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Serological specificity of thymidine kinase activity in herpes simplex virus-transformed L cells
69, 350-351
VIROLOGY
Serological MARGARET
(1976)
Specificity
of Thymidine Kinase Activity in Herpes Simplex Virus-Transformed L Cells
E. THOULESS,
KAILASH C. CHADHA’ MUNYON’
AND
WILLIAM
H.
Department of Virology, Medical School, University of Birmingham, B152TJ, England and Department of Medical Viral Oncology, Roswell Park Memorial Institute 666 Elm Street, Buffalo, New York 14263 Accepted August 21,1975 Thymidine kinase (TK) activities of L cells transformed by herpes simplex virus type 1 (HSV-1) or type 2 (HSV-2) were neutralized type specifically by antisera from rabbits immunized with these viruses. When compared with activities of lytically infected cells the specificity of neutralization was similar. The data strongly suggest the conclusion that the TK activity in HSV-transformed L cells is virus coded.
Thymidine kinase-deficient L cells (Ltk-) have been transformed, with ultraviolet-irradiated herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), into lines that produce thymidine kinase (TK) II). They were selected in a medium containing methotrexate, which only allowed the growth of cells that produced TK. There is some evidence that TK in these transformed cells is coded by viral DNA. The evidence is based on properties of the enzyme. TK present in transformed cells has the same electrophoretic mobility as TK in lytically infected cells and is different from the TK present in L cells (2). TK activity from HSV-Zinfected cells is much less stable at 40” than the enzyme from HSV-l-infected cells (3). The thermal stability at 41” of TK from HSV-l-transformed cells (Ltk- cells and bromodeoxyuridine (BUdR)-resistant HeLa cells) was the same as the stability of the enzyme from HSV-1 lytically infected cells. The same was true for HSV-2. The TK activities from HSV-a-transformed and HSV-2 lytically infected cells were equally unstable over 3 hr at 41” (4). Thouless (5) has given evidence for typespecific and cross-reacting antigenic determinants on TK from HSV-l- and HSV-2infected baby hamster kidney (BHK) cells. The demonstration of similar determi1 Roswell
Park
Memorial
nants on TK from transformed cells would therefore indicate that these enzymes were virus coded. We report here the results of antiserum neutralization tests on TK from such cells. The transformed cell lines used in this work are clone 139 and clone 212. Clone 139 is a HSV-l-transformed L cell line and is described elsewhere (2). Clone 212 is a newly isolated line of L cells transformed by strain 333 of HSV-2. The antisera used in this work were anti-HSV-1 (192) and anti-HSV-2 (409 and 367) hyperimmune rabbit serum as described and used by Thouless (5). The clone 139 and clone 212 cells were grown in HAT selective medium U ). Onehundred-million cells were washed in phosphate-buffered saline, resuspended in 1 ml of distilled water and sonicated. The sonicated preparation was centrifuged at 100,000 g and the supernatant fraction stored at -70” until used. The extracts containing lytically induced TK were prepared from HSV-infected BHK cells as reported by Klemperer et al. (6). Equal volumes of preimmune or hyperimmune serum and cell extract were mixed and assayed at 37” for 30 min for TK activity (6). The activities of extracts mixed with antisera were compared with those mixed with preimmune sera and are given as percentages (Table 11. The results show
Institute. 350
Copyright All rights
8 1976 by Academic Press, Inc. of reproduction in any form reserved.
SHORT
TABLE SEROLOGICAL
Virus type
SPECIFICITIES
OF THYMIDINE
351
COMMUNICATIONS
KINASES
1
FROM
TRANSFORMED
Source of TK activity
HSV-1 HSV-2 HSV-2
INFECTED
CELLS
Residual TK activity (o/cP Anti-HSV-1 serum 192b
HSV-1
AND LYTICALLY
HSV-l-transformed cells (clone 139) HSV-1 (HFEM) lytically infected BHK cells HSV-a-transformed cells (clone 212) HSV-2 (G-BRY) lytically infected BHK cells
Anti-HSV-2
serum
367*
409”
8
57
52
5
46
43
58
3
7
72
3
4
n Residual activity expressed as a percentage of the level of activity with preimmune serum. The values are the means of at least two experiments. Cell extracts gave about 20,000 cpm per assay when mixed with preimmune serum (5). * The preparation of these rabbit anti-HSV sera was described by Thouless (5).
that TK extracted from transformed cells behaves like that expected from lytically infected BHK cells when mixed with heterologous and homologous serum. These experiments show that the TK activities of L cells transformed by HSV-1 and HSV-2 are serologically similar to those induced by the viruses in lytically infected BHK cells and provide quite strong evidence that the TK in transformed cells is virus coded. ACKNOWLEDGMENTS This investigation was supported by a grant from the Medical Research Council of United Kingdom. The financial support by Public Health Service
Grant No. CA13114 from the National Cancer Institute is also acknowledged. REFERENCES 1. MUNYON,
W., KRAISELBURD, E., DAVIS, D., and J., J. Viral. 7,813-830 (1971). W., BUCHSBAUM, R., PAOLETTI, E., J., KRAISELBURD, E., and DAVIS, D., Virology 49, 683-689 (1972). THOULESS, M. E., and SKINNER, G. R. B., J. Gen. Vi&. 12, 195-197 (1971). DAVIS, D. B., MUNYON, W., BUCHSBAUM, R., and CHAWDA, R., J. Viral. 13,140-145 (19741. THOULESS, M. E., J. Gen. Viral. 17, 307-315 (1972). KLEMPERER, H. G., HAYNES, G. R., SHEDDEN, W. I. H., and WATFXIN, D. H., Virology 31, 120-128 (1967).
MANN, 2. MUNYON, MANN,
3. 4. 5. 6.
VIROLOGY
Serological MARGARET
(1976)
Specificity
of Thymidine Kinase Activity in Herpes Simplex Virus-Transformed L Cells
E. THOULESS,
KAILASH C. CHADHA’ MUNYON’
AND
WILLIAM
H.
Department of Virology, Medical School, University of Birmingham, B152TJ, England and Department of Medical Viral Oncology, Roswell Park Memorial Institute 666 Elm Street, Buffalo, New York 14263 Accepted August 21,1975 Thymidine kinase (TK) activities of L cells transformed by herpes simplex virus type 1 (HSV-1) or type 2 (HSV-2) were neutralized type specifically by antisera from rabbits immunized with these viruses. When compared with activities of lytically infected cells the specificity of neutralization was similar. The data strongly suggest the conclusion that the TK activity in HSV-transformed L cells is virus coded.
Thymidine kinase-deficient L cells (Ltk-) have been transformed, with ultraviolet-irradiated herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), into lines that produce thymidine kinase (TK) II). They were selected in a medium containing methotrexate, which only allowed the growth of cells that produced TK. There is some evidence that TK in these transformed cells is coded by viral DNA. The evidence is based on properties of the enzyme. TK present in transformed cells has the same electrophoretic mobility as TK in lytically infected cells and is different from the TK present in L cells (2). TK activity from HSV-Zinfected cells is much less stable at 40” than the enzyme from HSV-l-infected cells (3). The thermal stability at 41” of TK from HSV-l-transformed cells (Ltk- cells and bromodeoxyuridine (BUdR)-resistant HeLa cells) was the same as the stability of the enzyme from HSV-1 lytically infected cells. The same was true for HSV-2. The TK activities from HSV-a-transformed and HSV-2 lytically infected cells were equally unstable over 3 hr at 41” (4). Thouless (5) has given evidence for typespecific and cross-reacting antigenic determinants on TK from HSV-l- and HSV-2infected baby hamster kidney (BHK) cells. The demonstration of similar determi1 Roswell
Park
Memorial
nants on TK from transformed cells would therefore indicate that these enzymes were virus coded. We report here the results of antiserum neutralization tests on TK from such cells. The transformed cell lines used in this work are clone 139 and clone 212. Clone 139 is a HSV-l-transformed L cell line and is described elsewhere (2). Clone 212 is a newly isolated line of L cells transformed by strain 333 of HSV-2. The antisera used in this work were anti-HSV-1 (192) and anti-HSV-2 (409 and 367) hyperimmune rabbit serum as described and used by Thouless (5). The clone 139 and clone 212 cells were grown in HAT selective medium U ). Onehundred-million cells were washed in phosphate-buffered saline, resuspended in 1 ml of distilled water and sonicated. The sonicated preparation was centrifuged at 100,000 g and the supernatant fraction stored at -70” until used. The extracts containing lytically induced TK were prepared from HSV-infected BHK cells as reported by Klemperer et al. (6). Equal volumes of preimmune or hyperimmune serum and cell extract were mixed and assayed at 37” for 30 min for TK activity (6). The activities of extracts mixed with antisera were compared with those mixed with preimmune sera and are given as percentages (Table 11. The results show
Institute. 350
Copyright All rights
8 1976 by Academic Press, Inc. of reproduction in any form reserved.
SHORT
TABLE SEROLOGICAL
Virus type
SPECIFICITIES
OF THYMIDINE
351
COMMUNICATIONS
KINASES
1
FROM
TRANSFORMED
Source of TK activity
HSV-1 HSV-2 HSV-2
INFECTED
CELLS
Residual TK activity (o/cP Anti-HSV-1 serum 192b
HSV-1
AND LYTICALLY
HSV-l-transformed cells (clone 139) HSV-1 (HFEM) lytically infected BHK cells HSV-a-transformed cells (clone 212) HSV-2 (G-BRY) lytically infected BHK cells
Anti-HSV-2
serum
367*
409”
8
57
52
5
46
43
58
3
7
72
3
4
n Residual activity expressed as a percentage of the level of activity with preimmune serum. The values are the means of at least two experiments. Cell extracts gave about 20,000 cpm per assay when mixed with preimmune serum (5). * The preparation of these rabbit anti-HSV sera was described by Thouless (5).
that TK extracted from transformed cells behaves like that expected from lytically infected BHK cells when mixed with heterologous and homologous serum. These experiments show that the TK activities of L cells transformed by HSV-1 and HSV-2 are serologically similar to those induced by the viruses in lytically infected BHK cells and provide quite strong evidence that the TK in transformed cells is virus coded. ACKNOWLEDGMENTS This investigation was supported by a grant from the Medical Research Council of United Kingdom. The financial support by Public Health Service
Grant No. CA13114 from the National Cancer Institute is also acknowledged. REFERENCES 1. MUNYON,
W., KRAISELBURD, E., DAVIS, D., and J., J. Viral. 7,813-830 (1971). W., BUCHSBAUM, R., PAOLETTI, E., J., KRAISELBURD, E., and DAVIS, D., Virology 49, 683-689 (1972). THOULESS, M. E., and SKINNER, G. R. B., J. Gen. Vi&. 12, 195-197 (1971). DAVIS, D. B., MUNYON, W., BUCHSBAUM, R., and CHAWDA, R., J. Viral. 13,140-145 (19741. THOULESS, M. E., J. Gen. Viral. 17, 307-315 (1972). KLEMPERER, H. G., HAYNES, G. R., SHEDDEN, W. I. H., and WATFXIN, D. H., Virology 31, 120-128 (1967).
MANN, 2. MUNYON, MANN,
3. 4. 5. 6.