VIROLOGY
67,45@462
Induction
(1975)
of Cellular
DNA Synthesis
Mutant
of Herpes Simplex
K. YAMANISHI, Department
by a Temperature-Sensitive
T. OGIN0,2
of Virology, Research Institute for Microbial
Virus Type 2’ M. TAKAHASHI
AND
Diseases, Osaka University,
Suita,
Osaka, Japan
Accepted May 19, 1975 A temperature-sensitive mutant (ts 4) of herpes simplex virus type 2 (HSV-21, having the ability to transform hamster embryo (HaE) cells at the nonpermissive temperature of 38.5”, has been investigated in several aspects. It is defective in thymidine (TdR) kinase induction at both the permissive (34”) and the nonpermissive temperatures and defective in viral DNA synthesis at the nonpermissive temperature. However, stimulation of chromosomal DNA synthesis was detected at 16-28 hr after infection at the nonpermissive temperature in HaE cells arrested with low serum concentration. DNA synthesis was estimated by the incorporation of [3HJrdR or [‘H]CdR (deoxycytidine) into DNA, and differentiation of cellular from viral DNA was performed by buoyant density gradient centrifugation in CsCl or by DNA-DNA hybridization. By autoradiography with [‘H]TdR, it was found that the number of cells with grains in the nuclei increased in infected cultures at 16-28 hr after infection. Virus exposed to heat or uv light lost the ability to induce cellular DNA synthesis, indicating that active virus is responsible for stimulation of cellular DNA synthesis. INTRODUCTION
Oncogenic potential of herpes simplex virus (HSV) was first described by Duff and Rapp (1971). We reported that hamster embryo (HaE) and human embryo (HUE) cells were transformed with some of temperature-sensitive (ts) mutants of herpes simplex virus type 2 (HSV-2) at the nonpermissive temperature of 38.5” (Takahashi and Yamanishi, 1974). It has been recognized that the majority of oncogenic DNA virus have the capacity to stimulate host DNA synthesis (Dulbecco et al., 1965; Gershon et al., 1966; Yamashita and Shimojo, 1969; Takahashi et al., 1969). Recently Epstein-Barr (EB) virus and human cytomegalovirus, members of the herpesvirus group, were shown to induce synthe-
450 All rights of reproduction
in any form reserved.
MATERIALS
AND
METHODS
Cell culture and media. Hamster embryo (HaE) cells were prepared from Syrian hamster embryo and second- to fourthpassaged cells were used for the experiments. Human embryo lung (HEL) cells were obtained from aborted human em-
‘This investigation was supported by grants from the Ministry of Education and from the Princess Takamatsu Cancer Foundation. ‘Present address: Department of Microbiology, Hamamatsu University School of Medicine, Hamamatsu, Japan. Copyright 0 1975 by Academic Press, Inc.
sis of cellular DNA (Gerber and Hoyer, 1971; St. Jeor et al., 1974). With respect to the herpes simplex virus group, inhibition of host cell DNA synthesis by infection has been reported by several workers (Kaplan and Ben-Porat, 1963; Ben-Porat and Kaplan, 1963; Roizman and Roane, 1964; O’Callaghan et al., 1968). In the present experiments, we have found that ts 4, one of the ts mutants having the ability to transform HaE cells (Takahashi and Yamanishi, 1974), is defective in viral DNA synthesis and in induction of TdR kinase but stimulates host DNA synthesis of HaE cells at the nonpermissive temperature of 38.5”.
INDUCTION
OF HOST DNA SYNTHESIS
bryos and were used between the 5th and 20th passage. Eagle’s minimal essential medium (MEM) supplemented with 10% fetal bovine serum was used as growth medium, and 3% calf serum was employed for the maintenance medium. Virus and assay. Wild-type (UW-268 strain) and ts 4 of HSV-2 (Takahashi and Yamanishi, 1974) were prepared in HEL cells at 34”. When apparent cytopathic effect (CPE) occurred in infected cultures, cultures were subjected to rapid freezethawing three times and centrifuged at 3000 rpm for 10 min at 4”, and the supernatant fluids were harvested and stored at -80” as stock virus for the experiments. The presence of mycoplasma in the stock virus was checked as follows. Five-tenths of a milliliter of the virus material was incubated under 5% COZ for 2 weeks in mycoplasma broth (Difco) supplemented with 20% horse serum, 1% yeast extract (Difco). Then, 0.5-ml aliquots were poured onto plates of mycoplasma agar (Difco) supplemented with horse serum and yeast extract and incubated for 3 weeks as above. No colony formation of mycoplasma was observed during the incubation period. Plaque assay to determine infectious virus was carried out as follows. HEL cells grown in 60-mm-diameter glass plates were infected with 0.2-ml aliquots of virus and incubated at 34” for 1 hr for adsorption of virus. Then the cells were washed once with Hanks’ solution and overlaid with Medium 199 containing 10% calf serum and 0.6% agarose. Incubation proceeded at 34” and on the 7th day, the second overlay was done with the same medium containing 0.006% neutral red. The plaques were counted on the following day. of
Viral growth and morphological change infected cells. HaE cells grown in 50-ml
bottles (approximately 5 x lo5 cells) were infected with ts 4 or wild-type virus at an input multiplicity of 2-3 PFU/cell and incubated for 1 hr at room temperature. Then the cells were washed twice with Hanks’ solution, and 5 ml of maintenance medium was added and incubated at 34 or 38.5”. At the indicated times, cultures were harvested and stored at -80”. All the
BY HSV ts MUTANT
451
samples were subjected to rapid freezethawing three times and centrifuged at 3000 rpm for 10 min. Then the supernatant fluids were titrated by the plaque method at 34”. For morphological studies of infected HaE cells, coverslips in 60-mm plastic plates were used. Procedures of infection were the same as above, and after incubation for 24 hr, the cells were fixed and stained with hematoxylin-eosin. Assay of thymidine kinase. The methods of preparation of enzyme extract and the enzyme assay were the same as described by Ogino and Rapp (1971). Briefly, cells of HaE in 500-ml glass bottles (approximately 1 x lo7 cells) were infected at an input multiplicity of 2-3 PFU/cell. At 24 hr after infection, cells were washed three times with phosphate-buffered saline (pH 7.2), scraped loose with the aid of a rubber policeman and centrifuged. They were then suspended in 1 ml of 0.15 M KC1 in 0.05 M Tris-HCl (pH 8.0) containing 0.003 M 2-mercaptoethanol. The suspensions were subjected to ultrasonic disintegration and centrifuged at 36,190 g for 60 min at 4”. The supernatant-fraction was used as the source of enzyme. Protein determinations of cell extracts were carried out by the method of Lowry et al. (1951). The activity of the enzyme extract was assayed in a mixture consisting of: [2-‘“C]TdR, 0.2 &i (51.0 mCi/mmol); cold TdR, 10 nmol; ATP, 5 mM; MgCl,, 5 mM; cellular extract, 100-300 pg of protein; 0.05 M Tris-HCl (pH 8.0) up to 0.25 ml. The reaction was conducted at 38” for 15 min. The amount of TdR nucleotides was assayed by the DEAE-cellulose-disk method (Breitman, 1963). Specific activity of the enzyme was defined as nanomoles of TdR nucleotides produced in the reaction per 15 minutes per milligram of protein in the cellular extracts. Labeling of cells with radioisotope and analysis of DNA. Monolayers of HaE cells
in 60-mm plastic plates were infected with 0.2-ml aliquots of virus. Control cultures were exposed to an equal volume of inoculum which was prepared from uninfected HEL cells in the same way as the virus
452
YAMANISHI,
OGINO AND TAKAHASHI
inoculum. A small volume of [aH]TdR The total pellet, which contained cell de(specific activity, 12.0 Ci/mmol) or bris and HSV, was homogenized in 0.15 M [‘H]CdR (specific activity, 38.1 Ci/mmol) NaCl at 4” with 150 strokes in a tight-fitwas added to the incubation medium at ting Dounce homogenizer. The homogethe final concentration described in each nized suspension was made 50% (w/w) with experiment. After the labeling period, the respect to sucrose by addition of solid cells were scraped off the surface of the sucrose. Thirteen-milliliter samples of this plates, concentrated by centrifugation, solution were placed in 55-ml tubes, overwashed with 0.15 M NaCl buffered with 5 laid with 40, 30 and 20% (w/w) sucrose mA4 Tris-HCl (pH 7.0) containing 10 mM solutions (each 13 ml), centrifuged at EDTA, and the pelleted cells were stored 25,000 rpm for 24 hr at 4’ in a Beckman at -20” until tested. SW 25.2 rotor and the material banding in For the determination (counting) of total the 40-50s sucrose layer was removed. radioactivity in DNA, cells were solubi- After diluting six- to eightfold in TNE lized with 0.5% solution of sodium lauryl buffer (0.01 M Tris-HCl, pH 7.4, 0.1 M sulfate (SLS). Then cold trichloroacetic NaCl, and 0.001 M EDTA), the virus acid (TCA) was added to a final concentra- suspension was layered onto a 30-ml CsCl tion of 5% and the mixture was kept at 0” density gradient (density ranging from 1.19 for 1 hr. The precipitate was collected on to 1.50 g/ml) and centrifuged at 23,000 rpm 25mm Whatman 3 MM filter-paper disks for 2 hr at 4” in a Beckman SW 25.2 rotor. and washed with 5?&TCA, absolute alcohol A sharp band with a density of 1.28 g/ml and then dried. Radioactivity was assayed was collected. The purified virus was lysed in Beckman scintillator. by incubation for 2 hr at 37” in 0.01 M For the separation and assay of viral and Tris-HCl buffer containing 0.15 M NaCl, cellular DNA, 0.5% sodium N-lauryl sar- 0.004 M EDTA, pH 7.8, 1% sodium N-laucosinate and 50 &ml of Pronase in 0.1x ryl sarcosinate and 2 mg/ml of Pronase SSC (0.15 M NaCl + 0.015 M sodium which had been incubated at 80” for 10 min citrate) was added to the pelleted cells. to inactivate DNase activity prior to use. The mixture was kept at 37” for 1 hr and The clear viscous solution was then adcentrifuged at 3000 rpm for 15 min at 4”. justed to a density of 1.70 g/ml with CsCl Two-tenths of a milliliter of the superna- and subjected to isopycnic centrifugation tant fluid was mixed with 3.8 ml of CsCl in at 33,000 rpm in a Beckman rotor No. 40 0.1x SSC (density, 1.745 g/ml) and cen- for 60 hr. All fractions containing DNA trifuged at 33,000 rpm for 60 hr at 17” in a having a density greater than 1.717 g/ml preparative ultracentrifuge with a Beck- were pooled and dialyzed against 0.1x man rotor No. 40. The gradients were ssc. Extraction of DNA from cells. To the collected from the bottom on 25-mm Whatman 3 MM filter-paper disks and radioac- suspension of cells in SSC, Pronase was tivity was assayed in the same manner as added to a final concentration of 1 mg/ml, above. Samples of the gradient were re- and the mixture was incubated for 2 hr at moved at regular intervals, and the refrac- 37”, SLS was then added to a final concentive index was measured with a refractom- tration of 0.2%, and the suspension was incubated at 37” for several hours and then eter for density determinations. Virus purification and DNA extraction. extracted with phenol three times. After dialysis against SSC, pancreatic RNase Purification of HSV and DNA extraction thereof were carried out essentially follow- (heated at 80” for 10 min to inactivate ing the method of Wagner (1972) and contaminating DNase) was added (50 pg/ Graham et al. (1972). The supernatant ml) to degrade RNA. The mixture was medium from the infected HEL cells in incubated at room temperature for 1 hr, roller bottles was centrifuged at 19,000 rpm and RNase was removed by phenol extracfor 90 min in a Beckman rotor No. 19 at 4“. tion. The phenol was removed by dialysis The pelleted material was stored at -20’. as above.
INDUCTION
OF HOST DNA SYNTHESIS
DNA-DNA hybridization test. DNADNA hybridization was carried out essentially following the method described previously (Takahashi et al., 1969) with some modification (Collard et al., 1973). [3H]DNA was fragmented with an ultrasonic cleaner (Model OT-51, Sharp) for 3 min and denatured in water at 100” for 10 min. Hybridization was performed with 25-mm DNA-containing filters in 2 x SSC containing 0.1% SLS at 60” for 24 hr. Filters were washed with 100 ml of 3 mM Tris-HCl buffer (pH 9.1), dried and counted in a scintillation counter. Autoradiography. HaE cells were cultivated on the coverslips in Leighton tubes and arrested with low serum medium (lYo, 4 days). Duplicate coverslips were prepared for each sample. Cells were infected with ts 4 at an input multiplicity of 2-3 PFU/cell at 34”. After an adsorption period of 1 hr, the saved “conditioned medium” was added to the culture, and the incubation proceeded at 38.5”. [sH]TdR was added to the culture medium (2 pCi/ml) at the indicated times. After the labeling period, coverslips were taken out, rinsed with PBS and fixed with Bouin’s solution. After washing with 70% ethanol, coverslips were air dried, put in 0.2 N HClO, solution for 60 min at 4” to remove acid-soluble material and washed thoroughly with water. The coverslip was dipped in Sakura NR-MB emulsion and exposed for 3 days. After development, cells were stained with hematoxylin-eosin. Iododeoxyuridine (IUdR) treatment of cells and alkaline gradient analysis of DNA. Confluent HaE cultures were tryp-
sinized, and cells were collected by centrifugation. The cell pellet was suspended in MEM containing 10% calf serum. IUdR was added at a concentration of 100 pg/ml and distributed to the 60-mm plastic plates. The cultures were exposed to IUdR for 96 hr. Then, prior to infection, extracellular IUdR was removed by washing the cultures three times with Hanks’ solution. After an adsorption period of 1 hr at 34”, residual virus was washed off three times with Hanks’ solution and the maintenance medium of MEM containing 3% calf serum
BY HSV ts MUTANT
453
was added. [sH]TdR was put in the medium (2.5 &i/ml) for 24-28 hr p.i. After the labeling period, DNA was extracted using sodium N-lauryl sarcosinate and Pronase as described above. The DNA was then centrifuged to equilibrium in either neutral (density, 1.744 g/ml) or alkaline (density, 1.775 g/ml, pH 12.5, adjusted with KZO,) CsCl gradients. Both samples were centrifuged at 33,000 rpm for 60 hr at 17” in a preparative ultracentrifuge using Beckman rotor No. 40. The gradients were collected from the bottom and radioactivity was assayed. Cell marker DNA was prepared using growing HaE cells in the presence of [2-‘Clthymidine (0.2 &i/ml). Chemicals and reagents. Chemicals and reagents and their sources were as follows: CsCl (optical grade), SLS and sodium N-lauryl sarcosinate, Wako Pure Chemical Industries, Osaka; RNase, Worthington, Freehold, NJ; Pronase, Kyowa Chemical Industries, Tokyo; IUdR, Sigma, St. Louis, MO; [3H]TdR (specific activity, 12.0 Ci/mmol) and [‘H]CdR (specific activity, 38.1 Ci/mmol), Daiichi Chemical Company, Tokyo; [“C ]TdR (specific activity, 51.0 mCi/mmol), Radiochemical Centre, Amersham. RESULTS
1. Growth of ts 4 and Wild-Type HaE Cells and Morphological of Infected Cells
Virus in Change
The plating efficiencies of ts 4 and wildtype virus at 38.5 and 34” in HaE and HEL cells are given in Table 1. Although wildtype virus is slightly thermosensitive as previously reported by Crouch and Rapp (1972), ts 4 is distinctly thermosensitive in HaE cells as well as in HEL cells. The growth patterns of ts 4 and wildtype virus in HaE cells are given in Fig. 1. Both ts 4 and wild-type viruses replicated well at 34”. At the nonpermissive temperature of 38.5’, ts 4 could not replicate, while wild-type virus replicated less efficiently at 38.5O. Cytopathic effect (CPE) with characteristic intranuclear inclusions was prominent in cells infected eit.her with ts 4 or wildtype virus at 34”. However, little morpho-
454
YAMANISHI, TABLE
PLATING
OF TS 4 AND WILD TYPE OF ON HEL OR HAE CELLSa
Cell
Titer of virus (PFU/O.Z ml) 34”
Ts4
WildWe
1
EFFICIENCIES
HSV-2 Virus
OGINO
HEL HaE HEL HaE
1.5 1.2 1.5 8.0
x x x x
EOP” 38.5”/34”
38.5” 106
67,45@462
Induction
(1975)
of Cellular
DNA Synthesis
Mutant
of Herpes Simplex
K. YAMANISHI, Department
by a Temperature-Sensitive
T. OGIN0,2
of Virology, Research Institute for Microbial
Virus Type 2’ M. TAKAHASHI
AND
Diseases, Osaka University,
Suita,
Osaka, Japan
Accepted May 19, 1975 A temperature-sensitive mutant (ts 4) of herpes simplex virus type 2 (HSV-21, having the ability to transform hamster embryo (HaE) cells at the nonpermissive temperature of 38.5”, has been investigated in several aspects. It is defective in thymidine (TdR) kinase induction at both the permissive (34”) and the nonpermissive temperatures and defective in viral DNA synthesis at the nonpermissive temperature. However, stimulation of chromosomal DNA synthesis was detected at 16-28 hr after infection at the nonpermissive temperature in HaE cells arrested with low serum concentration. DNA synthesis was estimated by the incorporation of [3HJrdR or [‘H]CdR (deoxycytidine) into DNA, and differentiation of cellular from viral DNA was performed by buoyant density gradient centrifugation in CsCl or by DNA-DNA hybridization. By autoradiography with [‘H]TdR, it was found that the number of cells with grains in the nuclei increased in infected cultures at 16-28 hr after infection. Virus exposed to heat or uv light lost the ability to induce cellular DNA synthesis, indicating that active virus is responsible for stimulation of cellular DNA synthesis. INTRODUCTION
Oncogenic potential of herpes simplex virus (HSV) was first described by Duff and Rapp (1971). We reported that hamster embryo (HaE) and human embryo (HUE) cells were transformed with some of temperature-sensitive (ts) mutants of herpes simplex virus type 2 (HSV-2) at the nonpermissive temperature of 38.5” (Takahashi and Yamanishi, 1974). It has been recognized that the majority of oncogenic DNA virus have the capacity to stimulate host DNA synthesis (Dulbecco et al., 1965; Gershon et al., 1966; Yamashita and Shimojo, 1969; Takahashi et al., 1969). Recently Epstein-Barr (EB) virus and human cytomegalovirus, members of the herpesvirus group, were shown to induce synthe-
450 All rights of reproduction
in any form reserved.
MATERIALS
AND
METHODS
Cell culture and media. Hamster embryo (HaE) cells were prepared from Syrian hamster embryo and second- to fourthpassaged cells were used for the experiments. Human embryo lung (HEL) cells were obtained from aborted human em-
‘This investigation was supported by grants from the Ministry of Education and from the Princess Takamatsu Cancer Foundation. ‘Present address: Department of Microbiology, Hamamatsu University School of Medicine, Hamamatsu, Japan. Copyright 0 1975 by Academic Press, Inc.
sis of cellular DNA (Gerber and Hoyer, 1971; St. Jeor et al., 1974). With respect to the herpes simplex virus group, inhibition of host cell DNA synthesis by infection has been reported by several workers (Kaplan and Ben-Porat, 1963; Ben-Porat and Kaplan, 1963; Roizman and Roane, 1964; O’Callaghan et al., 1968). In the present experiments, we have found that ts 4, one of the ts mutants having the ability to transform HaE cells (Takahashi and Yamanishi, 1974), is defective in viral DNA synthesis and in induction of TdR kinase but stimulates host DNA synthesis of HaE cells at the nonpermissive temperature of 38.5”.
INDUCTION
OF HOST DNA SYNTHESIS
bryos and were used between the 5th and 20th passage. Eagle’s minimal essential medium (MEM) supplemented with 10% fetal bovine serum was used as growth medium, and 3% calf serum was employed for the maintenance medium. Virus and assay. Wild-type (UW-268 strain) and ts 4 of HSV-2 (Takahashi and Yamanishi, 1974) were prepared in HEL cells at 34”. When apparent cytopathic effect (CPE) occurred in infected cultures, cultures were subjected to rapid freezethawing three times and centrifuged at 3000 rpm for 10 min at 4”, and the supernatant fluids were harvested and stored at -80” as stock virus for the experiments. The presence of mycoplasma in the stock virus was checked as follows. Five-tenths of a milliliter of the virus material was incubated under 5% COZ for 2 weeks in mycoplasma broth (Difco) supplemented with 20% horse serum, 1% yeast extract (Difco). Then, 0.5-ml aliquots were poured onto plates of mycoplasma agar (Difco) supplemented with horse serum and yeast extract and incubated for 3 weeks as above. No colony formation of mycoplasma was observed during the incubation period. Plaque assay to determine infectious virus was carried out as follows. HEL cells grown in 60-mm-diameter glass plates were infected with 0.2-ml aliquots of virus and incubated at 34” for 1 hr for adsorption of virus. Then the cells were washed once with Hanks’ solution and overlaid with Medium 199 containing 10% calf serum and 0.6% agarose. Incubation proceeded at 34” and on the 7th day, the second overlay was done with the same medium containing 0.006% neutral red. The plaques were counted on the following day. of
Viral growth and morphological change infected cells. HaE cells grown in 50-ml
bottles (approximately 5 x lo5 cells) were infected with ts 4 or wild-type virus at an input multiplicity of 2-3 PFU/cell and incubated for 1 hr at room temperature. Then the cells were washed twice with Hanks’ solution, and 5 ml of maintenance medium was added and incubated at 34 or 38.5”. At the indicated times, cultures were harvested and stored at -80”. All the
BY HSV ts MUTANT
451
samples were subjected to rapid freezethawing three times and centrifuged at 3000 rpm for 10 min. Then the supernatant fluids were titrated by the plaque method at 34”. For morphological studies of infected HaE cells, coverslips in 60-mm plastic plates were used. Procedures of infection were the same as above, and after incubation for 24 hr, the cells were fixed and stained with hematoxylin-eosin. Assay of thymidine kinase. The methods of preparation of enzyme extract and the enzyme assay were the same as described by Ogino and Rapp (1971). Briefly, cells of HaE in 500-ml glass bottles (approximately 1 x lo7 cells) were infected at an input multiplicity of 2-3 PFU/cell. At 24 hr after infection, cells were washed three times with phosphate-buffered saline (pH 7.2), scraped loose with the aid of a rubber policeman and centrifuged. They were then suspended in 1 ml of 0.15 M KC1 in 0.05 M Tris-HCl (pH 8.0) containing 0.003 M 2-mercaptoethanol. The suspensions were subjected to ultrasonic disintegration and centrifuged at 36,190 g for 60 min at 4”. The supernatant-fraction was used as the source of enzyme. Protein determinations of cell extracts were carried out by the method of Lowry et al. (1951). The activity of the enzyme extract was assayed in a mixture consisting of: [2-‘“C]TdR, 0.2 &i (51.0 mCi/mmol); cold TdR, 10 nmol; ATP, 5 mM; MgCl,, 5 mM; cellular extract, 100-300 pg of protein; 0.05 M Tris-HCl (pH 8.0) up to 0.25 ml. The reaction was conducted at 38” for 15 min. The amount of TdR nucleotides was assayed by the DEAE-cellulose-disk method (Breitman, 1963). Specific activity of the enzyme was defined as nanomoles of TdR nucleotides produced in the reaction per 15 minutes per milligram of protein in the cellular extracts. Labeling of cells with radioisotope and analysis of DNA. Monolayers of HaE cells
in 60-mm plastic plates were infected with 0.2-ml aliquots of virus. Control cultures were exposed to an equal volume of inoculum which was prepared from uninfected HEL cells in the same way as the virus
452
YAMANISHI,
OGINO AND TAKAHASHI
inoculum. A small volume of [aH]TdR The total pellet, which contained cell de(specific activity, 12.0 Ci/mmol) or bris and HSV, was homogenized in 0.15 M [‘H]CdR (specific activity, 38.1 Ci/mmol) NaCl at 4” with 150 strokes in a tight-fitwas added to the incubation medium at ting Dounce homogenizer. The homogethe final concentration described in each nized suspension was made 50% (w/w) with experiment. After the labeling period, the respect to sucrose by addition of solid cells were scraped off the surface of the sucrose. Thirteen-milliliter samples of this plates, concentrated by centrifugation, solution were placed in 55-ml tubes, overwashed with 0.15 M NaCl buffered with 5 laid with 40, 30 and 20% (w/w) sucrose mA4 Tris-HCl (pH 7.0) containing 10 mM solutions (each 13 ml), centrifuged at EDTA, and the pelleted cells were stored 25,000 rpm for 24 hr at 4’ in a Beckman at -20” until tested. SW 25.2 rotor and the material banding in For the determination (counting) of total the 40-50s sucrose layer was removed. radioactivity in DNA, cells were solubi- After diluting six- to eightfold in TNE lized with 0.5% solution of sodium lauryl buffer (0.01 M Tris-HCl, pH 7.4, 0.1 M sulfate (SLS). Then cold trichloroacetic NaCl, and 0.001 M EDTA), the virus acid (TCA) was added to a final concentra- suspension was layered onto a 30-ml CsCl tion of 5% and the mixture was kept at 0” density gradient (density ranging from 1.19 for 1 hr. The precipitate was collected on to 1.50 g/ml) and centrifuged at 23,000 rpm 25mm Whatman 3 MM filter-paper disks for 2 hr at 4” in a Beckman SW 25.2 rotor. and washed with 5?&TCA, absolute alcohol A sharp band with a density of 1.28 g/ml and then dried. Radioactivity was assayed was collected. The purified virus was lysed in Beckman scintillator. by incubation for 2 hr at 37” in 0.01 M For the separation and assay of viral and Tris-HCl buffer containing 0.15 M NaCl, cellular DNA, 0.5% sodium N-lauryl sar- 0.004 M EDTA, pH 7.8, 1% sodium N-laucosinate and 50 &ml of Pronase in 0.1x ryl sarcosinate and 2 mg/ml of Pronase SSC (0.15 M NaCl + 0.015 M sodium which had been incubated at 80” for 10 min citrate) was added to the pelleted cells. to inactivate DNase activity prior to use. The mixture was kept at 37” for 1 hr and The clear viscous solution was then adcentrifuged at 3000 rpm for 15 min at 4”. justed to a density of 1.70 g/ml with CsCl Two-tenths of a milliliter of the superna- and subjected to isopycnic centrifugation tant fluid was mixed with 3.8 ml of CsCl in at 33,000 rpm in a Beckman rotor No. 40 0.1x SSC (density, 1.745 g/ml) and cen- for 60 hr. All fractions containing DNA trifuged at 33,000 rpm for 60 hr at 17” in a having a density greater than 1.717 g/ml preparative ultracentrifuge with a Beck- were pooled and dialyzed against 0.1x man rotor No. 40. The gradients were ssc. Extraction of DNA from cells. To the collected from the bottom on 25-mm Whatman 3 MM filter-paper disks and radioac- suspension of cells in SSC, Pronase was tivity was assayed in the same manner as added to a final concentration of 1 mg/ml, above. Samples of the gradient were re- and the mixture was incubated for 2 hr at moved at regular intervals, and the refrac- 37”, SLS was then added to a final concentive index was measured with a refractom- tration of 0.2%, and the suspension was incubated at 37” for several hours and then eter for density determinations. Virus purification and DNA extraction. extracted with phenol three times. After dialysis against SSC, pancreatic RNase Purification of HSV and DNA extraction thereof were carried out essentially follow- (heated at 80” for 10 min to inactivate ing the method of Wagner (1972) and contaminating DNase) was added (50 pg/ Graham et al. (1972). The supernatant ml) to degrade RNA. The mixture was medium from the infected HEL cells in incubated at room temperature for 1 hr, roller bottles was centrifuged at 19,000 rpm and RNase was removed by phenol extracfor 90 min in a Beckman rotor No. 19 at 4“. tion. The phenol was removed by dialysis The pelleted material was stored at -20’. as above.
INDUCTION
OF HOST DNA SYNTHESIS
DNA-DNA hybridization test. DNADNA hybridization was carried out essentially following the method described previously (Takahashi et al., 1969) with some modification (Collard et al., 1973). [3H]DNA was fragmented with an ultrasonic cleaner (Model OT-51, Sharp) for 3 min and denatured in water at 100” for 10 min. Hybridization was performed with 25-mm DNA-containing filters in 2 x SSC containing 0.1% SLS at 60” for 24 hr. Filters were washed with 100 ml of 3 mM Tris-HCl buffer (pH 9.1), dried and counted in a scintillation counter. Autoradiography. HaE cells were cultivated on the coverslips in Leighton tubes and arrested with low serum medium (lYo, 4 days). Duplicate coverslips were prepared for each sample. Cells were infected with ts 4 at an input multiplicity of 2-3 PFU/cell at 34”. After an adsorption period of 1 hr, the saved “conditioned medium” was added to the culture, and the incubation proceeded at 38.5”. [sH]TdR was added to the culture medium (2 pCi/ml) at the indicated times. After the labeling period, coverslips were taken out, rinsed with PBS and fixed with Bouin’s solution. After washing with 70% ethanol, coverslips were air dried, put in 0.2 N HClO, solution for 60 min at 4” to remove acid-soluble material and washed thoroughly with water. The coverslip was dipped in Sakura NR-MB emulsion and exposed for 3 days. After development, cells were stained with hematoxylin-eosin. Iododeoxyuridine (IUdR) treatment of cells and alkaline gradient analysis of DNA. Confluent HaE cultures were tryp-
sinized, and cells were collected by centrifugation. The cell pellet was suspended in MEM containing 10% calf serum. IUdR was added at a concentration of 100 pg/ml and distributed to the 60-mm plastic plates. The cultures were exposed to IUdR for 96 hr. Then, prior to infection, extracellular IUdR was removed by washing the cultures three times with Hanks’ solution. After an adsorption period of 1 hr at 34”, residual virus was washed off three times with Hanks’ solution and the maintenance medium of MEM containing 3% calf serum
BY HSV ts MUTANT
453
was added. [sH]TdR was put in the medium (2.5 &i/ml) for 24-28 hr p.i. After the labeling period, DNA was extracted using sodium N-lauryl sarcosinate and Pronase as described above. The DNA was then centrifuged to equilibrium in either neutral (density, 1.744 g/ml) or alkaline (density, 1.775 g/ml, pH 12.5, adjusted with KZO,) CsCl gradients. Both samples were centrifuged at 33,000 rpm for 60 hr at 17” in a preparative ultracentrifuge using Beckman rotor No. 40. The gradients were collected from the bottom and radioactivity was assayed. Cell marker DNA was prepared using growing HaE cells in the presence of [2-‘Clthymidine (0.2 &i/ml). Chemicals and reagents. Chemicals and reagents and their sources were as follows: CsCl (optical grade), SLS and sodium N-lauryl sarcosinate, Wako Pure Chemical Industries, Osaka; RNase, Worthington, Freehold, NJ; Pronase, Kyowa Chemical Industries, Tokyo; IUdR, Sigma, St. Louis, MO; [3H]TdR (specific activity, 12.0 Ci/mmol) and [‘H]CdR (specific activity, 38.1 Ci/mmol), Daiichi Chemical Company, Tokyo; [“C ]TdR (specific activity, 51.0 mCi/mmol), Radiochemical Centre, Amersham. RESULTS
1. Growth of ts 4 and Wild-Type HaE Cells and Morphological of Infected Cells
Virus in Change
The plating efficiencies of ts 4 and wildtype virus at 38.5 and 34” in HaE and HEL cells are given in Table 1. Although wildtype virus is slightly thermosensitive as previously reported by Crouch and Rapp (1972), ts 4 is distinctly thermosensitive in HaE cells as well as in HEL cells. The growth patterns of ts 4 and wildtype virus in HaE cells are given in Fig. 1. Both ts 4 and wild-type viruses replicated well at 34”. At the nonpermissive temperature of 38.5’, ts 4 could not replicate, while wild-type virus replicated less efficiently at 38.5O. Cytopathic effect (CPE) with characteristic intranuclear inclusions was prominent in cells infected eit.her with ts 4 or wildtype virus at 34”. However, little morpho-
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YAMANISHI, TABLE
PLATING
OF TS 4 AND WILD TYPE OF ON HEL OR HAE CELLSa
Cell
Titer of virus (PFU/O.Z ml) 34”
Ts4
WildWe
1
EFFICIENCIES
HSV-2 Virus
OGINO
HEL HaE HEL HaE
1.5 1.2 1.5 8.0
x x x x
EOP” 38.5”/34”
38.5” 106
