Archives of Virology 49, 249--259 (1975) © by Springer-Verlag 1975
Purification of Herpesvirus Saimiri and Properties of the Viral DNA ~ By J. A. SI~O~-DS2, W. G. RoB~Y 2, B. J. GaA~As~2, H. OIE3, and G. F. VA~Dn WOVDEe 2 Viral Biology Branch, National Cancer Institute, National Institutes of HeaIth. Bethesda, U.S.A. 3 Viral Leukemia and Lymphoma Branch, National Cancer Institute, NationM Institutes of Health, Bethesda, U.S.A. With 6 Figures Accepted July 14, 1975
Summary Conditions for growth , concentration, and purification of Herpesvirus sMmlri were determined. Optimal yields of infectious Herpesvirus sMmiri (HVS) were obtained from infected owl monkey kidney (OMK) cells grown at 32.5 ° C in medium containing 10 per cent fetal calf serum. Forty-five percent of the initial infectious HVS was recovered after an 18-fold concentration using 8 per cent polyethylene glycol 6000 in the presence of 0.5 ~ NaC1. Polyethylene glycol concentrated HVS was purified in an isopycnic-linear Renografin gradient (1.0--1.3 g/cm3). Ninetysix percent of the infectivity was recovered in a single 1.16 g/cm 3 density region. DNA extracted from purified HVS was resolved into two distinct density classes by CsC1 equilibrium centrifngation (1.727 and 1.709 g/cmS). DNase treated HVS virions yield four DNA species with densities of 1.727, 1.718, 1.712, and 1.706 g/ cm 3 in CsC1 centrifugation.
Introduction I-Ierpesvirus sMmiri (HVS) isolated from tile squirrel monkey Saimiri sciureus has shown the capacity to induce tymphomas in New World monkeys (11); this pathology and other biologieM properties of ttVS have recently been reviewed (3, 5). Some physieo-ehemieM properties of the virus have been characterized (2, 4, 5, 7, 9, t4). The density of the viral DNA in CsC1 is reported to be 1.729 g/ cms (7, 9). Recently FnECt(E~ST~IN and ~)VoLF (5) have presented evidence that HVS DNA exists as multiple density species in purified virions, and state t h a t two species, 1.729 and 1.694, represent heavy and light fragments of a third species (1.790 g/eroS). 1 This work was initially presented at the 1974 ASM meeting in Chicago, Illinois.
250
J . A . SIMO~DS et al. :
We n o w report a n i m p r o v e d procedure for c o n c e n t r a t i n g a n d p u r i f y i n g biologically active HVS a n d describe some physico-chemical characteristics of HVS DNA.
Materials and Methods Cell Culture Owl monkey kidney cells (OMK), a cell line established by Dr. L. V. MELENDEZ ( 11 ), were maintained as stationary monolayer cultures in 75 cm ~ plastic flasks (Bioquest, Oxnard, California). Cells were grown in t5 ml of McCoys 5A medium (modified, 25 mM Hepes buffer, Grand Island Biological Co., Grand Island, N.Y.), containing t0 per cent heat-inaetivited fetal calf serum (FCS) (Grand Island Biological Company) and 50 txg/ml of gentamiein (Sehering, Port Reading, N.J.). Roller cultures (690 cm2; Bellco, Vineland, New Jersey) of OMK cells were grown in 100 ml of Medium 199 (modified, Grand Island Biological Company) containing t0 per cent FCS and 50 ~xg/ml of gen~amicin. V E R 0 cells (American Type Culture Collection, t~ockville, Maryland) were grown and maintained in RPMI-1640 Medium containing 25 m ~ Itepes buffer (Grand Island Biological Co.) 10 per cent FCS, penicillin, streptomycin, and neomycin at 0.05, 0.05, and 0.1 mg/ml respectively (PSN, Grand Island Biological Company). Cultures were routinely tested for mycoplasma and found negative by L. I-Iayfliek, Department of Medical Microbiology, Stanford University School of Medicine, Stanford University, California 94305 and Norma tterrick, Mycoplasma Testing Service, Microbiological Associates, Bethesda, Maryland 20014. Virus Propagation Herpesvirus saimiri was added to freshly confluent monolayers in approximately one tenth of the normal volume of medium. After adsorption for 1 hour at 37 ° C, the cultures were re-fed and incubated for the remainder of the experiment at either 32.5 or 37 ° C. Virus and ceils were harvested when a 50--80 per cent cytopathie effect (CPE) was observed in the infected cells. Unless otherwise stated, all culture harvests were subjected to one freeze-thaw cycle, pooled, aliquoted, and stored at - - 1 3 0 ° C. Plaque Assay Virus infectivity was determined by plaque assay on OMK cell monolayers in 25 cm 2 flasks. Duplicate cultures, infected with serial log dilutions of ttSV as described above, were re-fed with 5 ml of growth medium and incubated at 37 ° C for 5--7 days. Plaques were read after staining the cell monolayer with 0.5 per cent crystal violet in 70 per cent methanol. Virus titer, expressed as plaque forming units (PFU)/ml, was the same whether cultures were overlaid with 0.5 per cent methyl cellulose containing growth medium or only with growth medium. Radioactive Labeling The DNA of }IVS was isotopically labeled by adding aH-thymidine (10--100 I~Ci/ ml; S.A. 53 Ci/mMole, Schwarz/Mann, Orangeburg, New York) to cell cultures 24 hours after infection. Samples were assayed for radioactivity by either counting directly in Aquasol (New England Nuclear, Boston, Massachusetts) or by trichloroaeetic acid (TCA) precipitation on membrane filters (Millipore, Bedford, Mass.) before counting in BBOT-toluene (Packard I n s t r u m e n t Company, Defamers Grove, Illinois).
Results Conditions/or Growth and Harvest o/ t I V S P r i m a r y cells from several s u b h u m a n p r i m a t e s have been reported to be susceptible to HVS infection as well as various established h u m a n a n d s u b h u m a n p r i m a t e cell lines (3). For the production of large a m o u n t s of tIVS the established
Purification of Herpesvirus Saimiri and Properties of the Viral D N A
251
cell lines, O M K a n d VEI%O, were t e s t e d for p r o d u c t i v i t y of infectious H V S u n d e r different conditions of growth. H e r p e s v i r u s saimiri yield from t h e O]}{K cell culture is four-fold g r e a t e r t h a n from V E R O ceils (Table 1). Moreover, i n c u b a t i o n of the infected cultures at 32.5° C w i t h m e d i u m containing 10 per cent FCS, favors r e c o v e r y of H V S from either cell line. I n f e c t e d O M K cells grown a t 32.5 ° C in t h e presence of 10 per cent FCS were therefore used for H V S production. Table 1. The e/]ect o/temperature and serum concentration on the yield o/ H VS in O M K and VERO cells a Cell line VERO
OMK
Temperature °C
Percent fetM calf serum
PFU/mI
37.0 37.0 32.5 32.5 37.0 37.0 32.5 32.5
2 10 2 10 2 10 2 10
1.4 × l04 1.Tx 104 3.5 X 10~ 3.9 × 10a 5.8 × 104 7.1 × 104 13.0 × 104 16.0× 104
Confluent monolayers were infected at a multiplicity of 0.01 PFU/cell and incubated as above until 50 per cent CPE (5--7 days). The cell cultures were then frozen and after thawing and filtration virus titer was determined on OMK monolayers.
E//ect o/Freeze- Thaw Cycles, Centri/ugation, and Filtration on Recovery o/In]ectious HVS Conditions for r e c o v e r y of H V S from infected O M K cells were d e t e r m i n e d (Table 2). Virus r e c o v e r e d from cells a n d s u p e r n a t a n t h a r v e s t e d at 50 per cent C P E w i t h vigorous p i p e t t i n g has a p p r o x i m a t e l y t h e same tiger as virus released from a similar cell cuspension after one f r e e z e - t h a w cycle. Moreover, t h e l a t t e r v i r u s tiger is n o t significantly r e d u c e d b y c e n t r i f u g a t i o n (or filtration) w i t h less t h a n 7 per cent of t h e t o t a l i n f e c t i v i t y r e m a i n i n g in t h e 1000 ;< g cell debris pellet. Multiple cycles of freeze a n d t h a w do n o t a p p e a r to increase H V S tiger. Table 2. E//ect o//reeze-thaw, centri/ugation and/iltration on the yield o/in/ectious H VS ~
Method of t{VS harvest
PFU/ml
1. Cells and supernatant vigorously pipetted
1.0 × l0 s
2. Cells and supernatant subjected to one freeze-thaw cycle a) No further procedures used b) Pellet from clarification at 1.000 x g, 10 minutes b c) Supernatant from clarification at 1000 x g, 10 minutes b d) Supernatant from clarification at 10,000 × g, 10 minutes b
t . 5 x 106 0.07 >
Purification of Herpesvirus Saimiri and Properties of the Viral DNA ~ By J. A. SI~O~-DS2, W. G. RoB~Y 2, B. J. GaA~As~2, H. OIE3, and G. F. VA~Dn WOVDEe 2 Viral Biology Branch, National Cancer Institute, National Institutes of HeaIth. Bethesda, U.S.A. 3 Viral Leukemia and Lymphoma Branch, National Cancer Institute, NationM Institutes of Health, Bethesda, U.S.A. With 6 Figures Accepted July 14, 1975
Summary Conditions for growth , concentration, and purification of Herpesvirus sMmlri were determined. Optimal yields of infectious Herpesvirus sMmiri (HVS) were obtained from infected owl monkey kidney (OMK) cells grown at 32.5 ° C in medium containing 10 per cent fetal calf serum. Forty-five percent of the initial infectious HVS was recovered after an 18-fold concentration using 8 per cent polyethylene glycol 6000 in the presence of 0.5 ~ NaC1. Polyethylene glycol concentrated HVS was purified in an isopycnic-linear Renografin gradient (1.0--1.3 g/cm3). Ninetysix percent of the infectivity was recovered in a single 1.16 g/cm 3 density region. DNA extracted from purified HVS was resolved into two distinct density classes by CsC1 equilibrium centrifngation (1.727 and 1.709 g/cmS). DNase treated HVS virions yield four DNA species with densities of 1.727, 1.718, 1.712, and 1.706 g/ cm 3 in CsC1 centrifugation.
Introduction I-Ierpesvirus sMmiri (HVS) isolated from tile squirrel monkey Saimiri sciureus has shown the capacity to induce tymphomas in New World monkeys (11); this pathology and other biologieM properties of ttVS have recently been reviewed (3, 5). Some physieo-ehemieM properties of the virus have been characterized (2, 4, 5, 7, 9, t4). The density of the viral DNA in CsC1 is reported to be 1.729 g/ cms (7, 9). Recently FnECt(E~ST~IN and ~)VoLF (5) have presented evidence that HVS DNA exists as multiple density species in purified virions, and state t h a t two species, 1.729 and 1.694, represent heavy and light fragments of a third species (1.790 g/eroS). 1 This work was initially presented at the 1974 ASM meeting in Chicago, Illinois.
250
J . A . SIMO~DS et al. :
We n o w report a n i m p r o v e d procedure for c o n c e n t r a t i n g a n d p u r i f y i n g biologically active HVS a n d describe some physico-chemical characteristics of HVS DNA.
Materials and Methods Cell Culture Owl monkey kidney cells (OMK), a cell line established by Dr. L. V. MELENDEZ ( 11 ), were maintained as stationary monolayer cultures in 75 cm ~ plastic flasks (Bioquest, Oxnard, California). Cells were grown in t5 ml of McCoys 5A medium (modified, 25 mM Hepes buffer, Grand Island Biological Co., Grand Island, N.Y.), containing t0 per cent heat-inaetivited fetal calf serum (FCS) (Grand Island Biological Company) and 50 txg/ml of gentamiein (Sehering, Port Reading, N.J.). Roller cultures (690 cm2; Bellco, Vineland, New Jersey) of OMK cells were grown in 100 ml of Medium 199 (modified, Grand Island Biological Company) containing t0 per cent FCS and 50 ~xg/ml of gen~amicin. V E R 0 cells (American Type Culture Collection, t~ockville, Maryland) were grown and maintained in RPMI-1640 Medium containing 25 m ~ Itepes buffer (Grand Island Biological Co.) 10 per cent FCS, penicillin, streptomycin, and neomycin at 0.05, 0.05, and 0.1 mg/ml respectively (PSN, Grand Island Biological Company). Cultures were routinely tested for mycoplasma and found negative by L. I-Iayfliek, Department of Medical Microbiology, Stanford University School of Medicine, Stanford University, California 94305 and Norma tterrick, Mycoplasma Testing Service, Microbiological Associates, Bethesda, Maryland 20014. Virus Propagation Herpesvirus saimiri was added to freshly confluent monolayers in approximately one tenth of the normal volume of medium. After adsorption for 1 hour at 37 ° C, the cultures were re-fed and incubated for the remainder of the experiment at either 32.5 or 37 ° C. Virus and ceils were harvested when a 50--80 per cent cytopathie effect (CPE) was observed in the infected cells. Unless otherwise stated, all culture harvests were subjected to one freeze-thaw cycle, pooled, aliquoted, and stored at - - 1 3 0 ° C. Plaque Assay Virus infectivity was determined by plaque assay on OMK cell monolayers in 25 cm 2 flasks. Duplicate cultures, infected with serial log dilutions of ttSV as described above, were re-fed with 5 ml of growth medium and incubated at 37 ° C for 5--7 days. Plaques were read after staining the cell monolayer with 0.5 per cent crystal violet in 70 per cent methanol. Virus titer, expressed as plaque forming units (PFU)/ml, was the same whether cultures were overlaid with 0.5 per cent methyl cellulose containing growth medium or only with growth medium. Radioactive Labeling The DNA of }IVS was isotopically labeled by adding aH-thymidine (10--100 I~Ci/ ml; S.A. 53 Ci/mMole, Schwarz/Mann, Orangeburg, New York) to cell cultures 24 hours after infection. Samples were assayed for radioactivity by either counting directly in Aquasol (New England Nuclear, Boston, Massachusetts) or by trichloroaeetic acid (TCA) precipitation on membrane filters (Millipore, Bedford, Mass.) before counting in BBOT-toluene (Packard I n s t r u m e n t Company, Defamers Grove, Illinois).
Results Conditions/or Growth and Harvest o/ t I V S P r i m a r y cells from several s u b h u m a n p r i m a t e s have been reported to be susceptible to HVS infection as well as various established h u m a n a n d s u b h u m a n p r i m a t e cell lines (3). For the production of large a m o u n t s of tIVS the established
Purification of Herpesvirus Saimiri and Properties of the Viral D N A
251
cell lines, O M K a n d VEI%O, were t e s t e d for p r o d u c t i v i t y of infectious H V S u n d e r different conditions of growth. H e r p e s v i r u s saimiri yield from t h e O]}{K cell culture is four-fold g r e a t e r t h a n from V E R O ceils (Table 1). Moreover, i n c u b a t i o n of the infected cultures at 32.5° C w i t h m e d i u m containing 10 per cent FCS, favors r e c o v e r y of H V S from either cell line. I n f e c t e d O M K cells grown a t 32.5 ° C in t h e presence of 10 per cent FCS were therefore used for H V S production. Table 1. The e/]ect o/temperature and serum concentration on the yield o/ H VS in O M K and VERO cells a Cell line VERO
OMK
Temperature °C
Percent fetM calf serum
PFU/mI
37.0 37.0 32.5 32.5 37.0 37.0 32.5 32.5
2 10 2 10 2 10 2 10
1.4 × l04 1.Tx 104 3.5 X 10~ 3.9 × 10a 5.8 × 104 7.1 × 104 13.0 × 104 16.0× 104
Confluent monolayers were infected at a multiplicity of 0.01 PFU/cell and incubated as above until 50 per cent CPE (5--7 days). The cell cultures were then frozen and after thawing and filtration virus titer was determined on OMK monolayers.
E//ect o/Freeze- Thaw Cycles, Centri/ugation, and Filtration on Recovery o/In]ectious HVS Conditions for r e c o v e r y of H V S from infected O M K cells were d e t e r m i n e d (Table 2). Virus r e c o v e r e d from cells a n d s u p e r n a t a n t h a r v e s t e d at 50 per cent C P E w i t h vigorous p i p e t t i n g has a p p r o x i m a t e l y t h e same tiger as virus released from a similar cell cuspension after one f r e e z e - t h a w cycle. Moreover, t h e l a t t e r v i r u s tiger is n o t significantly r e d u c e d b y c e n t r i f u g a t i o n (or filtration) w i t h less t h a n 7 per cent of t h e t o t a l i n f e c t i v i t y r e m a i n i n g in t h e 1000 ;< g cell debris pellet. Multiple cycles of freeze a n d t h a w do n o t a p p e a r to increase H V S tiger. Table 2. E//ect o//reeze-thaw, centri/ugation and/iltration on the yield o/in/ectious H VS ~
Method of t{VS harvest
PFU/ml
1. Cells and supernatant vigorously pipetted
1.0 × l0 s
2. Cells and supernatant subjected to one freeze-thaw cycle a) No further procedures used b) Pellet from clarification at 1.000 x g, 10 minutes b c) Supernatant from clarification at 1000 x g, 10 minutes b d) Supernatant from clarification at 10,000 × g, 10 minutes b
t . 5 x 106 0.07 >
