Vol. 15, No. 2 Printed in U.S.A.
JOURNAL OF VIROLOGY, Feb. 1975, p. 398-406 Copyright O 1975 American Society for Microbiology
Repetitive Sequences in Complete and Defective Genomes of Herpesvirus saimiri BERNHARD FLECKENSTEIN,* GEORG W. BORNKAMM, AND HANNS LUDWIG Institut fur Klinische Virologie der Universitat Erlangen-Nurnberg, 852 Erlangen, Germany* and Institut fur Virologie der Justus Liebig-Universitat, 63 Giessen, Germany
Received for publication 27 September 1974
Two types of Herpesvirus saimiri genomes can be isolated from purified virions: (i) the M genome is a double-stranded, linear DNA molecule with a mean contour length corresponding to 89 x 106 daltons. The M genome contains about 70% of unique sequences (light DNA, 36% guanine plus cytosine) and 30% reiterated sequences (heavy DNA, 71% guanine plus cytosine). (ii) The H genome is composed of heavy DNA only and is more heterogeneous in size. The sequences in the H genome are up to 40-fold reiterated, indicating defectiveness of this type of genome. The repetitions in the H genome and the M genome cross-hybridize almost completely and have identical kinetic complexity (2.8 x 106 daltons). DNA infectivity studies by using the calcium phosphate and the DEAE-dextran method gave further evidence that H genomes are defective: no infectious virus was recovered from permissive cells treated with heavy DNA, whereas M genome-infected cells developed cytopathic changes after 11 to 56 days. Defective H genomes were present in the progeny virus two passages after transfection.
Herpesvirus saimiri, an indigenous agent of squirrel monkeys (Saimiri sciureus) (19), induces malignant lymphomas or acute lymphocytic leukemias in several new world primate species (1, 14, 15, 20, 27). In a previous report (5) it has been shown that H. saimiri DNA consists of two species of double-stranded DNA, heavy (H) and light (L) DNA. Because of their wide difference in guanine plus cytosine (G+C) content, the separation by preparative equilibrium centrifugation in cesium chloride became possible. Non-fragmented H. saimiri DNA (medium [M ] DNA) which contains H and L sequences revealed an intermediate G+C content. In the present study, size and molecular weight, genetic complexity, and infectivity of the different H. saimiri DNA species have been analyzed. The results lead to the conclusion that reiterated sequences are present in complete and defective H. saimiri genomes. MATERIALS AND METHODS Viruses and cell culture. Two owl monkey kidney (OMK) monolayer cell lines, kindly supplied by M. D. Daniel, were used for propagation of H. saimiri. The cells were cultivated in minimal essential medium (MEM) supplemented with 8% heat-inactivated calf serum and 2%/c, heat-inactivated fetal calf serum without antibiotics under 8%c Co2. H. saimiri strain S 295 C, isolated by L. V. 398
Melendez, and strain no. 11, isolated by L. Falk, were plaque purified under 2% methylcellulose and used as stock virus. Infected OMK cells were refed after 2 to 3 days when the first cytopathogenic effects were observed. The virus was harvested 6 to 8 days later. Radioactive labeling of H. saimiri DNA was done as previously described (5). Purification of H- and L-H. saimiri DNA. H. saimiri was concentrated from the supernatant of infected OMK cells by subsequent pellet centrifugation in the type 19 rotor and in the SW27 rotor of a Spinco preparative centrifuge. Intracytoplasmic virus was recovered by incubating infected cells in virus standard buffer (VSB; 0.01 M Tris buffer [pH 7.4], 0.01 M KCl, 0.005 M EDTA) over 3 h at 4 C and by disrupting them with 12 strokes in a Dounce homogenizer. Virus obtained from the medium and from cells was pooled in VSB, homogenized once more by 20 strokes, and layered on a 15 to 30%: (wt/wt) sucrose gradient in VSB with 0.1% bovine serum albumin. After centrifugation at 20,000 rpm for 30 min at 4 C in the Spinco SW27 rotor, a band of enveloped particles was visible in the middle of the tube in most cases. The virus was collected by puncturing the side of the tube. The virus-containing fractions were diluted with VSB and sedimented by centrifugation. Virus concentrates were dissociated by incubation in 2% Sarkosyl NL, 0.01 M EDTA, 0.01 M KCl, 0.0015 M MgCl2, and 0.01 M Tris buffer (pH 7.4) over 5 h at 20 C and over 60 min at 60 C. Released DNA was purified by sedimentation in sucrose-sodium dodecyl sulfate (SDS) gradients and by isopycnic centrifuga-
VOL. 15, 1975
399
HERPES VIR US SAIMIRI GENOMES
tion in CsCl as described earlier (5). To obtain nonfragmented H. saimiri DNA, virus was lysed in 2 ml of 1% Sarkosyl NL, 0.005 M EDTA, and 0.1 x SSC (0.15 M NaCl, 0.015 M sodium citrate) by incubating it on top of 8.7 ml of 6.9 M CsCl over 5 h at 37 C and over 60 min at 60 C. Thereafter the DNA was centrifuged in the Spinco 50 Ti rotor at 30,000 rpm for 72 h at 23 C. Fractions were collected by piercing the bottom of the tube with a needle (40 by 1.2 mm). The flow rate was restricted to :1 m/min. Analytical ultracentrifugation. Centrifugation of the DNAs and calculation of the buoyant densities were done as described (17). H. saimiri, which was partially purified by one sedimentation run in 15 to 30% (wt/wt) sucrose, was suspended in TNE (0.01 M Tris buffer [pH 7.4 ], 0.1 M NaCl, and 0.001 M EDTA), lysed with 1% Sarkosyl NL, and adjusted with CsCl to give a refractive index of 1.4000. Approximately 5 qg of total DNA, calculated from the known specific activity, were carefully filled through a rubber tubing with an internal diameter of 0.3 mm into the centrifuge cell. Centrifugation was performed with a 6-place Ti ANG rotor in the Beckman analytical ultracentrifuge model E at 44,000 rpm for 20 h at 25 C. Determination of reassociation kinetics of viral DNA. Lyophilized viral DNA was dissolved at concentrations between 0.03 jg/ml and 1 Ag/ml in a solution of 0.1 M Tris buffer (pH 7.4), 0.01 M EDTA, 0.05% Sarkosyl NL, and 1 M NaCl. Samples of 50 Aliters were sealed in siliconized glass capillaries, sonically treated from the outside for 10 min with a Branson sonifier at 100 W, and heat denaturated at 120 C for 10 min. By this procedure, single-stranded DNA pieces of 5S (approximately 270 bases) were obtained, as determined by co-sedimentation with SV40 form II DNA in alkaline sucrose gradients (3). Reassociation was carried out at 73 C. After various intervals, specimens were chilled in ice water and chromatographed on 0.6-ml hydroxyapatite (DNA grade, BioRad Laboratories) columns. Singlestranded DNA was eluted at 60 C by 0.14 M sodium phosphate buffer (pH 6.8) containing 0.4% SDS; at least 97% of single-stranded DNA and less than 3% of doublezstranded DNA appeared in this fraction. Double-stranded DNA was eluted at the same temperature with 0.4 M sodium phosphate buffer (pH 6.8) containing 0.4% SDS. Eluates were adjusted to identical phosphate and SDS concentration in a total volume of 4 ml, mixed with 10 ml of toluene Triton X100 (2:1) scintillator, and counted at 2 C in the gel phase. For determination of the reassociation rate of a given DNA, the second order rate plot proposed by Wetmur and Davidson (26) was used. Determination of melting temperatures of H. saimiri DNA. Viral DNA in 0.01 M Tris-hydrochloride, 0.033 M sodium phosphate buffer (0.05 M Na+, pH 6.8) was filled in a denaturation cuvette of a Gilford 240 spectrophotometer and heated at a rate of 0.5 C/min using a Gilford type 2527 thermo-programmer. The absorbance at 260 nm was measured in 1-centigrade steps. Escherichia coli DNA was included as a standard.
To evaluate optimal conditions for reassociation the Tm values of fragmented H and L DNA in 1 M NaCl were determined, respectively. Tritium-labeled DNA was incubated at various temperatures for 10 min, followed by chilling in ice water. The radioactivity in single-stranded and double-stranded DNA was measured after chromatography on hydroxyapatite. The Tm value of H DNA in 1 M NaCl was found to be 103.5 C and that of L DNA to be 92 C. Preparation of H. saimiri DNA for electron microscopy. DNA was prepared for electron microscopy by the microdiffusion method described by Lang and Mitani (13). Droplets of 50 uliters containing 100 ng of viral DNA per ml, 0.2 M ammonium acetate, 1 mM EDTA, 2.7 x 10-So cytochrome c, 0.2% formaldehyde, and 50 mM Tris buffer (pH 7.4) were placed on plastic petri dishes. After 20 min at room temperature, the DNA-cytochrome c film was picked up by carbon-coated platinum grids, stained for 20 s in uranyl acetate according to Davis et al. (2), and dehydrated by treatment with ethanol for 10 s. The DNA was shadowed with Pt/Pd, and electron micrographs were taken with a Zeiss EM 10. Magnifications were calibrated by grating replicas (Zeiss, Oberkochen). Micrographs were enlarged 16-fold by a projection device and traced on paper, and the contour length was determined by a map measurer. In contour length measurements of phage PM2 DNA molecules the standard deviation was 2.0%. Infectivity assays with H. saimiri DNA. The calcium phosphate (9) and the DEAE-dextran (25) method which had been applied to demonstrate infectivity of herpes simplex virus DNA were slightly modified for transfection studies with H. saimiri DNA. H. saimiri DNA in 50 to 100 Aliters of 0.01 M Tris buffer, pH 7.0, was added to 2 ml Tris-buffered saline (8) containing 5 Ag of single-stranded salmon sperm DNA per ml. CaCl2 was added to a final concentration of 125 mM. Fine visible precipitates formed over a period of 20 min at 20 C. Almost confluent tissue cultures were infected with 0.5 ml of the slightly opaque fluid. After adsorption for 20 min at room temperature, cells were refed with MEM containing 10% fetal calf serum. For plaque assays, tissue culture fluids were removed from petri dishes 5 h after infection and replaced by 2% methyl-cellulose in MEM with 10% fetal calf serum. Using the DEAE-dextran method, OMK cells were washed with phosphate-buffered saline (pH 7.0) free of calcium and magnesium ions. Then, the cells were rinsed with 0.2 to 0.4 ml of a H. saimiri DNA solution containing 200 4g of DEAE-dextran per ml (mol wt 500,000; Pharmacia, Uppsala), and incubated for 60 min at ambient temperature. Thereafter the cultures were washed twice with phosphate-buffered saline and refed with MEM and 10% fetal calf serum or overlayed with 2% methyl-cellulose dissolved in the same medium. RESULTS
Base composition of H. saimiri DNA species. When partially purified H. saimiri was
400
FLECKENSTEIN, BORNKAMM, AND LUDWIG
gently lysed, immediately mixed with CsCl, and examined by analytical ultracentrifugation, three distinct DNA species became visible after equilibrium had been reached (Fig. 1A). Their relative amount was about 28% for L DNA, 40% for M DNA, and 20% for H DNA. Twelve percent of the DNA covered the area between M and H DNA. By co-centrifugation with E. coli DNA (p= 1.710 g/ml) or herpes simplex virus type 1 DNA 1.729
1.695 1705
1.6861
11.710 1.7251
(p
=
1.725 g/ml)
of 1.695,
4
as a
J. VIROL.
marker, buoyant densities
0.0002, 1.7045 + 0.0005, and 1.7292 i
0.0006 g/ml were calculated for the different DNAs (Fig. lA-C). Occasionally a DNA species of a density of 1.685, g/ml was detected (Fig. 1C-D) which corresponds to mycoplasma orale type 1 DNA (11). This agent had been isolated from OMK cells and typed serologically by W. Bredt. Thus, the H. saimiri DNA species have a G+C content of 70.6% (H DNA), 45.4% (M DNA), and 35.8% (L DNA) (24). When H. saimiri DNA was fragmented by squirting it vigorously 10 times through a needle (25 by 5/8 mm), only two DNA peaks at densities of 1.695, and 1.7292 g/ml could be seen after centrifugation. The M DNA peak disappeared completely. From the density shift of the DNA fragments of M DNA it can be concluded that M DNA is composed of 30% H and 70% L sequences.
LO
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JOURNAL OF VIROLOGY, Feb. 1975, p. 398-406 Copyright O 1975 American Society for Microbiology
Repetitive Sequences in Complete and Defective Genomes of Herpesvirus saimiri BERNHARD FLECKENSTEIN,* GEORG W. BORNKAMM, AND HANNS LUDWIG Institut fur Klinische Virologie der Universitat Erlangen-Nurnberg, 852 Erlangen, Germany* and Institut fur Virologie der Justus Liebig-Universitat, 63 Giessen, Germany
Received for publication 27 September 1974
Two types of Herpesvirus saimiri genomes can be isolated from purified virions: (i) the M genome is a double-stranded, linear DNA molecule with a mean contour length corresponding to 89 x 106 daltons. The M genome contains about 70% of unique sequences (light DNA, 36% guanine plus cytosine) and 30% reiterated sequences (heavy DNA, 71% guanine plus cytosine). (ii) The H genome is composed of heavy DNA only and is more heterogeneous in size. The sequences in the H genome are up to 40-fold reiterated, indicating defectiveness of this type of genome. The repetitions in the H genome and the M genome cross-hybridize almost completely and have identical kinetic complexity (2.8 x 106 daltons). DNA infectivity studies by using the calcium phosphate and the DEAE-dextran method gave further evidence that H genomes are defective: no infectious virus was recovered from permissive cells treated with heavy DNA, whereas M genome-infected cells developed cytopathic changes after 11 to 56 days. Defective H genomes were present in the progeny virus two passages after transfection.
Herpesvirus saimiri, an indigenous agent of squirrel monkeys (Saimiri sciureus) (19), induces malignant lymphomas or acute lymphocytic leukemias in several new world primate species (1, 14, 15, 20, 27). In a previous report (5) it has been shown that H. saimiri DNA consists of two species of double-stranded DNA, heavy (H) and light (L) DNA. Because of their wide difference in guanine plus cytosine (G+C) content, the separation by preparative equilibrium centrifugation in cesium chloride became possible. Non-fragmented H. saimiri DNA (medium [M ] DNA) which contains H and L sequences revealed an intermediate G+C content. In the present study, size and molecular weight, genetic complexity, and infectivity of the different H. saimiri DNA species have been analyzed. The results lead to the conclusion that reiterated sequences are present in complete and defective H. saimiri genomes. MATERIALS AND METHODS Viruses and cell culture. Two owl monkey kidney (OMK) monolayer cell lines, kindly supplied by M. D. Daniel, were used for propagation of H. saimiri. The cells were cultivated in minimal essential medium (MEM) supplemented with 8% heat-inactivated calf serum and 2%/c, heat-inactivated fetal calf serum without antibiotics under 8%c Co2. H. saimiri strain S 295 C, isolated by L. V. 398
Melendez, and strain no. 11, isolated by L. Falk, were plaque purified under 2% methylcellulose and used as stock virus. Infected OMK cells were refed after 2 to 3 days when the first cytopathogenic effects were observed. The virus was harvested 6 to 8 days later. Radioactive labeling of H. saimiri DNA was done as previously described (5). Purification of H- and L-H. saimiri DNA. H. saimiri was concentrated from the supernatant of infected OMK cells by subsequent pellet centrifugation in the type 19 rotor and in the SW27 rotor of a Spinco preparative centrifuge. Intracytoplasmic virus was recovered by incubating infected cells in virus standard buffer (VSB; 0.01 M Tris buffer [pH 7.4], 0.01 M KCl, 0.005 M EDTA) over 3 h at 4 C and by disrupting them with 12 strokes in a Dounce homogenizer. Virus obtained from the medium and from cells was pooled in VSB, homogenized once more by 20 strokes, and layered on a 15 to 30%: (wt/wt) sucrose gradient in VSB with 0.1% bovine serum albumin. After centrifugation at 20,000 rpm for 30 min at 4 C in the Spinco SW27 rotor, a band of enveloped particles was visible in the middle of the tube in most cases. The virus was collected by puncturing the side of the tube. The virus-containing fractions were diluted with VSB and sedimented by centrifugation. Virus concentrates were dissociated by incubation in 2% Sarkosyl NL, 0.01 M EDTA, 0.01 M KCl, 0.0015 M MgCl2, and 0.01 M Tris buffer (pH 7.4) over 5 h at 20 C and over 60 min at 60 C. Released DNA was purified by sedimentation in sucrose-sodium dodecyl sulfate (SDS) gradients and by isopycnic centrifuga-
VOL. 15, 1975
399
HERPES VIR US SAIMIRI GENOMES
tion in CsCl as described earlier (5). To obtain nonfragmented H. saimiri DNA, virus was lysed in 2 ml of 1% Sarkosyl NL, 0.005 M EDTA, and 0.1 x SSC (0.15 M NaCl, 0.015 M sodium citrate) by incubating it on top of 8.7 ml of 6.9 M CsCl over 5 h at 37 C and over 60 min at 60 C. Thereafter the DNA was centrifuged in the Spinco 50 Ti rotor at 30,000 rpm for 72 h at 23 C. Fractions were collected by piercing the bottom of the tube with a needle (40 by 1.2 mm). The flow rate was restricted to :1 m/min. Analytical ultracentrifugation. Centrifugation of the DNAs and calculation of the buoyant densities were done as described (17). H. saimiri, which was partially purified by one sedimentation run in 15 to 30% (wt/wt) sucrose, was suspended in TNE (0.01 M Tris buffer [pH 7.4 ], 0.1 M NaCl, and 0.001 M EDTA), lysed with 1% Sarkosyl NL, and adjusted with CsCl to give a refractive index of 1.4000. Approximately 5 qg of total DNA, calculated from the known specific activity, were carefully filled through a rubber tubing with an internal diameter of 0.3 mm into the centrifuge cell. Centrifugation was performed with a 6-place Ti ANG rotor in the Beckman analytical ultracentrifuge model E at 44,000 rpm for 20 h at 25 C. Determination of reassociation kinetics of viral DNA. Lyophilized viral DNA was dissolved at concentrations between 0.03 jg/ml and 1 Ag/ml in a solution of 0.1 M Tris buffer (pH 7.4), 0.01 M EDTA, 0.05% Sarkosyl NL, and 1 M NaCl. Samples of 50 Aliters were sealed in siliconized glass capillaries, sonically treated from the outside for 10 min with a Branson sonifier at 100 W, and heat denaturated at 120 C for 10 min. By this procedure, single-stranded DNA pieces of 5S (approximately 270 bases) were obtained, as determined by co-sedimentation with SV40 form II DNA in alkaline sucrose gradients (3). Reassociation was carried out at 73 C. After various intervals, specimens were chilled in ice water and chromatographed on 0.6-ml hydroxyapatite (DNA grade, BioRad Laboratories) columns. Singlestranded DNA was eluted at 60 C by 0.14 M sodium phosphate buffer (pH 6.8) containing 0.4% SDS; at least 97% of single-stranded DNA and less than 3% of doublezstranded DNA appeared in this fraction. Double-stranded DNA was eluted at the same temperature with 0.4 M sodium phosphate buffer (pH 6.8) containing 0.4% SDS. Eluates were adjusted to identical phosphate and SDS concentration in a total volume of 4 ml, mixed with 10 ml of toluene Triton X100 (2:1) scintillator, and counted at 2 C in the gel phase. For determination of the reassociation rate of a given DNA, the second order rate plot proposed by Wetmur and Davidson (26) was used. Determination of melting temperatures of H. saimiri DNA. Viral DNA in 0.01 M Tris-hydrochloride, 0.033 M sodium phosphate buffer (0.05 M Na+, pH 6.8) was filled in a denaturation cuvette of a Gilford 240 spectrophotometer and heated at a rate of 0.5 C/min using a Gilford type 2527 thermo-programmer. The absorbance at 260 nm was measured in 1-centigrade steps. Escherichia coli DNA was included as a standard.
To evaluate optimal conditions for reassociation the Tm values of fragmented H and L DNA in 1 M NaCl were determined, respectively. Tritium-labeled DNA was incubated at various temperatures for 10 min, followed by chilling in ice water. The radioactivity in single-stranded and double-stranded DNA was measured after chromatography on hydroxyapatite. The Tm value of H DNA in 1 M NaCl was found to be 103.5 C and that of L DNA to be 92 C. Preparation of H. saimiri DNA for electron microscopy. DNA was prepared for electron microscopy by the microdiffusion method described by Lang and Mitani (13). Droplets of 50 uliters containing 100 ng of viral DNA per ml, 0.2 M ammonium acetate, 1 mM EDTA, 2.7 x 10-So cytochrome c, 0.2% formaldehyde, and 50 mM Tris buffer (pH 7.4) were placed on plastic petri dishes. After 20 min at room temperature, the DNA-cytochrome c film was picked up by carbon-coated platinum grids, stained for 20 s in uranyl acetate according to Davis et al. (2), and dehydrated by treatment with ethanol for 10 s. The DNA was shadowed with Pt/Pd, and electron micrographs were taken with a Zeiss EM 10. Magnifications were calibrated by grating replicas (Zeiss, Oberkochen). Micrographs were enlarged 16-fold by a projection device and traced on paper, and the contour length was determined by a map measurer. In contour length measurements of phage PM2 DNA molecules the standard deviation was 2.0%. Infectivity assays with H. saimiri DNA. The calcium phosphate (9) and the DEAE-dextran (25) method which had been applied to demonstrate infectivity of herpes simplex virus DNA were slightly modified for transfection studies with H. saimiri DNA. H. saimiri DNA in 50 to 100 Aliters of 0.01 M Tris buffer, pH 7.0, was added to 2 ml Tris-buffered saline (8) containing 5 Ag of single-stranded salmon sperm DNA per ml. CaCl2 was added to a final concentration of 125 mM. Fine visible precipitates formed over a period of 20 min at 20 C. Almost confluent tissue cultures were infected with 0.5 ml of the slightly opaque fluid. After adsorption for 20 min at room temperature, cells were refed with MEM containing 10% fetal calf serum. For plaque assays, tissue culture fluids were removed from petri dishes 5 h after infection and replaced by 2% methyl-cellulose in MEM with 10% fetal calf serum. Using the DEAE-dextran method, OMK cells were washed with phosphate-buffered saline (pH 7.0) free of calcium and magnesium ions. Then, the cells were rinsed with 0.2 to 0.4 ml of a H. saimiri DNA solution containing 200 4g of DEAE-dextran per ml (mol wt 500,000; Pharmacia, Uppsala), and incubated for 60 min at ambient temperature. Thereafter the cultures were washed twice with phosphate-buffered saline and refed with MEM and 10% fetal calf serum or overlayed with 2% methyl-cellulose dissolved in the same medium. RESULTS
Base composition of H. saimiri DNA species. When partially purified H. saimiri was
400
FLECKENSTEIN, BORNKAMM, AND LUDWIG
gently lysed, immediately mixed with CsCl, and examined by analytical ultracentrifugation, three distinct DNA species became visible after equilibrium had been reached (Fig. 1A). Their relative amount was about 28% for L DNA, 40% for M DNA, and 20% for H DNA. Twelve percent of the DNA covered the area between M and H DNA. By co-centrifugation with E. coli DNA (p= 1.710 g/ml) or herpes simplex virus type 1 DNA 1.729
1.695 1705
1.6861
11.710 1.7251
(p
=
1.725 g/ml)
of 1.695,
4
as a
J. VIROL.
marker, buoyant densities
0.0002, 1.7045 + 0.0005, and 1.7292 i
0.0006 g/ml were calculated for the different DNAs (Fig. lA-C). Occasionally a DNA species of a density of 1.685, g/ml was detected (Fig. 1C-D) which corresponds to mycoplasma orale type 1 DNA (11). This agent had been isolated from OMK cells and typed serologically by W. Bredt. Thus, the H. saimiri DNA species have a G+C content of 70.6% (H DNA), 45.4% (M DNA), and 35.8% (L DNA) (24). When H. saimiri DNA was fragmented by squirting it vigorously 10 times through a needle (25 by 5/8 mm), only two DNA peaks at densities of 1.695, and 1.7292 g/ml could be seen after centrifugation. The M DNA peak disappeared completely. From the density shift of the DNA fragments of M DNA it can be concluded that M DNA is composed of 30% H and 70% L sequences.
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