JOURNAL OF VIROLoGY, Oct. 1977, p. 416-418 Copyright © 1977 American Society for Microbiology
Vol. 24, No. 1
Printed in U.S.A.
Temperature-Sensitive Mutants of Human Cytomegalovirus KOICHI YAMANISHI AND FRED RAPP* Department of Microbiology and Specialized Cancer Research Center, The Milton S. Hershey Medical Center, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033 Received for publication 28 April 1977
Eight temperature-sensitive mutants of human cytomegalovirus have been isolated after mutagenesis with nitrosoguanidine. Three of these mutants have been classified into three separate complementation groups and are capable of synthesizing virus DNA at the nonpermissive temperature (39.50C). Two others appear unable to synthesize virus DNA at the elevated temperature. Albrecht and Rapp (1) have shown that hamster embryo fibroblast cells can be transformed by UV-irradiated human cytomegalovirus. This finding was extended by Geder et al. (3), who succeeded in transforming human embryo lung (HEL) cells. The recognition of the genetic function of the virus necessary for cell transformation is required to understand the mechanisms involved. This can be achieved by isolation of appropriate virus mutants. The present work briefly describes the isolation of some temperature-sensitive (ts) mutants of cytomegalovirus and the preliminary characterization of those mutants. HEL cells, grown and maintained as described elsewhere (4), were used for virus propagation and for the plaque assay used to quantitate the virus preparations. For that assay, a 0.25% agarose overlay (5) was used. Permissive and nonpermissive temperatures, 33.5 and 39.50C, respectively, were used. The wild-type virus, strain AD169, was plaque-purified twice in HEL cells at 39.5CC. N-methyl-N'-nitro-N-nitrosoguanidine (NG) (Sigma Chemical Co., St. Louis, Mo.) was used as a mutagen. HEL cells in plastic bottles were infected with wild-type virus at a multiplicity of approximately 0.5 PFU/cell. Virus was adsorbed. at 33.50C for 2 h, and the cell sheets were washed with Tris buffer (pH 7.4) to remove unadsorbed virus. NG (10 pug/ ml) was added to the infected cultures at 0, 24, 48, or 72 h postinfection. Incubation was continued for 24 h, and the medium was then replaced with maintenance medium (Dulbeccomodified Eagle medium supplemented with 3% fetal calf serum and 0.15% NaHCO3). After 6 days of incubation at 33.5CC in a humidified mixture of 95% air and 5% CO2, the cultures were frozen and thawed three times. Cell debris was removed by centrifugation (Sorvall SS-34 rotor, 2,000 rpm, 10 min, 40C), and superna-
tants were stored at -80°C as "mutagenized" virus stock preparation. HEL cultures in 60mm plastic plates (Corning Co., Comning, N. Y.) were infected with approximately 10 PFU ofthe stock suspension of NG-treated cultures per plate. After 2 h of adsorption at 33.5°C, monolayers were overlaid with medium containing agarose and incubated at 33.5°C. Cultures were refed at weekly intervals with two overlays of agarose medium. Three weeks after infection, the cultures were examined microscopically, and the plaques were marked. The plaques were isolated with Pasteur pipettes and transferred to HEL monolayers grown in 24-well tissue culture panels (Linbro Disposo trays; Linbro Chemical Co., New Haven, Conn.) to amplify the virus titer. Cultures were incubated at 33.50C. The maximal cytopathic effect was observed after approximately 3 weeks. The virus of each well was then harvested separately and screened for temperature sensitivity in HEL monolayers. A 0.05-ml portion of each virus suspension was placed into one well in duplicate panels. After adsorption of virus for 2 h at 33.50C, 1 ml of maintenance medium was added to each well, and the panel was placed at either 33.5 or 39.5°C in a 5% C02 incubator. The cells at 39.5°C were incubated for 2 weeks and those at 33.5°C for 3 weeks. Isolates that produced CPE at 33.5°C and failed to produce CPE at 39.5°C were considered potential ts mutants. These potential ts mutants were plaque-purified twice to eliminate wild-type contaminants and revertants. Of 701 plaque isolates (Table 1) initially tested for temperature sensitivity, 3 isolates could be identified with a plating efficiency at least 300-fold higher at the permissive temperature than at the nonpermissive temperature (Table 2). Two ts mutants were isolated from 212 plaques examined from cultures treated with NG for 0 to 24 h, and 1 was isolated out of 416
VOL. 24, 1977
NOTES
84 plaques examined from the cultures treated for 24 to 48 h. No mutants were obtained from a total of 505 isolates from cultures treated for 48 to 72 h and 72 to 96 h postinfection. The virus yields obtained at 33.5 and 39.50C, the plating efficiencies, and virus DNA phenotypes at 39.50C of the three ts mutants are presented in Table 2. These ts mutants exhibited plating efficiencies (39.50C/33.50C) ranging from
Vol. 24, No. 1
Printed in U.S.A.
Temperature-Sensitive Mutants of Human Cytomegalovirus KOICHI YAMANISHI AND FRED RAPP* Department of Microbiology and Specialized Cancer Research Center, The Milton S. Hershey Medical Center, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033 Received for publication 28 April 1977
Eight temperature-sensitive mutants of human cytomegalovirus have been isolated after mutagenesis with nitrosoguanidine. Three of these mutants have been classified into three separate complementation groups and are capable of synthesizing virus DNA at the nonpermissive temperature (39.50C). Two others appear unable to synthesize virus DNA at the elevated temperature. Albrecht and Rapp (1) have shown that hamster embryo fibroblast cells can be transformed by UV-irradiated human cytomegalovirus. This finding was extended by Geder et al. (3), who succeeded in transforming human embryo lung (HEL) cells. The recognition of the genetic function of the virus necessary for cell transformation is required to understand the mechanisms involved. This can be achieved by isolation of appropriate virus mutants. The present work briefly describes the isolation of some temperature-sensitive (ts) mutants of cytomegalovirus and the preliminary characterization of those mutants. HEL cells, grown and maintained as described elsewhere (4), were used for virus propagation and for the plaque assay used to quantitate the virus preparations. For that assay, a 0.25% agarose overlay (5) was used. Permissive and nonpermissive temperatures, 33.5 and 39.50C, respectively, were used. The wild-type virus, strain AD169, was plaque-purified twice in HEL cells at 39.5CC. N-methyl-N'-nitro-N-nitrosoguanidine (NG) (Sigma Chemical Co., St. Louis, Mo.) was used as a mutagen. HEL cells in plastic bottles were infected with wild-type virus at a multiplicity of approximately 0.5 PFU/cell. Virus was adsorbed. at 33.50C for 2 h, and the cell sheets were washed with Tris buffer (pH 7.4) to remove unadsorbed virus. NG (10 pug/ ml) was added to the infected cultures at 0, 24, 48, or 72 h postinfection. Incubation was continued for 24 h, and the medium was then replaced with maintenance medium (Dulbeccomodified Eagle medium supplemented with 3% fetal calf serum and 0.15% NaHCO3). After 6 days of incubation at 33.5CC in a humidified mixture of 95% air and 5% CO2, the cultures were frozen and thawed three times. Cell debris was removed by centrifugation (Sorvall SS-34 rotor, 2,000 rpm, 10 min, 40C), and superna-
tants were stored at -80°C as "mutagenized" virus stock preparation. HEL cultures in 60mm plastic plates (Corning Co., Comning, N. Y.) were infected with approximately 10 PFU ofthe stock suspension of NG-treated cultures per plate. After 2 h of adsorption at 33.5°C, monolayers were overlaid with medium containing agarose and incubated at 33.5°C. Cultures were refed at weekly intervals with two overlays of agarose medium. Three weeks after infection, the cultures were examined microscopically, and the plaques were marked. The plaques were isolated with Pasteur pipettes and transferred to HEL monolayers grown in 24-well tissue culture panels (Linbro Disposo trays; Linbro Chemical Co., New Haven, Conn.) to amplify the virus titer. Cultures were incubated at 33.50C. The maximal cytopathic effect was observed after approximately 3 weeks. The virus of each well was then harvested separately and screened for temperature sensitivity in HEL monolayers. A 0.05-ml portion of each virus suspension was placed into one well in duplicate panels. After adsorption of virus for 2 h at 33.50C, 1 ml of maintenance medium was added to each well, and the panel was placed at either 33.5 or 39.5°C in a 5% C02 incubator. The cells at 39.5°C were incubated for 2 weeks and those at 33.5°C for 3 weeks. Isolates that produced CPE at 33.5°C and failed to produce CPE at 39.5°C were considered potential ts mutants. These potential ts mutants were plaque-purified twice to eliminate wild-type contaminants and revertants. Of 701 plaque isolates (Table 1) initially tested for temperature sensitivity, 3 isolates could be identified with a plating efficiency at least 300-fold higher at the permissive temperature than at the nonpermissive temperature (Table 2). Two ts mutants were isolated from 212 plaques examined from cultures treated with NG for 0 to 24 h, and 1 was isolated out of 416
VOL. 24, 1977
NOTES
84 plaques examined from the cultures treated for 24 to 48 h. No mutants were obtained from a total of 505 isolates from cultures treated for 48 to 72 h and 72 to 96 h postinfection. The virus yields obtained at 33.5 and 39.50C, the plating efficiencies, and virus DNA phenotypes at 39.50C of the three ts mutants are presented in Table 2. These ts mutants exhibited plating efficiencies (39.50C/33.50C) ranging from
