Rapid lysis of vaccinia virus on neutral sucrose gradients with release of intact DNA

ANALYTICAL

BIOCHEMISTRY

Rapid

71, 53-59 (1976)

Lysis of Vaccinia Virus on Neutral Sucrose Gradients with Release of intact DNA

J. RODNEY PARKHURST'AND CHARLES HEIDELBERGER~ McArdle

Laboratory

for Cancer Research, University Madison, Wisconsin 53706

of Wisconsin,

Received June 16, 1975: accepted October 15, 1975 A new and improved procedure has been developed for the isolation of intact DNA genomes from purified vaccinia virions. Purified virions are layered on a neutral sucrose gradient containing sodium dodecyl sulfate, 2-mercaptoethanol and sodium chloride at neutral pH. Intact viral DNA free from protein and fully sensitive to DNase I is rapidly released from the virions.

Vaccinia virus is a DNA containing Poxvirus with a genome of 120- 140 x lo6 daltons (l-5). The two DNA strands are held together by crosslinks (2, 4) located near the ends of the molecule (3). Several procedures have been employed in attempts to isolate vaccinia DNA, e.g., 2% sodium dodecyl sulfate; saturated guanidinium chloride; 8~ urea; 1% sodium deoxycholate; and phenol at 50°C (6). Most of these yield efficiencies of extraction of 1%. Phenol extraction of vaccinia virions at 20°C was somewhat more successful and resulted in lo-20% recovery of vaccinia DNA (6). A lengthy procedure employing 2mercaptoethanol and pronase gave 90-95% recovery of DNA (7). In 1967, Sarov and Becker (5) reported two procedures capable of releasing vaccinia DNA from vaccinia virions; they utilized sodium deoxycholate, pronase and sodium dodecyl sulfate. The virions were lysed either on top of preformed sucrose gradients for 8-12 hr (37°C) or in a glass tube followed by dialysis or phenol extraction and gentle layering onto a sucrose gradient. We previously described (4) a neutral sucrose gradient system consisting of 5-20% sucrose gradients containing 0.5% SDS, 0.10 M NaCl, and 5 mM Tris-HCl (pH 7.5). Vaccinia virus in 2 mM Tris-HCl (pH 7.5), 0.40 M NaCl, 2mM EDTA, 2% deoxycholic acid, 4% sucrose, 2% 2mercaptoethanol and 1% SDS was added to the top of the gradients, incubated for 30 min and centrifuged. Lysis of vaccinia vu-ions with release of intact viral DNA was achieved in the lysis layer. ‘Present address: The Medical College of Wisconsin, Department of Microbiology. Milwaukee, WI 53233 * American Cancer Society Professor of Oncology: to whom requests for reprints should be sent. 53 CopyrIght All right\

Q 1976 by Academtc Presr. Inc of reproduction I” any form reerved.

54

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HEIDELBERGER

The present communication describes an isolation of intact vaccinia viral DNA. No extraction is required to achieve complete procedure has been applied to the study of DNA on neutral sucrose gradients. MATERIALS

improved technique for rapid lysis layer, pronase, or DNA release of viral DNA. This the sedimentation of vaccinia

AND METHODS

Cells and virus. HeLa S, cells were grown in suspension in Eagle’s minimal essential medium for suspension culture (GIBCO, Grand Island, N.Y.) supplemented with 10% calf serum (GIBCO), 0.1% pluronic F68, 50 pg/ml of streptomycin and 50 units/ml of penicillin. Strain WR of vaccinia virus was used throughout. Vaccinia virus growth and purification. The purification of pox-viruses from sonicated cytoplasmic fractions of cells by sucrose gradient centrifugation (8,9) has served as the basis for our procedure. HeLa cells (1 X log, 5 x lo6 cells/ml) were resuspended in fresh Eagle’s medium containing 1% calf serum, IOmM MgCl, 0.1% pluronic F68 and antibiotics (adsorption medium) and infected with vaccinia virus at a multiplicity of infection (m.o.i.) of 3 to 5. The suspension was incubated for 1 hr at 37°C in a shaking incubator followed by sedimentation or dilution of the cells to 5 x IO5 cells/ml with fresh medium containing 10% calf serum, 0.1% pluronic F68 and antibiotics. Incubation was continued at 37°C for approximately 40 hr. The cells were collected by sedimentation at 500 g for 10 min, and washed with phosphate-buffered saline. The pellet was resuspended to a concentration of 2.5 x 10’ cells/ml in a hypotonic buffer containing 0.01 M Tris-HCI (pH 8.0), 0.005 M EDTA and 0.01 M KCI, and was incubated for 20 min on ice. The cells were Dounce homogenized and the nuclei were removed by centrifugation at 500 g for 10 min. The supematant fraction was removed and saved, while the pellet was resuspended in a small amount of hypotonic buffer and recentrifuged. Supernatant fractions were pooled and sonicated in an MSE disintegrator for 4 min at 1.5 A and 125 V in 5-6 ml aliquots, and the samples were kept cold by immersion in crushed ice. Seven to eight milliliters of the virus samples were layered onto 35% sucrose containing 0.01 M Tris-HCI (pH 7.5), and centrifuged at 15,000 rpm for 80 min in a Spinco SW25.1 rotor in a Spinco Model L ultracentrifuge at 4°C. The supernatant fraction was discarded and the pellet was resuspended in l-2 ml of hypotonic buffer per tube. The samples were sonicated as before and 3 ml aliquots were layered onto 35-55% sucrose gradients prepared in 0.01 M Tris-HCl (pH 7.5), and centrifuged at 15,000 rpm for 45 min at 4°C in SW25.1 nitrocellulose tubes. The virus band was removed through a hypodermic needle inserted through the side of the nitrocellulose tube. Virus bands were pooled and diluted 1:l with 0.01 Tris-HCI (pH 7.5), and centrifuged at 15,000 rpm for 45 min at 4°C in a SW25.1 rotor. The pelleted virus was resuspended

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in 0.01 M Tris-HCl (pH 7.5), and sonicated as before. The virus was stored at -20 to -70°C. The yield of virus can be significantly increased by rebanding the virus that pellets during gradient centrifugation. Rebanding of the virus in 35-55% sucrose increases the purity but decreases the yield. Preparation of double-labeled virions. HeLa cells were infected with vaccinia virus as described above. At 1 hr postinfection, 0.20 &i/ml of 14C-dThd (54 Ci/mol) and 2.0 PCilml of a 3H-amino acid mixture (algal profile, Schwarz-Mann) were added to the infected HeLa cell culture. Vaccinia virus was purified from the infected cells approximately 20 hr postinfection. Labeling and purification of bacteriophage T,BO,‘. Bacteriophage T4BOlr and its host Escherichia coli B23 were generous gifts from A. W. Kozinski. Radioactive phage was grown and purified by the method of Kozinski (10) utilizing 2.5 &i/ml of 3H-dThd (20 Ci/mol) or 0.40 &i/ml of 2-14C dThd (54 Ci/mol). DNA was extracted with phenol from the purified phage. Neutral sucrose gradients. Eleven milliliter neutral sucrose gradients (5-20%) containing 0.01 M Tris-HCl (pH 7.5), 0.10 M NaCl, 0.50% sodium dodecyl sulfate and 2% 2-mercaptoethanol were generated in No. 2842 polyallomer tubes (Damon, IEC). Vaccinia virus (5-1000 ~1 containing 0.12-0.64 pg of DNA) was carefully layered onto the top of the gradients, and incubated for 30 min at room temp. Centrifugation was for 132 min at 20°C and 35,000 rpm in an IEC SB-269 rotor and B-60 ultracentrifuge. Fractions, approximately 30, were collected from the bottom of the gradient through a small stainless steel tube inserted in the gradient. One milliliter of 100 pg/ml bovine serum albumin fraction V and 4 ml of 12.5% ice-cold trichloroacetic acid (TCA) were added to each fraction. The resulting precipitate was collected on Gelman 25 mm type E glass fiber filter discs, dried, and counted in 10 ml of a toluene-based scintillant . DNase treatment. Sucrose gradient fractions were pooled, dialyzed against 0.01 M Tris- HCl (pH 7.5), and incubated with 100 pg/ml of DNase I (2 x crystallized, Worthington) in the presence of 0.01 M Mg*+ at 37°C for 2 hr. Bovine serum albumin (1 ml of 100 pg/ml) and 10% TCA were added. The precipitate was collected on glass fiber filter discs, and the radioactivity was determined in a liquid scintillation spectrophotometer (Searle Analytic, Isocap). Calculation of the amount and molecular weight of viral DNA. The amount of vaccinia DNA was calculated assuming that 1 OD unit (at 260 nm) contains 1.2 x lOlo virions; the weight of one virion is about 5.5 x 10vg pg, and the weight of DNA per virion is 2.75 x lo-lo pg (7,9, 11). The molecular weight and sedimentation coefficient of vaccinia DNA were calculated by the method of Studier (12).

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AND HEIDELBERGER

RESULTS

Release of native vaccinia DNA on neutral sucrose gradients. When purified vaccinia virions are sedimented through 5-20% linear sucrose gradients containing 0.01 M Tris-HCl (pH 7.9, 0.10 M NaCl, 0.5% SDS, and 2% 2-mercaptoethanol, their DNA is completely released. No lysis layer is necessary. Figure 1 illustrates the sedimentation profile observed. Greater than 85% of the radioactivity applied to the gradient is recovered in TCA-precipitable material. The material in the peak fractions is fully sensitive to DNase. In this neutral sucrose gradient system, DNA from bacteriophage T, DNA (130 x lo6 daltons) cosediments with DNA from vaccinia vu-ions (Fig. 1). Since the above lysis procedure does not release T, DNA from phage, T4 DNA was extracted from the phage particles and layered on the gradients. Cosedimentation of vaccinia DNA with T, DNA confirms the molecular weight for vaccinia virus DNA of 120- 140 x lo6 daltons in agreement with the findings of Geshelin and Berns (3), and demonstrates that this lysis procedure releases intact vaccinia DNA. Vaccinia virions double-labeled with 14C-dThd and tritiated amino acids, as described in Materials and Methods: were analyzed in this neutral sucrose gradient system. A peak of 14C-dThd was observed at the loca-

I

5

IO 15 20 FRACTION NUMBER

25

x0 TOP

FIG. 1. Cosedimentation of r4C-dThd-labeled vaccinia virions, containing 0.15 pg of viral DNA, and 3H-dThd bacteriophage T4 DNA (1.0 Kg) on linear neutral sucrose gradients containing 0.50% SDS, 2% 2-mercaptoethanol and 0.10 M NaCl. Gradients were incubated at room temperature for 30 mitt, followed by centrifugation at 35,000 r-pm and 20°C for 3 hr; (0) 14C-dThd; (0) 3H-dThd. Bacteriophage T, DNA has a molecular weight of 130 x lo6 daltons (13).

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tion expected for viral DNA (Fig. Z), while all tritium was recovered at the top of the gradient. Thus, this procedure resulted in complete separation of labeled DNA and proteins, which was not previously obtained in similar systems (1). Minimal requirements for lysis of vaccinia virions. In order to determine the lowest concentration required for lysis of vaccinia vu-ions, the concentration of each component in the gradient system was altered while maintaining the others constant. The concentration of each component unless stated otherwise was as described in Materials and Methods. Varying the sodium chloride concentration from 0.001 to 0.10 M had no effect on the DNA profile. SDS was tested at 0.05, 0.50, and 1.0%. At 0.5 and 1% SDS vaccinia DNA was completely released, whereas 0.05% SDS resulted in recovery of only a portion (20-40%) of the DNA at the location expected of intact DNA. Five concentrations of 2-mercaptoethanol were tested: 0.01, 0.05, 0.20, 0.50 and 2.0%. At 0.01 and 0.05% 2-mercaptoethanol, minimal release (
FRACTION

NUMBER

TOP

FIG. 2. Independent sedimentation of DNA and proteins from double-labeled vaccinia virions (“C-dThd and 3H-amino acids). Virions containing 0.16 pg of viral DNA were applied to the top of the 5-20% sucrose gradient containing 0.50% SDS, 2% 2-mercaptoethanol and 0.10 M NaCI, as described in Materials and Methods. Centrifugation was for 132 min at 35,000 rpm, 20°C; (0) 14C-dThd; (0) 3H-amino acids.

58

PARKHURST

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HEIDELBERGER

judged by recovery of viral DNA (>80%) in the region in the gradient where native vaccinia DNA is expected. Effect of sample volume on release of vaccinia DNA. Double-labeled vaccinia vu-ions were layered onto preformed gradients in either 0.025, 0.20 or 1.00 ml of 0.01 M Tris-HCl (pH 7.5), incubated for 30 min, and centrifuged. For all sample volumes tested there was complete lysis of vii-ions with release of intact viral DNA. Tritiated proteins were recovered at the top of the gradients. DISCUSSION

An improved technique has been developed for the rapid on-gradient lysis of vaccinia virions with release of intact viral DNA at neutral pH. The viral DNA obtained with this procedure cosediments with bacteriophage T, DNA, confirming the molecular weight of 120-140 X lo6 daltons for vaccinia DNA (3). Vaccinia viral DNA thus obtained is free of protein, as judged by sedimentation of virions double-labeled with 14C-dThd and 3H-amino acids, and is fully sensitive to DNase I digestion. This procedure has been applied to the purification of viral DNA from purified vaccinia virions. The method is similar to that reported by Oda and Joklik (14), except that the overnight pronase treatment is eliminated and SDS and 2-mercaptoethanol are added simultaneously. The simplified procedure consists of adjusting virions to 0.5% SDS, 2% 2mercaptoethanol, 0.10 M NaCl and 0.01 M Tris-HCl (pH 7.5). After incubation at room temperature for 1 hr, the sample is twice extracted with phenol and dialyzed against buffer or precipitated with ethanol. Employing this procedure we have routinely recovered greater than 80% of the viral DNA. The resulting DNA has an OD 260/280 value B1.95. Virus loaded in sample volumes from 0.02 to 1.0 ml yield very similar DNA sedimentation profiles. In the larger sample volume most of the particles are not in contact with the lysing components of the gradient until centrifugation commences. The normal sedimentation of DNA in this case indicates that lysis probably occurs as soon as the virus comes in contact with the lysing components of the gradient. The technique described in this paper has several advantages over methods previously employed. First, it is rapid, and requires at most 10 min of incubation at room temperature prior to centrifugation. Second, it requires only a small amount of virus (1 x lo8 virions), since no extraction of viral DNA is required before centrifugation. Third, no shear of the DNA molecule occurs. Fourth, no proteolytic enzymes are required to achieve removal of protein from DNA. This simple technique may facilitate the study of DNA from other viruses as well.

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DNA

ACKNOWLEDGMENTS The invaluable technical assistance of Mrs. Virginia Garcia is greatly appreciated. This work was supported in part by Grants CA-7175 and CRTY-5006 from the National Cancer Institute. USPH.

REFERENCES 1. 2. 3. 4.

Becker, Y., Dym, H., and Sarov, I. (1968) Virology 36, 184-192. Berns, K. I., and Silverman, C. (1970) J. Viral. 5, 299-304. Geshelin, P.. and Berns, K. 1. (1974)5. Mol. Biol. 88, 785-796. Parkhurst, J. R., Peterson, A. R., and Heidelberger, C. (1973) Proc. USA.

5. 6. 7. 8. 9. IO. 11. 12. 13.

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Sarov, I., and Becker, Y. (1967) Virology 33, 369-375. Pfau, C. J.. and McCrea, J. F. (1962) Nature (London) 194, 894-895. Joklik, W. K. (1962)5. Mol. Biol. 5, 265-274. Fujiwara, Y., and Heidelberger, C. (1970) Mol. Pharmacof. 6, 281-291. Joklik, W. K. (1962) Biochim. Biophys. Acta 61, 290-301. Kozinski, A. W. (1961) Virology 13, 124-134. Joklik, W. K., and Becker, Y. (1964) J. Mol. Biol. 10, 425-474. Studier, F. W. (1965)J. Mol. Biol. 11, 373-390. Mathews, C. K. (1971) Bacteriophage Biochemistry, pp. 34-41, Van Nostrand Reinhold, New York. 14. Oda, K.-I., and Joklik, W. K. (1967)J. Mol. Biol. 27, 395-419.