Proc. Nat. Acad. Sci. USA Vol. 72, No. 8, pp. 3230-3234, August 1975
Microbiology
Recovery of infectious proviral DNA from mammalian cells infected with respiratory syncytial virus (ribovirus/temperature-sensitive mutant/nonvirogenic cells/DNA transfection)
ROBERT W. SIMPSON AND MASAO IINUMA* Waksman Institute of Microbiology, Rutgers University, The State University of New Jersey, New Brunswick, N.J. 08903
Communicated by Robert M. Chanock, May 21, 1975
The DNA fraction from a line of bovine emABSTRACT bryonic kidney cells originally exposed as primary cultures several months earlier to a temperature-sensitive (ts) mutant of respiratory syncytial (RS) virus could be used to transfect human HEp-2 cells with the production of infectious RS virus. The DNA donor cells, designated BEK/RS ts, retained their healthy fibroblastic appearance during continuous cultivation at a temperature (390) restrictive for growth of the original infecting mutant and showed no evidence for RS virus replication or viral antigen synthesis when directly examined for these activities by conventional methods. The infectious property of the DNA from BEK/RS ts cells was abolished by exposure of the nucleic acid preparation to DNase (but not RNase) or by pretreatment of recipient HEp-2 cells with actinomycin D or mitomycin C. The latter drug treatments substantially enhanced the replication of infecting wild-type RS virus in HEp-2 cells. Viral isolates derived from the progeny of a DNA transfection included clones possessing several genetic markers of the RS ts mutant originally used to infect BEK/RS ts cells and other virus clones tfat appeared to be either hybrid or wild-type for phenotypic properties such as their temperature sensitivity. An infectious proviral DNA was also detected in a line of virogenic HEp-2 cells (HEp-2/RS) persistently infected with respiratory syncytial virus after exposure to the wild-type strain 2 years earlier. An extensive literature exists attesting to the capacity of human and animal viruses to persist under conditions of natural infection or in cell culture systems and experimental animals (1-4). The pathogenetic mechanisms underlying these persistent infections are extremely diverse, as manifested by responses ranging from inapparent or latent infection, partial expression of viral gene functions on a continuous or intermittent basis, and noncytocidal "steady-state" production of infectious virus particles in apparent harmony with the resident host. Little is understood about the autonomy of viral nucleic acids associated with the persistence of conventional RNA-containing animal viruses in eukaryotes either in the absence of viral gene expression or between recrudescences of disease and virus production. This problem has occupied our interests for the past 2 years, during which time we have developed appropriate in vitro host-virus systems for studying molecular mechanisms which allow nononcogenic, enveloped riboviruses to coexist with the replicating host cell genome in a compatible manner. Respiratory syncytial (RS) Abbreviations: RS virus, respiratory syncytial virus; BEK cells, bovine embryonic kidney cells; R-MEM, reinforced Eagle's minimal essential medium; BSS, modified Hanks' balanced salt solution; SSC, standard saline-citrate; ts, temperature-sensitive; HR, heat-
resistant. * Visiting investigator from the Department of Bacteriology, Yamagata University School of Medicine, Yamagata, Japan.
virus was chosen for study, since consideration is being given to the use of live, temperature-sensitive virus vaccines of this important pathogen for large-scale immunoprophylaxis of human populations (5). This preliminary report chiefly concerns the important finding that RS virus can form infectious proviral DNA intermediates which persist in actively dividing mammalian cells independently of detectable viral gene expression.
MATERIALS AND METHODS Viruses and Cells. The A-2 strain of respiratory syncytial virus grown in bovine embryonic kidney (BEK) cells was originally obtained from a commercial laboratory (Flow Laboratories, Inc., Rockville, Md.) as the "wild-type" virus of this study. The origin of two mutants, HRS/50 and ts 4074, is briefly described under Results. Primary cultures of BEK cells used for the development of the BEK/RS ts line (see Results) were prepared with tissues from a local abattoir as recently described (6). We have also described the propagation of HEp-2 carcinoma cells in reinforced Eagle's minimal essential medium (RMEM) medium supplemented with 10% fetal calf serum (6). Petri dish, prescription bottle, or plastic T-flask cultures of these cells were incubated in a humidified CO2 incubator controlled at 350 until they formed confluent monolayers within 3-4 days. The BEK/RS ts cell line used in these experiments was maintained exclusively at 390 under conditions of transfer (about 1:3 split) at approximately weekly intervals. The chronically infected HEp-2/RS cells (Iinuma and Simpson, in preparation) were maintained in a similar manner over a continuous 2-year period except that the temperature of incubation was 350. The composition of modified Hanks' balanced salt solution (BSS), virus diluent containing gelatin (GBSS), atd other solutions used for cell culture work was reported earlier (7). Plaque Assays and Virus Cloning. All titrations for plaque forming units (PFU) were done in HEp-2 petri dish cultures as described elsewhere (Iinuma and Simpson, in preparation). For growth of virus clones, well-isolated plaques were sampled with pasteur pipettes (7) and the virus was suspended in 2 ml aliquots of BSS containing 20% fetal calf serum. Stocks of individual clones were prepared by infecting HEp-2 petri dish cultures (60 mm) with undiluted plaque suspension and utilizing cell lysates obtained by freezing/thawing of the cultures harvested after incubation at 350 for 6-7 days. Extraction of Cellular DNA and RNA Fractions. The cells extracted for nucleic acid included normal BEK (secondary cultures), normal HEp-2, the BEK/RS ts line, and the 3230
Microbiology: Simpson and finuma HEp-2/RS line maintained in vitro as described above. We employed the method of Marmur as modified by Hill"and Hillova (8) for DNA extraction with slight further modification. A total of about 108 cells (107/culture) attached as confluent monolayers were washed thoroughly with a buffer containing 0.15 M NaCl and 0.1 M ethylenediaminetetraacetate (EDTA), pH 8. Thereafter, the cells were lysed in situ by addition of 15 ml per 107 cells of the same buffer containing 1% sodium dodecyl sulfate and heating in a 600
Proc. Nat. Acad. Sci. USA 72 (1975)
2
3231
a
10
0
z
water bath for 10 min with intermittent agitation. After
cooling to room temperature, the lysates were pooled and combined with an equal volume of 24:1 (v/v) chloroform/ isoamyl alcohol and subjected to intermittent shaking during a 30-min incubation. The aqueous phase of this mixture was recovered following centrifugation at 6000 X g for 5 min
(Sorvall RC2-B centrifuge), taking care that the temperature setting was not low enough to precipitate the detergent present. Alcohol precipitation of the nucleic acid fraction was accomplished by adding two parts of 95% ethanol to one part aqueous phase. The precipitate formed after incubation at room temperature for 10 min was collected by low-speed centrifugation and dissolved over a period of 30 min or more in 15 ml of SSC buffer (0.15 M NaCI, 0.015 M sodium citrate, pH 7) with gentle to moderate mechanical agitation. An equal volume of RNase A (Worthington Biochemical Corp., Freehold, N.J.) in 0.15 M NaCl solution was added at a final concentration of 50 jig/ml and incubation was carried out at 370 for 60 min. Thereafter, an equal volume of Pronase (Calbiochem, La Jolla, Calif.) dissolved in 0.15 M NaCl solution was added at a concentration of 500 jig/ml and further incubation at 370 for 4 hr was carried out. Deproteinization with chloroform/isoamyl alcohol and subsequent precipitation of the aqueous phase with ethanol as described above was repeated. The precipitate was dissolved in 9 ml of SSC buffer within a period of 2-3 min. After re-precipitation with a one-half volume of absolute isopropanol for 10 min, the precipitate obtained was collected by low-speed centrifugation and sterilized by exposure to 15 ml of 75% ethanol for 24 hr at 4°. For the experiments described in this report, the final precipitate was dissolved in about 15 ml of Dulbecco's phosphate-buffered saline. The average 260/280 nm absorbance ratio of these preparations was 1.9 and yields of DNA approximated 200 jg from 108 cells. DNA prepared in this manner was stored at -70°. For RNA extraction, cells were washed thoroughly with a buffer containing 0.1 M NaCI, 0.01 M Tris-HCI (pH 8.5), and 0.001 M EDTA. The same buffer containing 1% sodium dodecyl sulfate was used to lyse the cells. The aqueous phase obtained after a single extraction with water-saturated phenol was subjected to two additional phenol extractions after which precipitation with 95% ethanol was carried out. The resulting precipitate was dissolved in SSC buffer and Pronase treatment at 370 was carried out as described for the DNA extraction technique. Thereafter, RNase-free DNase (Worthington) was added at a final concentration of 10 ,ug/ml and further incubation at 370 for 60 min was carried out. The final precipitate obtained after an additional alcohol precipitation was resuspended in high salt buffer consisting of 0.8 M NaCI, 0.03% EDTA, and 0.02 M sodium phosphate buffer at pH 7.2. DNA Transfection of Cells. Petri dish (60 mm) cultures of HEp-2 cells were thoroughly washed with BSS and inoculated with 1 ml of the appropriate DNA or control preparation diluted in high salt buffer. The inocula were removed by washing with BSS after a 10 min adsorption (250) and 5
4A
0.01
_ 30
60
90
120
150
MINUTES AT 500
FIG. 1. Heat inactivation kinetics of wild-type respiratory syncytial virus and mutants HRS/50 and ts 4074. Crude stocks of the tissue culture grown viruses containing 3% fetal calf serum as a constituent of the original growth medium (R-MEM) were heated undiluted in a 50° water bath for the times indicated. Heated samples were quickly chilled in ice and titrated for surviving infectious virus by standard plaque assay in HEp-2 cells at 35°. Mutant HRS/50 (0) was derived from wild-type RS (-) by selective enrichment, whereas mutant ts 4074 (-) was obtained by chemical mutagenesis of HRS/50 virus stocks (see text).
ml of R-MEM with 3% fetal calf serum was added to each culture. For this study, the cultures were incubated at 350. Cell-associated virus particles produced under these conditions were released by exposing cells to cycles of freezing/ thawing. The viral progeny contained in DNA transfection yields was assayed or cloned in HEp-2 cells as described above. For experiments involving drug-treated cells, the inhibitors were freshly prepared using cell culture medium as a suspending solution. The inhibitors used included actinomycin D (gift of Dr. J. 0. Lampen), mitomycin C (Calbiochem), and cytosine arabinonucleoside (Nutritional Biochemicals Co.). Immunofluorescent Staining. The y-globulin fraction of horse antiserum to RS virus (Flow Laboratories) was used to prepare a fluorescein isothiocyanate immune conjugate as recently described by us (6). Infected coverslip cultures of HEp-2 cells stained with this conjugate after fixation with cold acetone were examined by fluorescence microscopy. The samples were coded so that they could not be directly identified by the experimenter. RESULTS Origin and general properties of BEK/RS ts cells As part of a vaccine development program, we have isolated a collection of 25 ts (temperature-sensitive) mutants of RS virus using a regimen for chemical mutagenesis that is likely to produce multiple genetic lesions in genomes of the viable conditional lethal viruses recovered. The most stable of these RS mutants, ts 4074, was derived from a heat-resistant variant, HRS/50, by treatment of virus with N-methyl-N'-nitroN-nitrosoguanidine and subsequent growth of the surviving fraction in primary BEK cells in the presence of 5-fluorouridine. As illustrated in Fig. 1, ts 4074 shares the heat-resistant character of its HRS/50 progenitor by comparison with the inactivation kinetics observed with wild-type RS virus. Mutant ts 4074 has consistently failed to replicate or produce discernible plaques in BEK cells incubated at 390 or higher. A line of cells, hereafter designated BEK/RS ts, was
Proc. Nat. Acad. Sci. USA 72 (1975)
Microbiology: Simpson and Iinuma
3232
Table 1. Production of virus-specific antigen and infectious RS virus after transfection of HEp-2 cells with DNA from nonvirogenic BEK/RS ts cellsa Immunofluorescent staining for RS virus
Yield of infectious RS virus
antigenc
Transfection inoculum:
(PFU/ml) :d
Donor cells
Fraction testedb
Intensity
Positive cells (%)
350 assay
390assay
BEK/RS ts 4074 (passage 24 at 390)
0.8 Mg of DNA, untreated 0.8 Mg of DNA, plus DNase 0.8 jig of DNA, plus RNase 30 pg of RNA, untreated Crude cell lysate 5.2 jig of DNA, untreated
1-4+
-90
104.6
104.0
0
0
104.5 0 0
ND 0 0
0
0
Normal BEK (passage 2 at 390)
0
0
1-4+ 0 0
-90 0 0
0
0
cultures of bovine embryonic kidney (BEK) cells originally infected with RS ts mutant 4074 were maintained in vitro by cultivation at 390 and transfer at approximately weekly intervals. Total DNA was extracted (Materials and Methods) from these cells at the 24th passage level. Nd infectious RS virus could be detected in these cells by direct assay. Mock-infected BEK cells at the 2nd passage level served as a source of control cell DNA in this experiment. b The concentrations listed are the total amounts of DNA or RNA inoculated into HEp-2 monolayer cultures containing 2 x 106 cells. For DNase treatment, ribonuclease-free deoxyribonuclease was added to 42 fig of DNA in Dulbecco's PBS at a final concentration of 10 zg/ml and the mixture was incubated at 370 for 60 min. RNase treatment consisted of adding 50 ,g/ml (final concentration) of ribonuclease A to 42 ,ug of DNA and incubating this mixture at 370 for 60 min. Crude cell lysates were prepared by exposing 2 x 107 cells in 5 ml of phosphatebuffered saline to three cycles of freezing/thawing and removing the cellular debris by low speed centrifugation. HEp-2 cell monolayers were inoculated with 0.2 ml volumes of the undiluted lysate. c Coverslip cultures of attached HEp-2 cells were processed for immunofluorescent staining (Materials and Methods) 7 days after exposure to inoculum and subsequent incubation at 35°. d Confluent HEpr-2 monolayers incubated at 35° for 7 days after exposure to the inoculum indicated were lysed by freezing/thawing. Clarified cell lysates were assayed for plaque-forming virus in HEp-2 cells incubated at 350 and 39°. ND = not determined. a Primary
established by infection of primary BEK monolayer cultures with mutant ts 4074 which were incubated exclusively at nonpermissive temperature (390) during successive serial transfers to fresh culture vessels. Under these conditions, we were unable to detect virus-specific RS antigens in these
cells by immunofluorescent staining and no infectious RS virus could be found in cell lysates by direct plaque assay. However, late passage BEK/RS ts cells were consistently found to produce infectious RS virus when cocultivated with normal BEK cells or exposed to specific inducing agents,
Table 2. Phenotypic properties of RS virus clones isolated from the progeny of a DNA transfection experimenta
Source of clone
Controls Wild-type RS Mutant HRS/50 Mutant ts 4074e
Clone no.
RS-1
HRS-1
ts 4074 Test group: DNA transfection yieldf RS-DNA-1 RS-DNA-2
Syncytia. % Neutralization formation by RS virus at 350 antiserumb
Ratio of
390/350 PFU titerc
syn+ syn+ syn+
>99 >99 >99
99 >99
99
RS-DNA-4
syn+
>99
0.8-1.2 0.8-1.2
Microbiology
Recovery of infectious proviral DNA from mammalian cells infected with respiratory syncytial virus (ribovirus/temperature-sensitive mutant/nonvirogenic cells/DNA transfection)
ROBERT W. SIMPSON AND MASAO IINUMA* Waksman Institute of Microbiology, Rutgers University, The State University of New Jersey, New Brunswick, N.J. 08903
Communicated by Robert M. Chanock, May 21, 1975
The DNA fraction from a line of bovine emABSTRACT bryonic kidney cells originally exposed as primary cultures several months earlier to a temperature-sensitive (ts) mutant of respiratory syncytial (RS) virus could be used to transfect human HEp-2 cells with the production of infectious RS virus. The DNA donor cells, designated BEK/RS ts, retained their healthy fibroblastic appearance during continuous cultivation at a temperature (390) restrictive for growth of the original infecting mutant and showed no evidence for RS virus replication or viral antigen synthesis when directly examined for these activities by conventional methods. The infectious property of the DNA from BEK/RS ts cells was abolished by exposure of the nucleic acid preparation to DNase (but not RNase) or by pretreatment of recipient HEp-2 cells with actinomycin D or mitomycin C. The latter drug treatments substantially enhanced the replication of infecting wild-type RS virus in HEp-2 cells. Viral isolates derived from the progeny of a DNA transfection included clones possessing several genetic markers of the RS ts mutant originally used to infect BEK/RS ts cells and other virus clones tfat appeared to be either hybrid or wild-type for phenotypic properties such as their temperature sensitivity. An infectious proviral DNA was also detected in a line of virogenic HEp-2 cells (HEp-2/RS) persistently infected with respiratory syncytial virus after exposure to the wild-type strain 2 years earlier. An extensive literature exists attesting to the capacity of human and animal viruses to persist under conditions of natural infection or in cell culture systems and experimental animals (1-4). The pathogenetic mechanisms underlying these persistent infections are extremely diverse, as manifested by responses ranging from inapparent or latent infection, partial expression of viral gene functions on a continuous or intermittent basis, and noncytocidal "steady-state" production of infectious virus particles in apparent harmony with the resident host. Little is understood about the autonomy of viral nucleic acids associated with the persistence of conventional RNA-containing animal viruses in eukaryotes either in the absence of viral gene expression or between recrudescences of disease and virus production. This problem has occupied our interests for the past 2 years, during which time we have developed appropriate in vitro host-virus systems for studying molecular mechanisms which allow nononcogenic, enveloped riboviruses to coexist with the replicating host cell genome in a compatible manner. Respiratory syncytial (RS) Abbreviations: RS virus, respiratory syncytial virus; BEK cells, bovine embryonic kidney cells; R-MEM, reinforced Eagle's minimal essential medium; BSS, modified Hanks' balanced salt solution; SSC, standard saline-citrate; ts, temperature-sensitive; HR, heat-
resistant. * Visiting investigator from the Department of Bacteriology, Yamagata University School of Medicine, Yamagata, Japan.
virus was chosen for study, since consideration is being given to the use of live, temperature-sensitive virus vaccines of this important pathogen for large-scale immunoprophylaxis of human populations (5). This preliminary report chiefly concerns the important finding that RS virus can form infectious proviral DNA intermediates which persist in actively dividing mammalian cells independently of detectable viral gene expression.
MATERIALS AND METHODS Viruses and Cells. The A-2 strain of respiratory syncytial virus grown in bovine embryonic kidney (BEK) cells was originally obtained from a commercial laboratory (Flow Laboratories, Inc., Rockville, Md.) as the "wild-type" virus of this study. The origin of two mutants, HRS/50 and ts 4074, is briefly described under Results. Primary cultures of BEK cells used for the development of the BEK/RS ts line (see Results) were prepared with tissues from a local abattoir as recently described (6). We have also described the propagation of HEp-2 carcinoma cells in reinforced Eagle's minimal essential medium (RMEM) medium supplemented with 10% fetal calf serum (6). Petri dish, prescription bottle, or plastic T-flask cultures of these cells were incubated in a humidified CO2 incubator controlled at 350 until they formed confluent monolayers within 3-4 days. The BEK/RS ts cell line used in these experiments was maintained exclusively at 390 under conditions of transfer (about 1:3 split) at approximately weekly intervals. The chronically infected HEp-2/RS cells (Iinuma and Simpson, in preparation) were maintained in a similar manner over a continuous 2-year period except that the temperature of incubation was 350. The composition of modified Hanks' balanced salt solution (BSS), virus diluent containing gelatin (GBSS), atd other solutions used for cell culture work was reported earlier (7). Plaque Assays and Virus Cloning. All titrations for plaque forming units (PFU) were done in HEp-2 petri dish cultures as described elsewhere (Iinuma and Simpson, in preparation). For growth of virus clones, well-isolated plaques were sampled with pasteur pipettes (7) and the virus was suspended in 2 ml aliquots of BSS containing 20% fetal calf serum. Stocks of individual clones were prepared by infecting HEp-2 petri dish cultures (60 mm) with undiluted plaque suspension and utilizing cell lysates obtained by freezing/thawing of the cultures harvested after incubation at 350 for 6-7 days. Extraction of Cellular DNA and RNA Fractions. The cells extracted for nucleic acid included normal BEK (secondary cultures), normal HEp-2, the BEK/RS ts line, and the 3230
Microbiology: Simpson and finuma HEp-2/RS line maintained in vitro as described above. We employed the method of Marmur as modified by Hill"and Hillova (8) for DNA extraction with slight further modification. A total of about 108 cells (107/culture) attached as confluent monolayers were washed thoroughly with a buffer containing 0.15 M NaCl and 0.1 M ethylenediaminetetraacetate (EDTA), pH 8. Thereafter, the cells were lysed in situ by addition of 15 ml per 107 cells of the same buffer containing 1% sodium dodecyl sulfate and heating in a 600
Proc. Nat. Acad. Sci. USA 72 (1975)
2
3231
a
10
0
z
water bath for 10 min with intermittent agitation. After
cooling to room temperature, the lysates were pooled and combined with an equal volume of 24:1 (v/v) chloroform/ isoamyl alcohol and subjected to intermittent shaking during a 30-min incubation. The aqueous phase of this mixture was recovered following centrifugation at 6000 X g for 5 min
(Sorvall RC2-B centrifuge), taking care that the temperature setting was not low enough to precipitate the detergent present. Alcohol precipitation of the nucleic acid fraction was accomplished by adding two parts of 95% ethanol to one part aqueous phase. The precipitate formed after incubation at room temperature for 10 min was collected by low-speed centrifugation and dissolved over a period of 30 min or more in 15 ml of SSC buffer (0.15 M NaCI, 0.015 M sodium citrate, pH 7) with gentle to moderate mechanical agitation. An equal volume of RNase A (Worthington Biochemical Corp., Freehold, N.J.) in 0.15 M NaCl solution was added at a final concentration of 50 jig/ml and incubation was carried out at 370 for 60 min. Thereafter, an equal volume of Pronase (Calbiochem, La Jolla, Calif.) dissolved in 0.15 M NaCl solution was added at a concentration of 500 jig/ml and further incubation at 370 for 4 hr was carried out. Deproteinization with chloroform/isoamyl alcohol and subsequent precipitation of the aqueous phase with ethanol as described above was repeated. The precipitate was dissolved in 9 ml of SSC buffer within a period of 2-3 min. After re-precipitation with a one-half volume of absolute isopropanol for 10 min, the precipitate obtained was collected by low-speed centrifugation and sterilized by exposure to 15 ml of 75% ethanol for 24 hr at 4°. For the experiments described in this report, the final precipitate was dissolved in about 15 ml of Dulbecco's phosphate-buffered saline. The average 260/280 nm absorbance ratio of these preparations was 1.9 and yields of DNA approximated 200 jg from 108 cells. DNA prepared in this manner was stored at -70°. For RNA extraction, cells were washed thoroughly with a buffer containing 0.1 M NaCI, 0.01 M Tris-HCI (pH 8.5), and 0.001 M EDTA. The same buffer containing 1% sodium dodecyl sulfate was used to lyse the cells. The aqueous phase obtained after a single extraction with water-saturated phenol was subjected to two additional phenol extractions after which precipitation with 95% ethanol was carried out. The resulting precipitate was dissolved in SSC buffer and Pronase treatment at 370 was carried out as described for the DNA extraction technique. Thereafter, RNase-free DNase (Worthington) was added at a final concentration of 10 ,ug/ml and further incubation at 370 for 60 min was carried out. The final precipitate obtained after an additional alcohol precipitation was resuspended in high salt buffer consisting of 0.8 M NaCI, 0.03% EDTA, and 0.02 M sodium phosphate buffer at pH 7.2. DNA Transfection of Cells. Petri dish (60 mm) cultures of HEp-2 cells were thoroughly washed with BSS and inoculated with 1 ml of the appropriate DNA or control preparation diluted in high salt buffer. The inocula were removed by washing with BSS after a 10 min adsorption (250) and 5
4A
0.01
_ 30
60
90
120
150
MINUTES AT 500
FIG. 1. Heat inactivation kinetics of wild-type respiratory syncytial virus and mutants HRS/50 and ts 4074. Crude stocks of the tissue culture grown viruses containing 3% fetal calf serum as a constituent of the original growth medium (R-MEM) were heated undiluted in a 50° water bath for the times indicated. Heated samples were quickly chilled in ice and titrated for surviving infectious virus by standard plaque assay in HEp-2 cells at 35°. Mutant HRS/50 (0) was derived from wild-type RS (-) by selective enrichment, whereas mutant ts 4074 (-) was obtained by chemical mutagenesis of HRS/50 virus stocks (see text).
ml of R-MEM with 3% fetal calf serum was added to each culture. For this study, the cultures were incubated at 350. Cell-associated virus particles produced under these conditions were released by exposing cells to cycles of freezing/ thawing. The viral progeny contained in DNA transfection yields was assayed or cloned in HEp-2 cells as described above. For experiments involving drug-treated cells, the inhibitors were freshly prepared using cell culture medium as a suspending solution. The inhibitors used included actinomycin D (gift of Dr. J. 0. Lampen), mitomycin C (Calbiochem), and cytosine arabinonucleoside (Nutritional Biochemicals Co.). Immunofluorescent Staining. The y-globulin fraction of horse antiserum to RS virus (Flow Laboratories) was used to prepare a fluorescein isothiocyanate immune conjugate as recently described by us (6). Infected coverslip cultures of HEp-2 cells stained with this conjugate after fixation with cold acetone were examined by fluorescence microscopy. The samples were coded so that they could not be directly identified by the experimenter. RESULTS Origin and general properties of BEK/RS ts cells As part of a vaccine development program, we have isolated a collection of 25 ts (temperature-sensitive) mutants of RS virus using a regimen for chemical mutagenesis that is likely to produce multiple genetic lesions in genomes of the viable conditional lethal viruses recovered. The most stable of these RS mutants, ts 4074, was derived from a heat-resistant variant, HRS/50, by treatment of virus with N-methyl-N'-nitroN-nitrosoguanidine and subsequent growth of the surviving fraction in primary BEK cells in the presence of 5-fluorouridine. As illustrated in Fig. 1, ts 4074 shares the heat-resistant character of its HRS/50 progenitor by comparison with the inactivation kinetics observed with wild-type RS virus. Mutant ts 4074 has consistently failed to replicate or produce discernible plaques in BEK cells incubated at 390 or higher. A line of cells, hereafter designated BEK/RS ts, was
Proc. Nat. Acad. Sci. USA 72 (1975)
Microbiology: Simpson and Iinuma
3232
Table 1. Production of virus-specific antigen and infectious RS virus after transfection of HEp-2 cells with DNA from nonvirogenic BEK/RS ts cellsa Immunofluorescent staining for RS virus
Yield of infectious RS virus
antigenc
Transfection inoculum:
(PFU/ml) :d
Donor cells
Fraction testedb
Intensity
Positive cells (%)
350 assay
390assay
BEK/RS ts 4074 (passage 24 at 390)
0.8 Mg of DNA, untreated 0.8 Mg of DNA, plus DNase 0.8 jig of DNA, plus RNase 30 pg of RNA, untreated Crude cell lysate 5.2 jig of DNA, untreated
1-4+
-90
104.6
104.0
0
0
104.5 0 0
ND 0 0
0
0
Normal BEK (passage 2 at 390)
0
0
1-4+ 0 0
-90 0 0
0
0
cultures of bovine embryonic kidney (BEK) cells originally infected with RS ts mutant 4074 were maintained in vitro by cultivation at 390 and transfer at approximately weekly intervals. Total DNA was extracted (Materials and Methods) from these cells at the 24th passage level. Nd infectious RS virus could be detected in these cells by direct assay. Mock-infected BEK cells at the 2nd passage level served as a source of control cell DNA in this experiment. b The concentrations listed are the total amounts of DNA or RNA inoculated into HEp-2 monolayer cultures containing 2 x 106 cells. For DNase treatment, ribonuclease-free deoxyribonuclease was added to 42 fig of DNA in Dulbecco's PBS at a final concentration of 10 zg/ml and the mixture was incubated at 370 for 60 min. RNase treatment consisted of adding 50 ,g/ml (final concentration) of ribonuclease A to 42 ,ug of DNA and incubating this mixture at 370 for 60 min. Crude cell lysates were prepared by exposing 2 x 107 cells in 5 ml of phosphatebuffered saline to three cycles of freezing/thawing and removing the cellular debris by low speed centrifugation. HEp-2 cell monolayers were inoculated with 0.2 ml volumes of the undiluted lysate. c Coverslip cultures of attached HEp-2 cells were processed for immunofluorescent staining (Materials and Methods) 7 days after exposure to inoculum and subsequent incubation at 35°. d Confluent HEpr-2 monolayers incubated at 35° for 7 days after exposure to the inoculum indicated were lysed by freezing/thawing. Clarified cell lysates were assayed for plaque-forming virus in HEp-2 cells incubated at 350 and 39°. ND = not determined. a Primary
established by infection of primary BEK monolayer cultures with mutant ts 4074 which were incubated exclusively at nonpermissive temperature (390) during successive serial transfers to fresh culture vessels. Under these conditions, we were unable to detect virus-specific RS antigens in these
cells by immunofluorescent staining and no infectious RS virus could be found in cell lysates by direct plaque assay. However, late passage BEK/RS ts cells were consistently found to produce infectious RS virus when cocultivated with normal BEK cells or exposed to specific inducing agents,
Table 2. Phenotypic properties of RS virus clones isolated from the progeny of a DNA transfection experimenta
Source of clone
Controls Wild-type RS Mutant HRS/50 Mutant ts 4074e
Clone no.
RS-1
HRS-1
ts 4074 Test group: DNA transfection yieldf RS-DNA-1 RS-DNA-2
Syncytia. % Neutralization formation by RS virus at 350 antiserumb
Ratio of
390/350 PFU titerc
syn+ syn+ syn+
>99 >99 >99
99 >99
99
RS-DNA-4
syn+
>99
0.8-1.2 0.8-1.2
