JOURNAL OF VIROLOGY, JUlY 1975, p. 192-202 Copyright O 1975 American Society for Microbiology
Vol. 16, No. 1 Printed in U.S.A.
Isolation and Characterization of Temperature-Sensitive Mutants of Measles Virus CAROLYN M. BERGHOLZ,1 MICHAEL P. KILEY,2 AND FRANCIS E. PAYNE* Department of Epidemiology, School of Public Health, The University of Michigan, Ann Arbor, Michigan 48104 Received for publication 26 February 1975
Nine temperature-sensitive (ts) mutants of nonattenuated Edmonston strain measles virus were isolated from wild-type virus which was grown in the presence of 5-fluorouracil. Adsorption, temperature shift, and complementation experiments indicated that all these mutants were restricted at an intracellular stage of infection. However, all the mutants were more rapidly inactivated at 41 C than was wild-type virus, suggesting that the ts product of each mutant either influences or is a structural component of the virus. Three complementation groups were found to be respresented among the mutants. Group A contained one mutant and it did not induce synthesis of detectable amounts of viral antigen at the nonpermissive temperature (39 C). Group B consisted of six mutants which did not induce viral antigen synthesis at 39 C and one mutant which did. Group C was represented by one mutant and it induced viral antigen synthesis at 39 C. The two mutants which induced synthesis of viral antigen also induced synthesis of relatively small amounts of virus-specific RNA at 39 C. These mutants, while producing cytoplasmic and nuclear accumulations of viral antigen at 39 C, were restricted in production of syncytia and hemadsorption. All the mutants were less neurovirulent than wild-type virus, as indicated by their inability to produce acute disease in newborn hamsters.
Interest in the molecular aspects of measles essential medium supplemented with non-essential virus replication has been stimulated by obser- amino acids and 10% fetal calf serum. All media vation of measles antigen but no detectable contained penicillin (100 U/ml), streptomycin (100 infectious virus in cell cultures of brain from ,gg/ml), kanamycin (100 ug/ml), and amphotericin B (0.5 Ag/ml). Cultures were incubated in a humidified patients with subacute sclerosing panencephali- atmosphere of 5% CO, and air. tis (SSPE) (2, 4, 11) and by isolation of measles Virus. Wild-type virus, which plated with equal or a closely related virus from such cultures efficiency at 33 and 39 C, was plaque-purified from after prolonged cultivation or co-cultivation (1, non-attenuated Edmonston strain measles virus, orig9, 18, 19). Temperature-sensitive (ts) condi- inally obtained from H. M. Meyer, Jr. Wild-type virus tional-lethal mutants of animal viruses are of produced plaques which were 2 to 3 mm in diameter. great potential value for studying virus replica- Pools of virus were produced by infecting Vero cells at tion. Ts mutants of measles virus should also be 33 C, rapidly freezing and thawing the cultures when useful for analyzing restrictions in virus replica- 80% or more of the cells showed cytopathology, and the culture fluids by low-speed centrifugation tion, such as observed in SSPE. In this commu- clearing before storage at - 60 C. nication we report the isolation and prelimiPlaque assay. Vero cell monolayers, grown in 2-oz nary characterization of nine ts mutants of (ca. 0.06-liter) prescription bottles, were inoculated measles virus. with 0.5 ml of virus diluted in Eagle minimal essential medium supplemented with non-essential amino acids containing 2% fetal calf serum. After a 2-h adsorption period, monolayers were rinsed with Hanks balanced salt solution (BSS) and overlayed with 5 ml of Eagle minimal essential medium supplemented with non-essential amino acids containing 3.9% fetal calf serum and 1.8% agar. Plaque bottles 'Present address: Microbiology Department, Rush-Pres- were incubated for 7 days, at which time 5 ml of agar byterian-St. Luke's Medical Center, Chicago, Ill. 60612. 2Present address: Division of Biomedical Sciences, School medium containing 0.06% neutral red was added. of Medical Sciences, University of Nevada, Reno, Nev. 89507. Plaques were counted on day 8. 192
MATERIALS AND METHODS Cells. Monolayer cultures of Vero cells (26), a continuous line of green monkey kidney cells, were used. Growth medium consisted of Eagle minimal
VoL. 16, 1975
Mutagenesis. Cell cultures were inoculated with wild-type virus at a multiplicity of approximately 1.0. After a 1-h adsorption period, the cultures were rinsed with BSS. Growth medium containing 50 or 100 ug of 5-fluorouracil (Sigma) per ml was then added, and the cultures were incubated further at 33 C. Virus was harvested from cultures treated with 50 ug of 5-fluorouracil per ml on day 4, when 90% of the cells in infected control cultures showed cytopathic effects (CPE), and from cultures treated with 100 gg of 5-fluorouracil per ml on day 3, when cells treated with mutagen were coming off the glass. Apparently because of the relatively early harvest, 100 Mg of 5-fluorouracil per ml decreased virus yield by 1.2 log1. whereas 50 ug/ml decreased it by 3.0 log,1 Isolation of ts mutants. Well-isolated plaques of mutagenized virus were picked, and each agar plug was inoculated into a tube culture containing 1 ml of growth medium. Yields from these cultures served as initial pools of the plaque isolates. Each isolate was screened for temperature sensitivity by plating a 101-I dilution at 39 C and a 10-3 dilution at 33 C. Those isolates that yielded few or no plaques at 39 C and titers of 104 to 10' at 33 C were considered potentially usable mutants. Several minipools (1 ml each) of these potential mutants were produced at 33 C by inoculating tube cultures at varying dilutions, as described by Cooper (5). Minipools which gave ratios of plating efficiencies (39 C/33 C) of 10- 4 or less were then used at varying input multiplicities to produce 5-ml pools which were again tested for reversion. Pools with
a
ratio of plating efficiencies (39 C/33 C) of 10-4
less, or rarely of 10-', were used in the present study. Immunofluorescent staining. Measles virus antigen was detected by immunofluorescence using the indirect staining technique. Cell monolayers on cover slips were fixed in acetone. The sensitizing serum used was an unadsorbed serum from an SSPE patient and was diluted 1:10. The staining serum used was fluorescein-conjugated horse anti-human serum (Progressive Laboratories) diluted 1:20. Hemadsorption. Infected tube cultures were tested for hemadsorption by adding 0.1 ml of a 0.4% suspension of simian erythrocytes to the 1 ml of growth medium. Cultures were incubated further for 1 h and then observed for hemadsorption by light microscopy. Uninfected cultures served as controls. Complementation experiments. Mutants which did not produce viral antigen at 39 C were initially screened for complementation using immunofluorescence to test for synthesis of antigen. All mutants were subsequently tested for complementation by measuring virus yield in singly and doubly infected cultures. Vero cells (2 x 10') in small (well area, 1.2 cm2) Leighton tubes were infected with 0.2 ml of virus inoculum at a multiplicity of approximately 4. To control for possible multiplicity-dependent leakiness, single infections were carried out at a multiplicity of infection equal to the combined multiplicity in the doubly infected cultures. After a 2-h adsorption period at 33 C, the cell monolayers were rinsed with BSS and 0.3 ml of growth medium was added. or
ts MUTANTS OF MEASLES VIRUS
193
Preliminary experiments indicated that shift-up as late as 6 h postinfection did not allow the mutants to produce CPE or significant amounts of virus. For this reason, infected cultures were incubated at 33 C for 5 h after adsorption and then shifted to 39 C, in the hope of enhancing the probability of complementation. For immunofluorescence assay, cover slips were fixed at 72 h postinfection. For virus yield assays the following procedure was used. When a doubly infected culture showed 75% CPE (day 3 or 4), it was rapidly frozen along with its corresponding singly infected cultures. Virus yields were measured by plaque assay at 33 and 39 C. Complementation indexes were calculated as the ratio Jyield of (x + y),, titered at 33 C yield of (x + y) ,, titered at 39 C ]: [yield of (x),, titered at 33 C + yield of (y),9 titered at 33 C ], where x and y are any pair of mutants and subscript 39 indicates that the virus yield was from an infected culture incubated at 39 C. A complementation index greater than 1 was considered positive. Analysis of virus-specific RNA of infected cells. The procedures for labeling and analysis of RNA from infected cells were essentially as described previously (14). Virus added to cell monolayers in 75-cm2 plastic tissue culture flasks at a multiplicity of 0.05 PFU/cell was adsorbed for 2 h at 33 C. Cultures were then rinsed with 5 ml of BSS, and 10 ml of Eagle minimal essential medium supplemented with non-essential amino acids containing 2% fetal calf serum was added. Cultures were incubated further at 39 C. Mock-infected cultures served as controls. At 60 h postinfection, actinomycin D (Calbiochem) and cycloheximide (Sigma) were added at a final concentration of 40 and 75 jsg/ml, respectively. Two hours later [5-'H]uridine (20 Ci/mM, Nuclear Dynamics) was added to give a final concentration of 60 uCi/ml. After a 5-h labeling period, medium was removed, and the cultures were rinsed with 5 ml of cold Tris-buffered saline (0.01 M Tris, 0.14 M NaCl, pH 7.4). Cells were suspended in cold Tris-buffered saline by shaking with glass beads, washed twice in cold Tris-buffered saline, and then resuspended in reticulocyte standard buffer (0.01 M Tris, 0.01 M NaCl, 0.0015 M MgCl,, pH 7.4). Cytoplasmic extracts were prepared by Dounce homogenization, and RNA was extracted from these extracts by the phenol-sodium dodecyl sulfate method. After ethanol precipitation RNA pellets were resuspended in NTE buffer (0.1 M NaCl, 0.01 M Tris, 0.001 M EDTA, pH 7.4) containing 0.025% sodium dodecyl sulfate. RNA was analyzed in 5 to 20% (wt/vol) sucrose gradients in 0.02 M acetate buffer, pH 5.0. The acid-insoluble radioactivity in each fraction was determined as previously described (13). The amounts of 18 and 28S ribosomal marker RNA and their positions in the gradients were determined by continuously monitoring the gradient effluent at 254 nm. Inoculation of newborn hamsters. Multiparous pregnant Golden Syrian hamsters were obtained from Lakeview Hamster Colony, Newfield, N.J. Offspring were inoculated intracerebrally 12 to 24 h after birth with approximately 0.05 ml of virus suspension.
194
RESULTS Efficiency of plating. Nine ts mutants were isolated which had ratios of plating efficiencies (39 C/33 C) of 10-4 or less (Table 1). With one exception (ts-1), all ts mutants were isolated from the pool of virus which had been mutagenized with 5-fluorouracil at 100,ug/ml. Mutants represented 1.8% of the plaques tested from this pool. Attempts to measure true reversion frequencies of the mutants were precluded by the fact that insufficient infectious virus could be extracted from agar plugs of picked plaques. However, the frequency of isolation of mutants from the mutagenized virus pool and the frequent appearance of revertant-type virus in pools of the mutants suggested that the conditional-lethal characteristics of these viruses are attributable to single-step mutations. Kinetics of virus production. Growth of ts mutants and wild-type virus at 33 and 39 C was determined. Growth curves at 33 and 39 C of wild-type virus and mutants ts-1 and ts-3 are illustrated in Fig. 1. Wild-type virus yielded 1 log less infectious virus by 72 h at 39 than at 33 C. This lower yield was probably a reflection of heat inactivation of progeny virus, since at 48 h postinfection the yield was higher at 39 than at 33 C. At 33 C these ts mutants, like wild-type virus, produced 106 PFU/ml. When virus production by the ts mutants at 39 C was examined, titers were less than 10 PFU/ml throughout the 72-h incubation. All ts mutants, except ts-6, failed to yield significant amounts of infectious virus by 72 h postinfection at 39 C, while producing 103 to 105 PFU/ml at the permissive temperature (Table 2). Virus produced by ts-6 at 39 C was found to be temperature sensitive, indicating that this yield was due to leak rather than reversion. Ts-2 grows slowly TABLE 1. Ratios of plating efficiencies (39 C/33 C) of wild-type and ts mutants of measles virus Virus
Wild-type ts- 1 ts-2 ts-3 ts-4 ts-5 ts-6 ts-7 ts-8 ts-9
J. VIROL.
BERGHOLZ, KILEY, AND PAYNE
PFU/ml at 33C 39C
4.0 x 1.3 x 3.6 x 7.2 x 8.6 x 8.8 x 3.4 x 9.8 x 4.8 x 8.0 x
105 105
10' 105
10' 105
10' 105
10' 105
4.8 x 105 101
Vol. 16, No. 1 Printed in U.S.A.
Isolation and Characterization of Temperature-Sensitive Mutants of Measles Virus CAROLYN M. BERGHOLZ,1 MICHAEL P. KILEY,2 AND FRANCIS E. PAYNE* Department of Epidemiology, School of Public Health, The University of Michigan, Ann Arbor, Michigan 48104 Received for publication 26 February 1975
Nine temperature-sensitive (ts) mutants of nonattenuated Edmonston strain measles virus were isolated from wild-type virus which was grown in the presence of 5-fluorouracil. Adsorption, temperature shift, and complementation experiments indicated that all these mutants were restricted at an intracellular stage of infection. However, all the mutants were more rapidly inactivated at 41 C than was wild-type virus, suggesting that the ts product of each mutant either influences or is a structural component of the virus. Three complementation groups were found to be respresented among the mutants. Group A contained one mutant and it did not induce synthesis of detectable amounts of viral antigen at the nonpermissive temperature (39 C). Group B consisted of six mutants which did not induce viral antigen synthesis at 39 C and one mutant which did. Group C was represented by one mutant and it induced viral antigen synthesis at 39 C. The two mutants which induced synthesis of viral antigen also induced synthesis of relatively small amounts of virus-specific RNA at 39 C. These mutants, while producing cytoplasmic and nuclear accumulations of viral antigen at 39 C, were restricted in production of syncytia and hemadsorption. All the mutants were less neurovirulent than wild-type virus, as indicated by their inability to produce acute disease in newborn hamsters.
Interest in the molecular aspects of measles essential medium supplemented with non-essential virus replication has been stimulated by obser- amino acids and 10% fetal calf serum. All media vation of measles antigen but no detectable contained penicillin (100 U/ml), streptomycin (100 infectious virus in cell cultures of brain from ,gg/ml), kanamycin (100 ug/ml), and amphotericin B (0.5 Ag/ml). Cultures were incubated in a humidified patients with subacute sclerosing panencephali- atmosphere of 5% CO, and air. tis (SSPE) (2, 4, 11) and by isolation of measles Virus. Wild-type virus, which plated with equal or a closely related virus from such cultures efficiency at 33 and 39 C, was plaque-purified from after prolonged cultivation or co-cultivation (1, non-attenuated Edmonston strain measles virus, orig9, 18, 19). Temperature-sensitive (ts) condi- inally obtained from H. M. Meyer, Jr. Wild-type virus tional-lethal mutants of animal viruses are of produced plaques which were 2 to 3 mm in diameter. great potential value for studying virus replica- Pools of virus were produced by infecting Vero cells at tion. Ts mutants of measles virus should also be 33 C, rapidly freezing and thawing the cultures when useful for analyzing restrictions in virus replica- 80% or more of the cells showed cytopathology, and the culture fluids by low-speed centrifugation tion, such as observed in SSPE. In this commu- clearing before storage at - 60 C. nication we report the isolation and prelimiPlaque assay. Vero cell monolayers, grown in 2-oz nary characterization of nine ts mutants of (ca. 0.06-liter) prescription bottles, were inoculated measles virus. with 0.5 ml of virus diluted in Eagle minimal essential medium supplemented with non-essential amino acids containing 2% fetal calf serum. After a 2-h adsorption period, monolayers were rinsed with Hanks balanced salt solution (BSS) and overlayed with 5 ml of Eagle minimal essential medium supplemented with non-essential amino acids containing 3.9% fetal calf serum and 1.8% agar. Plaque bottles 'Present address: Microbiology Department, Rush-Pres- were incubated for 7 days, at which time 5 ml of agar byterian-St. Luke's Medical Center, Chicago, Ill. 60612. 2Present address: Division of Biomedical Sciences, School medium containing 0.06% neutral red was added. of Medical Sciences, University of Nevada, Reno, Nev. 89507. Plaques were counted on day 8. 192
MATERIALS AND METHODS Cells. Monolayer cultures of Vero cells (26), a continuous line of green monkey kidney cells, were used. Growth medium consisted of Eagle minimal
VoL. 16, 1975
Mutagenesis. Cell cultures were inoculated with wild-type virus at a multiplicity of approximately 1.0. After a 1-h adsorption period, the cultures were rinsed with BSS. Growth medium containing 50 or 100 ug of 5-fluorouracil (Sigma) per ml was then added, and the cultures were incubated further at 33 C. Virus was harvested from cultures treated with 50 ug of 5-fluorouracil per ml on day 4, when 90% of the cells in infected control cultures showed cytopathic effects (CPE), and from cultures treated with 100 gg of 5-fluorouracil per ml on day 3, when cells treated with mutagen were coming off the glass. Apparently because of the relatively early harvest, 100 Mg of 5-fluorouracil per ml decreased virus yield by 1.2 log1. whereas 50 ug/ml decreased it by 3.0 log,1 Isolation of ts mutants. Well-isolated plaques of mutagenized virus were picked, and each agar plug was inoculated into a tube culture containing 1 ml of growth medium. Yields from these cultures served as initial pools of the plaque isolates. Each isolate was screened for temperature sensitivity by plating a 101-I dilution at 39 C and a 10-3 dilution at 33 C. Those isolates that yielded few or no plaques at 39 C and titers of 104 to 10' at 33 C were considered potentially usable mutants. Several minipools (1 ml each) of these potential mutants were produced at 33 C by inoculating tube cultures at varying dilutions, as described by Cooper (5). Minipools which gave ratios of plating efficiencies (39 C/33 C) of 10- 4 or less were then used at varying input multiplicities to produce 5-ml pools which were again tested for reversion. Pools with
a
ratio of plating efficiencies (39 C/33 C) of 10-4
less, or rarely of 10-', were used in the present study. Immunofluorescent staining. Measles virus antigen was detected by immunofluorescence using the indirect staining technique. Cell monolayers on cover slips were fixed in acetone. The sensitizing serum used was an unadsorbed serum from an SSPE patient and was diluted 1:10. The staining serum used was fluorescein-conjugated horse anti-human serum (Progressive Laboratories) diluted 1:20. Hemadsorption. Infected tube cultures were tested for hemadsorption by adding 0.1 ml of a 0.4% suspension of simian erythrocytes to the 1 ml of growth medium. Cultures were incubated further for 1 h and then observed for hemadsorption by light microscopy. Uninfected cultures served as controls. Complementation experiments. Mutants which did not produce viral antigen at 39 C were initially screened for complementation using immunofluorescence to test for synthesis of antigen. All mutants were subsequently tested for complementation by measuring virus yield in singly and doubly infected cultures. Vero cells (2 x 10') in small (well area, 1.2 cm2) Leighton tubes were infected with 0.2 ml of virus inoculum at a multiplicity of approximately 4. To control for possible multiplicity-dependent leakiness, single infections were carried out at a multiplicity of infection equal to the combined multiplicity in the doubly infected cultures. After a 2-h adsorption period at 33 C, the cell monolayers were rinsed with BSS and 0.3 ml of growth medium was added. or
ts MUTANTS OF MEASLES VIRUS
193
Preliminary experiments indicated that shift-up as late as 6 h postinfection did not allow the mutants to produce CPE or significant amounts of virus. For this reason, infected cultures were incubated at 33 C for 5 h after adsorption and then shifted to 39 C, in the hope of enhancing the probability of complementation. For immunofluorescence assay, cover slips were fixed at 72 h postinfection. For virus yield assays the following procedure was used. When a doubly infected culture showed 75% CPE (day 3 or 4), it was rapidly frozen along with its corresponding singly infected cultures. Virus yields were measured by plaque assay at 33 and 39 C. Complementation indexes were calculated as the ratio Jyield of (x + y),, titered at 33 C yield of (x + y) ,, titered at 39 C ]: [yield of (x),, titered at 33 C + yield of (y),9 titered at 33 C ], where x and y are any pair of mutants and subscript 39 indicates that the virus yield was from an infected culture incubated at 39 C. A complementation index greater than 1 was considered positive. Analysis of virus-specific RNA of infected cells. The procedures for labeling and analysis of RNA from infected cells were essentially as described previously (14). Virus added to cell monolayers in 75-cm2 plastic tissue culture flasks at a multiplicity of 0.05 PFU/cell was adsorbed for 2 h at 33 C. Cultures were then rinsed with 5 ml of BSS, and 10 ml of Eagle minimal essential medium supplemented with non-essential amino acids containing 2% fetal calf serum was added. Cultures were incubated further at 39 C. Mock-infected cultures served as controls. At 60 h postinfection, actinomycin D (Calbiochem) and cycloheximide (Sigma) were added at a final concentration of 40 and 75 jsg/ml, respectively. Two hours later [5-'H]uridine (20 Ci/mM, Nuclear Dynamics) was added to give a final concentration of 60 uCi/ml. After a 5-h labeling period, medium was removed, and the cultures were rinsed with 5 ml of cold Tris-buffered saline (0.01 M Tris, 0.14 M NaCl, pH 7.4). Cells were suspended in cold Tris-buffered saline by shaking with glass beads, washed twice in cold Tris-buffered saline, and then resuspended in reticulocyte standard buffer (0.01 M Tris, 0.01 M NaCl, 0.0015 M MgCl,, pH 7.4). Cytoplasmic extracts were prepared by Dounce homogenization, and RNA was extracted from these extracts by the phenol-sodium dodecyl sulfate method. After ethanol precipitation RNA pellets were resuspended in NTE buffer (0.1 M NaCl, 0.01 M Tris, 0.001 M EDTA, pH 7.4) containing 0.025% sodium dodecyl sulfate. RNA was analyzed in 5 to 20% (wt/vol) sucrose gradients in 0.02 M acetate buffer, pH 5.0. The acid-insoluble radioactivity in each fraction was determined as previously described (13). The amounts of 18 and 28S ribosomal marker RNA and their positions in the gradients were determined by continuously monitoring the gradient effluent at 254 nm. Inoculation of newborn hamsters. Multiparous pregnant Golden Syrian hamsters were obtained from Lakeview Hamster Colony, Newfield, N.J. Offspring were inoculated intracerebrally 12 to 24 h after birth with approximately 0.05 ml of virus suspension.
194
RESULTS Efficiency of plating. Nine ts mutants were isolated which had ratios of plating efficiencies (39 C/33 C) of 10-4 or less (Table 1). With one exception (ts-1), all ts mutants were isolated from the pool of virus which had been mutagenized with 5-fluorouracil at 100,ug/ml. Mutants represented 1.8% of the plaques tested from this pool. Attempts to measure true reversion frequencies of the mutants were precluded by the fact that insufficient infectious virus could be extracted from agar plugs of picked plaques. However, the frequency of isolation of mutants from the mutagenized virus pool and the frequent appearance of revertant-type virus in pools of the mutants suggested that the conditional-lethal characteristics of these viruses are attributable to single-step mutations. Kinetics of virus production. Growth of ts mutants and wild-type virus at 33 and 39 C was determined. Growth curves at 33 and 39 C of wild-type virus and mutants ts-1 and ts-3 are illustrated in Fig. 1. Wild-type virus yielded 1 log less infectious virus by 72 h at 39 than at 33 C. This lower yield was probably a reflection of heat inactivation of progeny virus, since at 48 h postinfection the yield was higher at 39 than at 33 C. At 33 C these ts mutants, like wild-type virus, produced 106 PFU/ml. When virus production by the ts mutants at 39 C was examined, titers were less than 10 PFU/ml throughout the 72-h incubation. All ts mutants, except ts-6, failed to yield significant amounts of infectious virus by 72 h postinfection at 39 C, while producing 103 to 105 PFU/ml at the permissive temperature (Table 2). Virus produced by ts-6 at 39 C was found to be temperature sensitive, indicating that this yield was due to leak rather than reversion. Ts-2 grows slowly TABLE 1. Ratios of plating efficiencies (39 C/33 C) of wild-type and ts mutants of measles virus Virus
Wild-type ts- 1 ts-2 ts-3 ts-4 ts-5 ts-6 ts-7 ts-8 ts-9
J. VIROL.
BERGHOLZ, KILEY, AND PAYNE
PFU/ml at 33C 39C
4.0 x 1.3 x 3.6 x 7.2 x 8.6 x 8.8 x 3.4 x 9.8 x 4.8 x 8.0 x
105 105
10' 105
10' 105
10' 105
10' 105
4.8 x 105 101
