embryo fibroblasts infected with N D virus strain N were cultured in the presence of [3H]leucine. Non-infected cells were cultured with [14C]leucine. Uptake of label into cell polypeptides revealed, by polyacrylamide-gel electrophoresis, that the precursor protein Fo was not cleaved in NDvirus-strain-N-infected cells. Addition of trypsin to these infected cells during culture resulted in a loss of 3H label in the Fo glycoprotein on gels, whereas radioactivity appeared in a 56000-dalton band, where F (as F,) glycoprotein migrates. The results of these experiments show that in chick-embryo-fibroblasts infected with N D virus strain N the Fo glycoprotein is not cleaved by proteineases within the cell. It is incorporated into the plasma membrane of the cells in an uncleaved precursor form and is then cleaved to form Fl,* by trypsin to activate haemolytic activity. In this way, therefore, N D virus strain N is similar to N D virus strain La Sota (Nagai et al., 1976). This work was supported by a research studentship to J. H. D. from the Medical Research Council. Nagai, Y., Klenk, H. D. & Rott, R. (1976) Virology 72,494-508 Samson, A. C.R. & Fox, C.F.(1973)J . Virol. 12,579-587
Sequencing the 5’-End of Foot-and-Mouth-Disease Virus T. J. R. HARRIS The Animal Virus Research Institute, Pirbright, Woking, Surrey CU24 ONF, U.K.
Comparison of Translation Products in vivo and in vitro of Cowpea-Mosaic Virus R. W. GOLDBACH Department of Molecular Biology, Agricultural University, D e Dreijen, Wageningen, The Netherlands
Vol. 7
BIOCHEMICAL SOCIETY TRANSACTIONS
Activation of Haemolytic Activity in Chick-Embryo Fibroblast Cells Infected with Newcastle-Disease Virus Strain N J. HELEN DICKINSON* and A. C. R. SAMSON Department of Genetics, University of Newcastle upon Tyne, Newcastle upon Tyne N E l 7RU, U.K.
The glycoprotein F of N D virus is synthesized in infected cells in a precursor form (Samson & Fox, 1973) now designated F,,. In virulent strains of N D virus this precursor protein is cleaved by proteinases within the host cell to give the F glycoprotein in the form Fl,z. In avirulent strains this sequence of events does not occur; the precursor protein Fo is incorporated uncleaved into the plasma membrane of the host cell (Nagai et al., 1976). In this position it can be cleaved by incubating infected cells with trypsin. Nagai et al. (1976) have also shown that virions purified from chick-embryo fibroblasts infected with N D virus strain La Sota do not possess haemolytic activity. This property is restored when virions are treated with trypsin. The activation of haemolytic activity in chick-embryo fibroblasts infected with the avirulent strain N of N D virus cultured in the presence of trypsin has been examined in this present work. Chick-embryo fibroblasts infected with the avirulent N D virus strain N or the mesovirulent strain Baudette (Bd) were cultured in 199 medium with or without trypsin at 37°C for 7 h before assay for haemolytic activity. Where trypsin was added to cultures this was at the start of infection and was at a concentration of O.Spg/rnl. For the haemolysis assay monolayers of cells were washed with phosphate-buffered saline, pH7, and were disrupted with a glass spreader. The cell suspension (in 1ml of phosphate-buffered saline) was frozen at -2O"C, thawed, sonicated and mixed with 1ml of erythrocytes ( l x 10' cells/ml in phosphate-buffered saline, pH7). Incubation Abbreviation used: ND virus. Newcastle-disease virus * Present address: Department of Biology, University of York, Heslington,York YO1 5DD, U.K.
Table 1. Comparison of haemolysis and haemadsorption assays in cells infected with Newcastle-disease virus strain N o r Bd in thepresence or absence 0j"trypsin Readings are absorbance at 415nm. Where readings are > 2 the sample was diluted in phosphate-buffered saline, pH 7, for the haemolysis assay and with distilled water for haemadsorption assays. This value was then multiplied by the appropriate dilution factor. Mock-infected controls gave readings of 0.027 absorbance units for haemolysis and 0.00 for haemadsorption activity. Further details of the experimental procedure are in the text. N D virus strain Bd
N D virus strain N
, Assay Haemolysis
\
,
No tryspin 0.59 0.55 0.62
With trypsin 2.43 2.73 2.31
No trypsin 6.04 6.08 5.04
With trypsin 4.72 4.64 3.62
Mean Haemadsorpt ion
..
0.60 2.8 2.8 3.4
2.49 3.0 3.44 3.44
5.12 3.36 3.54 3.66
4.33 3.04 3.45 3.64
Mean
..
3.0
3.29
3.52
3.38 1979
583rd MEETING, CAMBRIDGE
1085
was for 1h at 37°C. Subsequently cell debris was removed by centrifugation and the absorbance of the supernatant was monitored at 415 nm. Haemadsorption activity in infected cultures was examined to test whether trypsin would affect this viral glycoprotein activity. To assay haemadsorption monolayers of cells were washed with phosphate-buffered saline, pH7, covered with 1ml of erythrocytes ( 5 x lo7 cells/ml in phosphate-buffered saline) and placed at 4°C for 30 min for adsorption to occur. Monolayers were washed to remove unabsorbed erythrocytes until mock-infected monolayers were clear as viewed by microscopy. Adsorbed erythrocytes were then lysed by adding 1 ml of distilled water. The lysate was removed and the cells were then washed with a further 1 ml of distilled water. The two washings were combined and centrifuged to remove cell debris, and the absorbance of the lysate was monitored at 415nm. Results are shown in Table 1. Addition of trypsin to the culture medium does not affect the haemadsorption activity of cultured cells infected with either strain of virus. Also, the addition of trypsin does not stimulate the haemolytic activity of ND-virusstrain-Bd-infected cells. The small decrease in haemolytic activity (5.72 down to 4.33) is because some cells begin to detach from the monolayer before the assay as a result of the action of the trypsin. Incubation of infected cells with trypsin did, however, induce a stimulation of haemolytic activity for N D virus strain N (Table 1). In another approach to the problem chick-embryo fibroblasts infected with N D virus strain N were cultured in the presence of [3H]leucine. Non-infected cells were cultured with [14C]leucine. Uptake of label into cell polypeptides revealed, by polyacrylamide-gel electrophoresis, that the precursor protein Fo was not cleaved in NDvirus-strain-N-infected cells. Addition of trypsin to these infected cells during culture resulted in a loss of 3H label in the Fo glycoprotein on gels, whereas radioactivity appeared in a 56000-dalton band, where F (as F,) glycoprotein migrates. The results of these experiments show that in chick-embryo-fibroblasts infected with N D virus strain N the Fo glycoprotein is not cleaved by proteineases within the cell. It is incorporated into the plasma membrane of the cells in an uncleaved precursor form and is then cleaved to form Fl,* by trypsin to activate haemolytic activity. In this way, therefore, N D virus strain N is similar to N D virus strain La Sota (Nagai et al., 1976). This work was supported by a research studentship to J. H. D. from the Medical Research Council. Nagai, Y., Klenk, H. D. & Rott, R. (1976) Virology 72,494-508 Samson, A. C.R. & Fox, C.F.(1973)J . Virol. 12,579-587
Sequencing the 5’-End of Foot-and-Mouth-Disease Virus T. J. R. HARRIS The Animal Virus Research Institute, Pirbright, Woking, Surrey CU24 ONF, U.K.
Comparison of Translation Products in vivo and in vitro of Cowpea-Mosaic Virus R. W. GOLDBACH Department of Molecular Biology, Agricultural University, D e Dreijen, Wageningen, The Netherlands
Vol. 7
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