JOURNAL OF VIROLOGY, July 1976, p. 43-53 Copyright C 1976 American Society for Microbiology
Vol. 19, No. 1 Printed in U.S.A.
Isolation and Characterization of Temperature-Sensitive Mutants of Adenovirus Type 2 PETER KATHMANN, JOCHEN SCHICK, ERNST-L. WINNACKER, AND WALTER DOERFLER* Institute of Genetics, University of Cologne, Cologne, Germany
Received for publication 18 July 1975
Fourteen temperature-sensitive mutants of human adenovirus type 2, which differed in their plaquing efficiencies at the permissive and nonpermissive temperatures by 4 to 5 orders of magnitude, were isolated. These mutants, which could be assigned to seven complementation groups, were tested for their capacity to synthesize adenovirus DNA at the nonpermissive temperature. Three mutants in three different complementation groups proved deficient in viral DNA synthesis. The DNA-negative mutant H2ts2O6 complemented the DNA-negative mutants H5ts36 and H5ts125, whereas mutant H2ts201 complemented H5ts36 only. Among the DNA-negative mutants, H2ts206 synthesized the smallest amount of viral DNA at the nonpermissive temperature (39.5 C). Data obtained in temperature shift experiments indicated that a very early function was involved in temperature sensitivity. In keeping with this observation, early virus-specific mRNA was not detected in cells infected with H2ts206 and maintained at 39.5 C. Prolonged (52 h) incubation of cells infected with H2ts206 at the nonpermissive temperature led to the synthesis of a highmolecular-weight form of viral DNA. In numerous virus systems, conditional lethal mutants of the temperature-sensitive (ts) class proved useful in the analysis of viral gene functions (13). For several adenovirus types, ts mutants were isolated and partly characterized, as in adenovirus type 2 (Ad2) (2), type 5 (Ad5) (12, 27, 32), type 12 (18, 19, 24) and type 31 (25, 26) and in chicken embryo lethal orphan virus (17). In the present report we describe the isolation of 14 ts mutants of Ad2 from virus stocks inactivated by nitrous acid, hydroxylamine, or ethane methane sulfonate (EMS). The mutants could be divided into seven complementation groups. To localize their genetic defects within either the early or the late phase of the lytic infection cycle, each of the mutants was examined for its capacity to synthesize viral DNA at the nonpermissive temperature. Among the 14 mutants, three showed defects in viral DNA synthesis, whereas 11 mutants seemed to replicate their DNA equally well at the permissive (32.5 C) and nonpermissive (39.5 C) temperatures. One of the DNA-negative mutants, H2ts206, complemented the DNA-negative mutants of Ad5, H5ts36 (30) and H5ts125 (12). This mutant was characterized further in temperature shift-up and shift-down experiments. The results suggested that the defect was located in a very early function required within the first few hours postinfection. The
finding that Ad2-specific mRNA could not be detected in cells infected with this mutant supports this conclusion. In addition, it was observed that upon infection and maintenance at the nonpermissive temperature a fast-sedimenting form of viral DNA was synthesized at late times after infection. MATERIALS AND METHODS Media and sera. The composition of Eagle medium for suspension cultures (11) and of reinforced Eagle medium (Dulbecco medium) (1, 9) have been described. Calf and horse sera were purchased from Flow Laboratories. Solutions. PBS-d is phosphate-buffered saline (10) without Mg2+ and Ca2+. The compositions of Tris-saline (33), the alkaline sucrose (Schwarz/ Mann) solutions (4), and the toluene methanolbased scintillator (4) have been described elsewhere. EMS (Eastman Kodak Co.) was dissolved in Trissaline at a concentration of 20 mg/ml. Solutions of 1.4 M nitrous acid and 2 M hydroxylamine were prepared in 1 M sodium acetate, pH 4.6, and in 0.1 M phosphate buffer, pH 7.5, respectively. Agarose (Sigma) was used as a 1.6% solution in water. The neutral red solution (0.3%, wt/vol) was obtained from Flow Laboratories. All other chemicals were reagent grade and were used without further purification. Cells and virus. HeLa cells used in this study were the gift of J. F. Williams, Glasgow. KB cells were obtained from the American Type Culture Collection. Wallace Rowe, National Institutes of 43
44
KATHMANN ET AL.
Health, Bethesda, Md., kindly supplied seed preparations of human Ad2. The Ad5 mutants, H5ts36 (32) and H5ts125 (12), were gifts of Jim Williams, Glasgow, and Harry Ginsberg, New York, respectively. They were obtained from John Sussenbach, Utrecht, and Jim Williams, Glasgow. Mycoplasma assay. All cell cultures were monitored weekly for mycoplasma contamination by the microbiological methods developed by Hayflick (16). Cultures used in the experiments reported proved free of mycoplasma contamination. Radioactive isotopes. [6-3H]thymidine (22 to 30 Ci/mmol) and [14Clformic acid, sodium salt (50 to 60 mCi/mmol), were purchased from the AmershamBuchler Co., Braunschweig, Germany. Preparation and purification of Ad2 and viral DNA. KB cells growing in suspension culture were inoculated with CsCl-purified Ad2 at a multiplicity of infection (MOI) of 10 to 100 PFU/cell. The cells were collected by centrifugation, and the virus was harvested between 30 and 36 h postinfection (p.i.), when virus yields were optimal (21). The methods for the purification of virus and of labeled and unlabeled viral DNA have been described in detail elsewhere (6, 8). Infectivity assay with Ad2. Infectivity of Ad2 was determined by plaque assay on HeLa cells growing in monolayers on 30-mm Falcon plastic dishes. The procedure developed by Williams (31) was used with minor modifications. HeLa cells were grown to confluent monolayers in Dulbecco medium supplemented with 10% horse serum. After inoculation of the cells with different virus dilutions and after a 60min adsorption period, the cells.were overlaid with 4 ml of 0.8% agarose in Dulbecco medium supplemented with 2.5% horse serum, 25 mM MgCl2, and 1 mM arginine. A second overlay of 2 ml, containing 0.01% neutral red, was added 4 or 9 days after inoculation depending on whether incubation was at 32.5 or 39.5 C, respectively. Plaques were counted 1 and 2 days after the addition of the second overlay. Wild-type Ad2 as well as each of the ts mutants were purified by four single-plaque isolations at either 39.5 C (wild-type virus) or 32.5 C (ts mutants). Confluent monolayers of HeLa cells were grown in Dulbecco medium containing 10% calf serum (DC medium) in 75-cm2 Falcon plastic flasks and inoculated with plaque isolates of wild-type virus or ts mutants at an MOI of 0.1 PFU/cell. Cultures were harvested either after 2 days at 39.5 C or after 5 days at 32.5 C. After disruption of infected cells by ultrasonic treatment, extracts were clarified by low-speed centrifugation and kept frozen at -80 C. Crude extracts of this type were used as inocula throughout the study. The same procedures were followed for the propagation of the Ad5 mutants. Inactivation and mutagenesis of Ad2. Three different mutagens were used. (i) For nitrous acid (HNO2) mutagenesis the procedure of Williams et al. (32) was followed. One part of crude extract from Ad2-infected HeLa cells was treated with 2 parts of 1.4 M NaNO2 in I M sodium acetate, pH 4.6, at 25 C. At various times after mixing these components, the
J. V IROL.
reaction was stopped by a 100-fold dilution of aliquots in 1 M Tris-hydrochloride, pH 8, at 4 C. Further dilutions were carried out in Tris-saline, and the samples were immediately assayed for plaqueforming activity on HeLa cells at 32.5 C (permissive temperature). (ii) Mutagenesis by hydroxylamine was performed according to Williams et al. (32). One part of crude extract from Ad2-infected HeLa cells was mixed with 2 parts of 2 N hydroxylamine in 0.1 M sodium phosphate, pH 7.5, at 25 C. At various times after starting the reaction, samples were diluted 100-fold in PBS containing 2% calf serum at 4 C. The dilutions were dialyzed against PBS for 12 h at 4 C prior to titer determinations on HeLa cell monolayers.
(iii) Mutagenesis by EMS (3) was performed by mixing 1 part of crude extract from Ad2-infected HeLa cells with 2 parts of 2% EMS in Tris-saline at 37 C. At various times after starting the reaction, samples were diluted 100-fold in Tris-saline at 4 C and assayed for infectivity titers in HeLa cells. Isolation of ts mutants. Wild-type Ad2 selected after four single-plaque isolations at 39.5 C and propagated as described above was inactivated by 3 to 4 logs by three different mutagens, as outlined in the preceding section, and immediately plated on HeLa cells at 32.5 C. After a 9- to 10-day incubation period, solitary, well-isolated plaques were punched out at random with a Pasteur pipette and transferred to a tube containing DC medium. The virus from each plaque was liberated by five cycles of freezing and thawing and retitered on HeLa cells at both 32.5 and 39.5 C. Isolates with a differential plaquing efficiency of 102 and higher were scored as mutants and further purified by three single-plaque isolations. The 14 ts mutants described in this report were derived from five different mutagenesis experiments, three with HNO2 and one each with EMS and hydroxylamine. Complementation tests. HeLa cells growing in monolayers were inoculated at the nonpermissive temperature with pairs of ts mutants and with each of the mutants separately. The MOI was 10 PFU/cell in single infections and 5 PFU/cell for each of the mutants in double infections. Adsorption was allowed to proceed for 1 h. Subsequently, the cells were washed with 1 ml of PBS and then incubated for 15 min with Ad2-specific antiserum (rabbit) diluted 1/100 with PBS. The antiserum was removed, the cells were washed again with PBS, and medium was added. The infected cells were incubated at 39.5 C. At 36 h p.i., the cells were scraped off the plate and resuspended in 1 ml of PBS. The cells were disrupted by five cycles of freezing and thawing, and virus yields were determined by plaque assay on HeLa cell monolayers at 32.5 C. The same procedure was followed in complementation experiments between Ad2 and Ad5. Complementation indexes were calculated from the formula: complementation index = [virus yield (mutant a x mutant b)]/[virus yield mutant a + virus yield mutant b]. DNA synthesis in HeLa cells infected with wild
VOL. 19, 1976
type and ts mutants of Ad2. Dense monolayers of HeLa cells (approximately 106 cells) in 30-mm-diameter plastic petri dishes were inoculated with wildtype or mutant Ad2 at an MOI of 20 PFU/cell. Adsorption was allowed to proceed for 60 min at 32.5 C; subsequently, the inoculum was removed, 1.5 ml of fresh medium (DC) was added, and incubation was continued at 32.5 or 39.5 C. The newly synthesized DNA in the Ad2-infected cells was labeled with 5 pCi of [3H]thymidine per ml from 12 to 24 h p.i. and from 60 to 120 h p.i. at 39.5 or 32.5 C, respectively. In some of the complementation experiments using the DNA-negative mutants of Ad2 or Ad5, HeLa cells were singly or doubly infected with various combinations of mutants, and the ability of the infected cells to synthesize viral DNA was scored as described below. Analysis of intracellular DNA by zonal sedimentation in alkaline sucrose density gradients. Previously described procedures were used (4, 6) with minor modifications. At the end of the labeling period, the infected cells were washed and scraped off the dish, and 10,000 to 20,000 cells were lysed in an 0.1-ml layer of 0.5 N NaOH on top of an alkaline sucrose density gradient (4 ml, 5 to 20%) in SW56 centrifuge tubes containing a 0.3-ml cushion of 70% sucrose solution in alkali. To each gradient a small amount of 14C-labeled Ad2 DNA was added to the 0. 1-ml lysis layer as marker. Lysis of the cells proceeded at 4 C for at least 14 h before the gradients were centrifuged in the SW56 rotor of the L2-65B Spinco ultracentrifuge at 4 C and 55,000 rpm for 90 min. After centrifugation, 4- or 6-drop fractions were collected and counted directly in 5 ml of toluene methanol scintillator in a Packard Tri-Carb liquid scintillation spectrometer, model 3330. Conditions for the preparation and centrifugation of SW41 sucrose gradients have been described (4). Analysis of DNA from Ad2-infected HeLa cells by DNA-DNA hybridization. The procedure of Denhardt (5) was used, as previously described (6, 7). Schleicher and Schuell (Dassel, Germany) nitrocellulose filters were used, and each new lot of filters was pretested with authentic Ad2 DNA. Gradient fractions from SW41 alkaline sucrose gradients were pooled (Fig. 3), neutralized, and dialyzed against 4 x SSC prior.to DNA-DNA hybridization. Thermostability of ts206. The thermal stability of the mutant H2ts206 was determined by incubating, at 39 to 40 C, a 1:10 dilution in PBS of crude extracts of HeLa cells that had been infected with the mutant. Incubation was for 0, 1, 2, 4, and 6 h. Subsequently, the samples were chilled and diluted 102_ fold in ice-cold PBS, and the plaque titer was determined. Isolation and detection of Ad2-specific mRNA in cells infected with mutant ts206. HeLa cells growing in monolayers were infected with wild-type Ad2 or with mutant ts206. Adsorption was allowed to proceed for 60 min at either 32.5 or 39.5 C. Cells inoculated with ts206 at either temperature were then incubated at 32.5 and 39.5 C, whereas cells inoculated with wild-type Ad2 at either temperature were incubated only at those respective temperatures. The infected cells were labeled with 100 ,uCi of
ts MUTANTS OF ADENOVIRUS TYPE 2
45
[3H]uridine per ml between 2 and 4 h p.i. (at 39.5 C) and between 11 and 16 h p.i. (at 32.5 C). Subsequently, polysomes and mRNA were isolated and analyzed by filter hybridization to Ad2 DNA as described earlier (20).
RESULTS Ad2. Ad2 is inactivated by of Inactivation treatment in vitro with nitrous acid, hydroxylamine, or EMS. In all three cases, the rate of inactivation follows single-hit kinetics. Nitrous acid is considerably more efficient than hydroxylamine or EMS as an inactivating agent. For the isolation of mutants, wild-type virus stocks were inactivated with each of three mutagens by 2 to 4 orders of magnitude. Isolation of ts mutants. In Table 1 the plaque-forming capacities at the permissive and nonpermissive temperatures have been summarized for the 14 ts mutant isolates. The ratio of the plaque-forming activities at 39.5 C over that at 32.5 C is close to 1 for wild-type Ad2 and 10-4 to 10-s for the ts mutants. When virus isolated from single plaques grown at 32.5 C was titrated at the permissive temperature, the yields ranged from 5 x 103 (ts213) to 7 x 105 (ts214) PFU/ml, whereas at the nonpermissive temperature the yield never exceeded 102 PFU/ml. It was noticed that ts213 formed only tiny plaques at the permissive temperature, whereas the plaque size of each of the other mutants at 32.5 C was comparable to that of wild type. The three agents applied, nitrous acid, hydroxylamine, and EMS, proved about equally efficient mutagens with Ad2 (Table 2). The value for EMS obviously was based on too small a number of isolates to be statistically meaningful. Characterization of ts mutants for defects in viral DNA synthesis. To determine whether the defects were in an early or late function, the 14 ts mutants were investigated for their ability to synthesize viral DNA in HeLa cells at the nonpermissive temperature. As a first step, the time of maximal Ad2 DNA synthesis had to be determined for the wild-type virus at the permissive and nonpermissive temperatures. After several experiments, labeling periods with [3H]thymidine (5 ,uCi/ml) between 12 and 24 h p.i. and between 60 and 120 h p.i. were chosen for the higher and lower temperatures, respectively. Accordingly, confluent monolayers of HeLa cells were inoculated in duplicate with 20 PFU of each of the 14 ts mutants per cell, incubated either at 32.5 or at 39.5 C, and labeled with [3H]thymidine at the time intervals indicated above. At the end of the labeling period, cells were lysed, and the newly synthe-
46
J . V IROL .
KATHMANN ET AL. TABLE 1. Plaque-forming ability of 14 ts mutants of Ad2 at 32.5 and 39.5 C
Ratio of PFU/ml at 39.5/
Infectivity (PFU/mlP Virus
Mutagenic agent
32.5 C 6.9 x 108
Wild type H2ts2Old 202 203 204 205 206 207 208 209 214
Nitrous acid
5.0 5.0 10.0 5.0 4.0 1.0 7.0 3.0 2.0 4.0
108 108 108 108 108 108 108 108 108 108
1.0 x 108 5.0 x 108 5.0 x 108
210 211 212
Hydroxylamine
x x x x x x x x x x
39.5 O 6.3 x 108
Vol. 19, No. 1 Printed in U.S.A.
Isolation and Characterization of Temperature-Sensitive Mutants of Adenovirus Type 2 PETER KATHMANN, JOCHEN SCHICK, ERNST-L. WINNACKER, AND WALTER DOERFLER* Institute of Genetics, University of Cologne, Cologne, Germany
Received for publication 18 July 1975
Fourteen temperature-sensitive mutants of human adenovirus type 2, which differed in their plaquing efficiencies at the permissive and nonpermissive temperatures by 4 to 5 orders of magnitude, were isolated. These mutants, which could be assigned to seven complementation groups, were tested for their capacity to synthesize adenovirus DNA at the nonpermissive temperature. Three mutants in three different complementation groups proved deficient in viral DNA synthesis. The DNA-negative mutant H2ts2O6 complemented the DNA-negative mutants H5ts36 and H5ts125, whereas mutant H2ts201 complemented H5ts36 only. Among the DNA-negative mutants, H2ts206 synthesized the smallest amount of viral DNA at the nonpermissive temperature (39.5 C). Data obtained in temperature shift experiments indicated that a very early function was involved in temperature sensitivity. In keeping with this observation, early virus-specific mRNA was not detected in cells infected with H2ts206 and maintained at 39.5 C. Prolonged (52 h) incubation of cells infected with H2ts206 at the nonpermissive temperature led to the synthesis of a highmolecular-weight form of viral DNA. In numerous virus systems, conditional lethal mutants of the temperature-sensitive (ts) class proved useful in the analysis of viral gene functions (13). For several adenovirus types, ts mutants were isolated and partly characterized, as in adenovirus type 2 (Ad2) (2), type 5 (Ad5) (12, 27, 32), type 12 (18, 19, 24) and type 31 (25, 26) and in chicken embryo lethal orphan virus (17). In the present report we describe the isolation of 14 ts mutants of Ad2 from virus stocks inactivated by nitrous acid, hydroxylamine, or ethane methane sulfonate (EMS). The mutants could be divided into seven complementation groups. To localize their genetic defects within either the early or the late phase of the lytic infection cycle, each of the mutants was examined for its capacity to synthesize viral DNA at the nonpermissive temperature. Among the 14 mutants, three showed defects in viral DNA synthesis, whereas 11 mutants seemed to replicate their DNA equally well at the permissive (32.5 C) and nonpermissive (39.5 C) temperatures. One of the DNA-negative mutants, H2ts206, complemented the DNA-negative mutants of Ad5, H5ts36 (30) and H5ts125 (12). This mutant was characterized further in temperature shift-up and shift-down experiments. The results suggested that the defect was located in a very early function required within the first few hours postinfection. The
finding that Ad2-specific mRNA could not be detected in cells infected with this mutant supports this conclusion. In addition, it was observed that upon infection and maintenance at the nonpermissive temperature a fast-sedimenting form of viral DNA was synthesized at late times after infection. MATERIALS AND METHODS Media and sera. The composition of Eagle medium for suspension cultures (11) and of reinforced Eagle medium (Dulbecco medium) (1, 9) have been described. Calf and horse sera were purchased from Flow Laboratories. Solutions. PBS-d is phosphate-buffered saline (10) without Mg2+ and Ca2+. The compositions of Tris-saline (33), the alkaline sucrose (Schwarz/ Mann) solutions (4), and the toluene methanolbased scintillator (4) have been described elsewhere. EMS (Eastman Kodak Co.) was dissolved in Trissaline at a concentration of 20 mg/ml. Solutions of 1.4 M nitrous acid and 2 M hydroxylamine were prepared in 1 M sodium acetate, pH 4.6, and in 0.1 M phosphate buffer, pH 7.5, respectively. Agarose (Sigma) was used as a 1.6% solution in water. The neutral red solution (0.3%, wt/vol) was obtained from Flow Laboratories. All other chemicals were reagent grade and were used without further purification. Cells and virus. HeLa cells used in this study were the gift of J. F. Williams, Glasgow. KB cells were obtained from the American Type Culture Collection. Wallace Rowe, National Institutes of 43
44
KATHMANN ET AL.
Health, Bethesda, Md., kindly supplied seed preparations of human Ad2. The Ad5 mutants, H5ts36 (32) and H5ts125 (12), were gifts of Jim Williams, Glasgow, and Harry Ginsberg, New York, respectively. They were obtained from John Sussenbach, Utrecht, and Jim Williams, Glasgow. Mycoplasma assay. All cell cultures were monitored weekly for mycoplasma contamination by the microbiological methods developed by Hayflick (16). Cultures used in the experiments reported proved free of mycoplasma contamination. Radioactive isotopes. [6-3H]thymidine (22 to 30 Ci/mmol) and [14Clformic acid, sodium salt (50 to 60 mCi/mmol), were purchased from the AmershamBuchler Co., Braunschweig, Germany. Preparation and purification of Ad2 and viral DNA. KB cells growing in suspension culture were inoculated with CsCl-purified Ad2 at a multiplicity of infection (MOI) of 10 to 100 PFU/cell. The cells were collected by centrifugation, and the virus was harvested between 30 and 36 h postinfection (p.i.), when virus yields were optimal (21). The methods for the purification of virus and of labeled and unlabeled viral DNA have been described in detail elsewhere (6, 8). Infectivity assay with Ad2. Infectivity of Ad2 was determined by plaque assay on HeLa cells growing in monolayers on 30-mm Falcon plastic dishes. The procedure developed by Williams (31) was used with minor modifications. HeLa cells were grown to confluent monolayers in Dulbecco medium supplemented with 10% horse serum. After inoculation of the cells with different virus dilutions and after a 60min adsorption period, the cells.were overlaid with 4 ml of 0.8% agarose in Dulbecco medium supplemented with 2.5% horse serum, 25 mM MgCl2, and 1 mM arginine. A second overlay of 2 ml, containing 0.01% neutral red, was added 4 or 9 days after inoculation depending on whether incubation was at 32.5 or 39.5 C, respectively. Plaques were counted 1 and 2 days after the addition of the second overlay. Wild-type Ad2 as well as each of the ts mutants were purified by four single-plaque isolations at either 39.5 C (wild-type virus) or 32.5 C (ts mutants). Confluent monolayers of HeLa cells were grown in Dulbecco medium containing 10% calf serum (DC medium) in 75-cm2 Falcon plastic flasks and inoculated with plaque isolates of wild-type virus or ts mutants at an MOI of 0.1 PFU/cell. Cultures were harvested either after 2 days at 39.5 C or after 5 days at 32.5 C. After disruption of infected cells by ultrasonic treatment, extracts were clarified by low-speed centrifugation and kept frozen at -80 C. Crude extracts of this type were used as inocula throughout the study. The same procedures were followed for the propagation of the Ad5 mutants. Inactivation and mutagenesis of Ad2. Three different mutagens were used. (i) For nitrous acid (HNO2) mutagenesis the procedure of Williams et al. (32) was followed. One part of crude extract from Ad2-infected HeLa cells was treated with 2 parts of 1.4 M NaNO2 in I M sodium acetate, pH 4.6, at 25 C. At various times after mixing these components, the
J. V IROL.
reaction was stopped by a 100-fold dilution of aliquots in 1 M Tris-hydrochloride, pH 8, at 4 C. Further dilutions were carried out in Tris-saline, and the samples were immediately assayed for plaqueforming activity on HeLa cells at 32.5 C (permissive temperature). (ii) Mutagenesis by hydroxylamine was performed according to Williams et al. (32). One part of crude extract from Ad2-infected HeLa cells was mixed with 2 parts of 2 N hydroxylamine in 0.1 M sodium phosphate, pH 7.5, at 25 C. At various times after starting the reaction, samples were diluted 100-fold in PBS containing 2% calf serum at 4 C. The dilutions were dialyzed against PBS for 12 h at 4 C prior to titer determinations on HeLa cell monolayers.
(iii) Mutagenesis by EMS (3) was performed by mixing 1 part of crude extract from Ad2-infected HeLa cells with 2 parts of 2% EMS in Tris-saline at 37 C. At various times after starting the reaction, samples were diluted 100-fold in Tris-saline at 4 C and assayed for infectivity titers in HeLa cells. Isolation of ts mutants. Wild-type Ad2 selected after four single-plaque isolations at 39.5 C and propagated as described above was inactivated by 3 to 4 logs by three different mutagens, as outlined in the preceding section, and immediately plated on HeLa cells at 32.5 C. After a 9- to 10-day incubation period, solitary, well-isolated plaques were punched out at random with a Pasteur pipette and transferred to a tube containing DC medium. The virus from each plaque was liberated by five cycles of freezing and thawing and retitered on HeLa cells at both 32.5 and 39.5 C. Isolates with a differential plaquing efficiency of 102 and higher were scored as mutants and further purified by three single-plaque isolations. The 14 ts mutants described in this report were derived from five different mutagenesis experiments, three with HNO2 and one each with EMS and hydroxylamine. Complementation tests. HeLa cells growing in monolayers were inoculated at the nonpermissive temperature with pairs of ts mutants and with each of the mutants separately. The MOI was 10 PFU/cell in single infections and 5 PFU/cell for each of the mutants in double infections. Adsorption was allowed to proceed for 1 h. Subsequently, the cells were washed with 1 ml of PBS and then incubated for 15 min with Ad2-specific antiserum (rabbit) diluted 1/100 with PBS. The antiserum was removed, the cells were washed again with PBS, and medium was added. The infected cells were incubated at 39.5 C. At 36 h p.i., the cells were scraped off the plate and resuspended in 1 ml of PBS. The cells were disrupted by five cycles of freezing and thawing, and virus yields were determined by plaque assay on HeLa cell monolayers at 32.5 C. The same procedure was followed in complementation experiments between Ad2 and Ad5. Complementation indexes were calculated from the formula: complementation index = [virus yield (mutant a x mutant b)]/[virus yield mutant a + virus yield mutant b]. DNA synthesis in HeLa cells infected with wild
VOL. 19, 1976
type and ts mutants of Ad2. Dense monolayers of HeLa cells (approximately 106 cells) in 30-mm-diameter plastic petri dishes were inoculated with wildtype or mutant Ad2 at an MOI of 20 PFU/cell. Adsorption was allowed to proceed for 60 min at 32.5 C; subsequently, the inoculum was removed, 1.5 ml of fresh medium (DC) was added, and incubation was continued at 32.5 or 39.5 C. The newly synthesized DNA in the Ad2-infected cells was labeled with 5 pCi of [3H]thymidine per ml from 12 to 24 h p.i. and from 60 to 120 h p.i. at 39.5 or 32.5 C, respectively. In some of the complementation experiments using the DNA-negative mutants of Ad2 or Ad5, HeLa cells were singly or doubly infected with various combinations of mutants, and the ability of the infected cells to synthesize viral DNA was scored as described below. Analysis of intracellular DNA by zonal sedimentation in alkaline sucrose density gradients. Previously described procedures were used (4, 6) with minor modifications. At the end of the labeling period, the infected cells were washed and scraped off the dish, and 10,000 to 20,000 cells were lysed in an 0.1-ml layer of 0.5 N NaOH on top of an alkaline sucrose density gradient (4 ml, 5 to 20%) in SW56 centrifuge tubes containing a 0.3-ml cushion of 70% sucrose solution in alkali. To each gradient a small amount of 14C-labeled Ad2 DNA was added to the 0. 1-ml lysis layer as marker. Lysis of the cells proceeded at 4 C for at least 14 h before the gradients were centrifuged in the SW56 rotor of the L2-65B Spinco ultracentrifuge at 4 C and 55,000 rpm for 90 min. After centrifugation, 4- or 6-drop fractions were collected and counted directly in 5 ml of toluene methanol scintillator in a Packard Tri-Carb liquid scintillation spectrometer, model 3330. Conditions for the preparation and centrifugation of SW41 sucrose gradients have been described (4). Analysis of DNA from Ad2-infected HeLa cells by DNA-DNA hybridization. The procedure of Denhardt (5) was used, as previously described (6, 7). Schleicher and Schuell (Dassel, Germany) nitrocellulose filters were used, and each new lot of filters was pretested with authentic Ad2 DNA. Gradient fractions from SW41 alkaline sucrose gradients were pooled (Fig. 3), neutralized, and dialyzed against 4 x SSC prior.to DNA-DNA hybridization. Thermostability of ts206. The thermal stability of the mutant H2ts206 was determined by incubating, at 39 to 40 C, a 1:10 dilution in PBS of crude extracts of HeLa cells that had been infected with the mutant. Incubation was for 0, 1, 2, 4, and 6 h. Subsequently, the samples were chilled and diluted 102_ fold in ice-cold PBS, and the plaque titer was determined. Isolation and detection of Ad2-specific mRNA in cells infected with mutant ts206. HeLa cells growing in monolayers were infected with wild-type Ad2 or with mutant ts206. Adsorption was allowed to proceed for 60 min at either 32.5 or 39.5 C. Cells inoculated with ts206 at either temperature were then incubated at 32.5 and 39.5 C, whereas cells inoculated with wild-type Ad2 at either temperature were incubated only at those respective temperatures. The infected cells were labeled with 100 ,uCi of
ts MUTANTS OF ADENOVIRUS TYPE 2
45
[3H]uridine per ml between 2 and 4 h p.i. (at 39.5 C) and between 11 and 16 h p.i. (at 32.5 C). Subsequently, polysomes and mRNA were isolated and analyzed by filter hybridization to Ad2 DNA as described earlier (20).
RESULTS Ad2. Ad2 is inactivated by of Inactivation treatment in vitro with nitrous acid, hydroxylamine, or EMS. In all three cases, the rate of inactivation follows single-hit kinetics. Nitrous acid is considerably more efficient than hydroxylamine or EMS as an inactivating agent. For the isolation of mutants, wild-type virus stocks were inactivated with each of three mutagens by 2 to 4 orders of magnitude. Isolation of ts mutants. In Table 1 the plaque-forming capacities at the permissive and nonpermissive temperatures have been summarized for the 14 ts mutant isolates. The ratio of the plaque-forming activities at 39.5 C over that at 32.5 C is close to 1 for wild-type Ad2 and 10-4 to 10-s for the ts mutants. When virus isolated from single plaques grown at 32.5 C was titrated at the permissive temperature, the yields ranged from 5 x 103 (ts213) to 7 x 105 (ts214) PFU/ml, whereas at the nonpermissive temperature the yield never exceeded 102 PFU/ml. It was noticed that ts213 formed only tiny plaques at the permissive temperature, whereas the plaque size of each of the other mutants at 32.5 C was comparable to that of wild type. The three agents applied, nitrous acid, hydroxylamine, and EMS, proved about equally efficient mutagens with Ad2 (Table 2). The value for EMS obviously was based on too small a number of isolates to be statistically meaningful. Characterization of ts mutants for defects in viral DNA synthesis. To determine whether the defects were in an early or late function, the 14 ts mutants were investigated for their ability to synthesize viral DNA in HeLa cells at the nonpermissive temperature. As a first step, the time of maximal Ad2 DNA synthesis had to be determined for the wild-type virus at the permissive and nonpermissive temperatures. After several experiments, labeling periods with [3H]thymidine (5 ,uCi/ml) between 12 and 24 h p.i. and between 60 and 120 h p.i. were chosen for the higher and lower temperatures, respectively. Accordingly, confluent monolayers of HeLa cells were inoculated in duplicate with 20 PFU of each of the 14 ts mutants per cell, incubated either at 32.5 or at 39.5 C, and labeled with [3H]thymidine at the time intervals indicated above. At the end of the labeling period, cells were lysed, and the newly synthe-
46
J . V IROL .
KATHMANN ET AL. TABLE 1. Plaque-forming ability of 14 ts mutants of Ad2 at 32.5 and 39.5 C
Ratio of PFU/ml at 39.5/
Infectivity (PFU/mlP Virus
Mutagenic agent
32.5 C 6.9 x 108
Wild type H2ts2Old 202 203 204 205 206 207 208 209 214
Nitrous acid
5.0 5.0 10.0 5.0 4.0 1.0 7.0 3.0 2.0 4.0
108 108 108 108 108 108 108 108 108 108
1.0 x 108 5.0 x 108 5.0 x 108
210 211 212
Hydroxylamine
x x x x x x x x x x
39.5 O 6.3 x 108
