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Rapid small-scale isolation of SV40 virions and SV40 DNA
Journal of Virological Methods 90 (2000) 109 – 114 www.elsevier.com/locate/jviromet
Rapid small-scale isolation of SV40 virions and SV40 DNA Salvatore J. Orlando, Maziar Nabavi, Editte Gharakhanian * Department of Biological Sciences, California State Uni6ersity Long Beach, 1250 Bellflower Bl6d, Long Beach, CA 90840, USA Received 13 December 1999; received in revised form 24 March 2000; accepted 27 March 2000
Abstract A rapid method for the small-scale isolation of SV40 virions and SV40 DNA is presented. CV-1 monkey epithelial cells are transfected with linear SV40 DNA. After the onset of transfection, cells are lysed by several freeze/thaw cycles and virions are isolated using polyethylene glycol (PEG) precipitation of DNase I treated lysates. Viral DNA is released by proteinase K and dithiothreitol treatment of the isolated virions followed by phenol/chloroform extraction and ethanol precipitation. This method yields on average 7.5 ×104 plaque forming units (PFUs) and DNA of adequate purity and concentration to be used for restriction analysis on ethidium bromide agarose gels from a single 35-mm tissue culture dish. © 2000 Elsevier Science B.V. All rights reserved. Keywords: SV40 virus; Transfection; Virion isolation; Viral DNA isolation
1. Introduction Simian virus 40 (SV40) is a DNA tumor virus in the Papovaviridae family. Much is known about the SV40 virus and its structure (Tooze and Acheson, 1980; Liddington et al., 1991; Stehle et al., 1996). It is not only studied in basic virological research, but is also used as a model system for several cellular/molecular processes (Graven et al., 1999; Kohli and Jorgensen, 1999), and as a potential gene delivery vector (Oppenheim et al., 1986; Sandalon et al., 1997). Conventional methods for the isolation of virus have involved largescale infections followed by cesium chloride * Corresponding author. Tel.: +1-562-9854803; fax: +1562-9858878. E-mail address: [email protected] (E. Gharakhanian).
gradients or radioactive labeling of DNA (Pignatti et al., 1979; Darville, 1983). Such methods, while effective, require time, labor, and expensive reagents, and they are not efficient for screening a large number of small samples. A rapid smallscale isolation of herpes simplex virus DNA has been previously presented that begins with infections and does not remove completely the cellular DNA (Kintner and Brandt, 1994). Our method describes a simple miniprep to rapidly isolate SV40 virions and pure SV40 DNA following DNA transfections. DNA recovered from one 35-mm dish of transfected cells is sufficient in amount and purity for molecular biological manipulations including restriction analysis on ethidium bromide gels and polymerase chain reaction (PCR). This procedure can be used to screen for mutant virions, and regions of mutations can be
0166-0934/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 - 0 9 3 4 ( 0 0 ) 0 0 1 7 6 - 2
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S.J. Orlando et al. / Journal of Virological Methods 90 (2000) 109–114
easily amplified by PCR and sequenced. It may also be possible to use this method to screen and harvest other viruses. This procedure does not require expensive chemicals or radioactivity and allows for rapid screening of a large number of transfected samples.
2. Materials and methods
2.1. Plasmid 6ector and cell cultures The mammalian expression vector pSV40, which contains the entire SV40 genome, has been described previously (Clever and Kasamatsu, 1993). BamHI (Roche Molecular Biochemicals) was used to remove the SV40 genome from the pSV40 plasmid. The DNA was electrophoresed on a 1% agarose gel containing 1 mg/ml ethidium bromide in Tris-borate buffer (TBE) (0.09 M Tris base, 0.09 M boric acid, 0.002 M EDTA) (Maniatis et al., 1989) and the SV40 band was excised and gel extracted with a QIAquick gel extraction kit (Qiagen) using the recommended protocol. CV-1 African green monkey kidney epithelial cells (ATCC) were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum (FCS) and 1× penicillin-streptomycinFungizone (PSF) (10 U/ml penicillin, 10 mg/ml streptomycin, and 0.25 mg/ml amphotericin B) (Gibco/BRL).
2.2. Transfections CV-1 cells were seeded onto 35-mm dishes and grown to 50–70% confluency. DNA transfections were performed using Lipofectamine, a cationic lipid, and PLUS reagent (Gibco/BRL). For each dish, 3 mg of linear SV40 DNA was diluted in 200 ml Opti-MEM reduced serum media (Gibco/BRL) and was incubated at room temperature for 20 min with 3 ml PLUS reagent. Concurrently, 12 ml Lipofectamine was diluted in 200 ml Opti-MEM and incubated at room temperature for 20 min. Both mixes were then combined and incubated at room temperature for another 30 min. Cells were rinsed twice with Opti-MEM, overlaid with the DNA-lipid mixture plus an additional 1 ml of
Opti-MEM, and incubated for 5 h at 37°C in 5.5% CO2. After 5 h, 1 ml of DMEM plus 20% FCS was added to the cells and incubation was continued for 2 h more. At hour 7, all media was removed, the cells were rinsed once with 1 × phosphate buffered saline (PBS) (0.137 M NaCl, 2.68 mM KCl, 10.14 mM Na2HPO4, 1.76 mM KH2PO4), and 2 ml of DMEM with 10% FCS and 1 × PSF was added to each dish. Cells were kept at 37°C with 5.5% CO2 until 100% of the cells showed cytopathic effect (CPE).
2.3. Virion and 6irus DNA isolation Transfected cells were scraped and rinsed with an additional 500 ml PBS and were then subjected to four freeze/thaw cycles in a dry ice/ethanol bath. Cellular debris was removed by centrifugation at 11 000 rpm for 10 min. To the supernatant, DNase I (Roche Molecular Biochemicals) was added to a final concentration of 0.5 U/ml in the presence of 42 mM MgCl2 and the sample was incubated on ice for 10 min. The reaction was stopped by the addition of EDTA to a final concentration of 5 mM. In initial isolations, samples were also treated with 0.4 mg/ml RNase for 45 min at 37°C. Solid NaCl was then added to a final concentration of 1 M and incubation was continued on ice for an additional hour. The sample was then centrifuged at 7500 rpm for 10 min and any pellet was discarded. To precipitate virion particles, polyethylene glycol (PEG, MW 4000, BDH Laboratory Supplies) was added (10% w/v) to the supernatant and dissolved by gentle vortexing. The sample was then incubated on ice for 1 h followed by centrifugation at 7500 rpm for 10 min. The supernatant was discarded and the pellet was resuspended in 50 ml 10 mM Tris–HCl pH 8.0. The sample was stored at 4°C as virion stock or was used to isolate viral DNA. To recover viral DNA, proteinase K (Qiagen) was added to a final concentration of 50 mg/ml and the sample was incubated at 56°C for 1 h. Dithiothreitol (DTT) was then added to a final concentration of 25 mM and incubation continued at 56°C for an additional 30 min. Viral DNA was extracted with phenol/chloroform and was ethanol precipitated.
S.J. Orlando et al. / Journal of Virological Methods 90 (2000) 109–114
2.4. Infection and plaque assays CV-1 cells were infected to test the integrity and infectivity of the recovered virions. To 70% confluent cells, 10 − 3 – 10 − 4 dilutions of virion stock in serum free DMEM were added. Plates were rocked every 15 min for 1.5 h at 37°C in 5.5% CO2. Cells were then overlaid with an additional 2 ml of DMEM with 3% FCS and were incubated for 2 days. The media were then removed and the cells were rinsed once with 1× PBS followed by the addition of DMEM containing 0.8% agarose, 10% FCS, and 1× PSF. Upon solidification of the agarose overlay an additional milliliter of liquid media was added. To visualize plaques, agarose was removed gently by inversion and cells were rinsed once with 1 × PBS and stained with crystal violet (Fendrick and Hallick, 1983).
2.5. Restriction and Southern blot analysis of 6iral DNA
111
gen) to a final concentration of 1 mg/ml and 1× reaction buffer. Samples were cycled 30 times in a Promega PCR machine and results were visualized on a 2% agarose gel containing 1 mg/ml ethidium bromide.
3. Results The general procedure for SV40 virion and viral DNA isolation is outlined in Fig. 1. Viral DNA can be harvested in under 6 h. We examined the viability of DNase I treated, PEG precipitated virus. If virions were compromised by DNase I treatment, we would see reduced or lack of plaque formation. Plaque assays from DNase I treated samples showed no reduction in plaque formation compared with non-DNase I treated ones (Fig. 2a). This confirmed the integrity of the viral capsid. Based on the number of plaques formed, the average titer of the isolated virions was calculated to be 75 000 PFUs from one
One half of the viral DNA yield from a 35-mm dish was digested with NdeI, or EcoRV (Roche Molecular Biochemicals) for 2 h and was electrophoresed in a 1% agarose gel containing 1 mg/ml ethidium bromide in TBE. For Southern blot analysis, one fifth of DNase untreated, DNase treated, or DNase and RNase treated viral yield from a single 35-mm dish was subjected to DNA isolation and electrophoresed as mentioned above. Electrophoresed DNA was transferred to nitrocellulose by capillary transfer and baked in a vacuum oven for 1.5 h at 80°C (Maniatis et al., 1989). Blots were probed with two 32P end-labeled SV40 oligonucleotides (Operon Technologies).
2.6. PCR Viral DNA was subjected to PCR using forward and reverse SV40 primers, 5%-TCCTAATGTGCAGTCAGGTG-3% and 5%-GATGTTCATCAGGATTGCCC-3%, with expected amplified DNA of 326 bp (SV40 1366 – 1692). Samples contained 50–500 ng of template DNA, 5 pmol each of forward and reverse primers, dNTPs to a final concentration of 2.4 mM, Taq polymerase (Qia-
Fig. 1. Flow chart of rapid small-scale SV40 and SV40 DNA isolation.
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Fig. 2. Virions and packaged viral DNA are resistant to DNase I treatment. (a) The infectivity of DNase I treated, PEG precipitated virions was tested. CV-1 cells were infected with recovered virions, overlaid with 0.8% agarose, and monitored for plaque formation. Both DNase I treated and untreated virions produced plaques. (b) Southern blot analysis of extracted DNA from DNase I treated and untreated PEG precipitated virions probed with two 32P end-labeled SV40 oligonucleotides. An equivalent amount of sample (one fifth of a single 35-mm harvest) was loaded into each lane. Lane 1 contains PEG precipitated virions without DNase I and RNase treatment. Lane 2 contains PEG precipitated virions treated with only DNase I. Lane 3 contains PEG precipitated virions treated with both DNase I and RNase.
35-mm dish. Extracted DNA from DNase I treated and untreated samples were subjected to Southern blot analysis using SV40 derived oligonucleotides (Fig. 2b). Results from Southerns, together with DNA profiles seen in ethidium bromide gels (Fig. 3a), confirmed SV40 DNA as the only recovered DNA species. A portion of the recovered DNA is DNase I sensitive (Fig. 2b), yet plaque formation is not (Fig. 2a), suggesting degradation of unpackaged SV40 DNA by DNase I treatment. Up to 1 mg of naked DNA containing SV40 sequences was completely degraded under the same conditions (data not shown). Thus, DNase I was effective in eliminating cellular and/or unpackaged viral DNA but did not compromise virion infectivity. RNase treatment of samples did not result in any differ-
ences in Southern profiles (Fig. 2b), and no evidence of RNA contamination was detected in the ethidium bromide gels (Fig. 3a). Therefore, RNase treatment was not routinely included. DNA recovered from each sample dish ranged from a low of 100 ng to a high of 1.5 mg with most samples containing 500–700 ng DNA. Recovered SV40 DNA was clean enough for restriction digest and for subsequent restriction analysis directly on ethidium bromide gels (as in Fig. 3a). Consistently, there was no cellular DNA contamination in samples. Extracted DNA was also a sufficient template for PCR using SV40 derived primers (Fig. 3b). PCR products were sequenced (CSU Northridge, DNA Sequencing Facility) and further confirmed SV40 sequences.
S.J. Orlando et al. / Journal of Virological Methods 90 (2000) 109–114
4. Discussion A method is described for rapid small-scale isolation of SV40 virus and SV40 DNA. The entire procedure can be carried out in 1.5-ml microcentrifuge tubes. DNA isolation can be completed in under 6 h for a large number of samples, yields an average of 500 ng DNA from a single transfected 35-mm dish, is cost-effective, and does not require the use of radioactivity. The
113
viral isolation method can be used to obtain initial recombinant SV40 virus from recombinant SV40 DNA for further plaque purification. The DNA isolation method can be used to screen SV40 mutants or to harvest packaged viral DNA. Thus, the rapid small-scale procedure is ideal for screening SV40 packaging and/or assembly mutants (Gharakhanian et al., 2000). Although the amount of DNA recovered varies between samples, consistently enough viral DNA, devoid of cellular DNA or RNA contamination, is obtained for direct examination in ethidium bromide gels. This procedure has been repeated several times and found it can be scaled up using a 60-mm dish and can begin with an infection versus a transfection to recover more virus and/or DNA. This procedure may also be adapted for isolation of other DNA viruses with permissive modes of infection. Limiting factors would be the size of the viral genome since transfection efficiency is reduced as the size of the DNA is increased, and the transfection efficiency of the host cells. If beginning from an infection, however, one should find the harvesting procedure very useful for rapid isolation of a large variety of DNA viruses and their genomes from permissive host cells. Acknowledgements
Fig. 3. Restriction and PCR analysis of SV40 DNA minipreparation. (a) One half of viral DNA from a single 35-mm dish was digested with NdeI (lane 3) or EcoRV (lane 4) and electrophoresed on a 1% agarose gel containing 1 mg/ml ethidium bromide. Bacteriophage l DNA digested with HindIII (lane 1) and linearized SV40 (lane 2) were used as size markers. The arrows in lane 3 denote the two expected fragments produced from the digestion with NdeI, (4.2 and 1.0 kb). Lane 4 contains viral DNA restricted once with EcoRV and matches the linearized SV40 DNA (lane 2) at 5.2 kb. (b) PCR analysis to test the quality of extracted viral DNA. Using SV40 derived primers 100 ng viral DNA was amplified with PCR. The size marker used was a 100-bp ladder (lane 1). Lane 2 contains the PCR products at an expected size of 326 bp.
This work was made possible by a grant from the National Science Foundation-Research for Undergraduate Institutions (MCB-9630904) to E.G. S.J.O. was supported by the California State University at Long Beach, College of Natural Sciences and Mathematics. M.N. was partially supported by MCB-9630904. References Clever, J.L., Kasamatsu, H., 1993. Identification of a DNAbinding domain in simian virus 40 capsid proteins Vp2 and Vp3. J. Biol. Chem. 268, 20877 – 20882. Darville, J.M., 1983. A miniaturised and simplified technique for typing and subtyping herpes simplex virus. J. Clin. Pathol. 36, 929 – 934. Fendrick, J.L., Hallick, L.M., 1983. Optimal conditions for titration of SV40 by the plaque assay method. J. Virol. Methods 7, 93 – 102.
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Gharakhanian, E., Orlando, S.J., Fasching, C.L., 2000. Cys9 Ser mutant SV40 Vp1 has an altered epitope at its C-terminal arm but continues to form infectious virions. J. Gen. Virol., manuscript submitted. Graven, K.K., Yu, Q., Pan, D., Roncarati, J.S., Farber, H.W., 1999. Identification of an oxygen responsive enhancer element in the glyceraldehyde-3-phosphate dehydrogenase gene. Biochim. Biophys. Acta 1447, 208–218. Kintner, R.L., Brandt, C.R., 1994. Rapid small-scale isolation of herpes simplex virus DNA. J. Virol. Methods 48, 189– 196. Kohli, M., Jorgensen, T.J., 1999. The influence of SV40 immortalization of human fibroblasts on p53-dependent radiation responses. Biochem. Biophys. Res. Commun. 257, 168–176. Liddington, R.C., Yan, Y., Moulai, J., Sahli, R., Benjamin, T.L., Harrison, S.C., 1991. Structure of simian virus 40 at 3.8-A resolution. Nature 354, 278–284. Maniatis, T., Fritsch, E.F., Sambrook, J., 1989. Molecular
.
Cloning: A Laboratory Manual, second ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Oppenheim, A., Peleg, A., Fibach, E., Rachmilewitz, E.A., 1986. Efficient introduction of plasmid DNA into human hemopoietic cells by encapsidation in simian virus 40 pseudovirions. Proc. Natl. Acad. Sci. USA 82, 6925 – 6929. Pignatti, P.F., Cassai, E., Bertazzoni, U., 1979. Herpes simplex virus DNA synthesis in a partially purified soluble extract from infected cells. J. Virol. 32, 1033 – 1036. Sandalon, Z., Dalyot-Herman, N., Oppenheim, A.B., Oppenheim, A., 1997. In vitro assembly of SV40 virions and pseudovirions: vector development for gene therapy. Hum. Gene Ther. 8, 843 – 849. Stehle, T., Gamblin, S.J., Yan, Y., Harrison, S.C., 1996. The structure of simian virus 40 refined at 3.1-A resolution. Structure 4, 165 – 182. Tooze, J., Acheson, N.H., 1980. DNA Tumor Viruses. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Rapid small-scale isolation of SV40 virions and SV40 DNA Salvatore J. Orlando, Maziar Nabavi, Editte Gharakhanian * Department of Biological Sciences, California State Uni6ersity Long Beach, 1250 Bellflower Bl6d, Long Beach, CA 90840, USA Received 13 December 1999; received in revised form 24 March 2000; accepted 27 March 2000
Abstract A rapid method for the small-scale isolation of SV40 virions and SV40 DNA is presented. CV-1 monkey epithelial cells are transfected with linear SV40 DNA. After the onset of transfection, cells are lysed by several freeze/thaw cycles and virions are isolated using polyethylene glycol (PEG) precipitation of DNase I treated lysates. Viral DNA is released by proteinase K and dithiothreitol treatment of the isolated virions followed by phenol/chloroform extraction and ethanol precipitation. This method yields on average 7.5 ×104 plaque forming units (PFUs) and DNA of adequate purity and concentration to be used for restriction analysis on ethidium bromide agarose gels from a single 35-mm tissue culture dish. © 2000 Elsevier Science B.V. All rights reserved. Keywords: SV40 virus; Transfection; Virion isolation; Viral DNA isolation
1. Introduction Simian virus 40 (SV40) is a DNA tumor virus in the Papovaviridae family. Much is known about the SV40 virus and its structure (Tooze and Acheson, 1980; Liddington et al., 1991; Stehle et al., 1996). It is not only studied in basic virological research, but is also used as a model system for several cellular/molecular processes (Graven et al., 1999; Kohli and Jorgensen, 1999), and as a potential gene delivery vector (Oppenheim et al., 1986; Sandalon et al., 1997). Conventional methods for the isolation of virus have involved largescale infections followed by cesium chloride * Corresponding author. Tel.: +1-562-9854803; fax: +1562-9858878. E-mail address: [email protected] (E. Gharakhanian).
gradients or radioactive labeling of DNA (Pignatti et al., 1979; Darville, 1983). Such methods, while effective, require time, labor, and expensive reagents, and they are not efficient for screening a large number of small samples. A rapid smallscale isolation of herpes simplex virus DNA has been previously presented that begins with infections and does not remove completely the cellular DNA (Kintner and Brandt, 1994). Our method describes a simple miniprep to rapidly isolate SV40 virions and pure SV40 DNA following DNA transfections. DNA recovered from one 35-mm dish of transfected cells is sufficient in amount and purity for molecular biological manipulations including restriction analysis on ethidium bromide gels and polymerase chain reaction (PCR). This procedure can be used to screen for mutant virions, and regions of mutations can be
0166-0934/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 - 0 9 3 4 ( 0 0 ) 0 0 1 7 6 - 2
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S.J. Orlando et al. / Journal of Virological Methods 90 (2000) 109–114
easily amplified by PCR and sequenced. It may also be possible to use this method to screen and harvest other viruses. This procedure does not require expensive chemicals or radioactivity and allows for rapid screening of a large number of transfected samples.
2. Materials and methods
2.1. Plasmid 6ector and cell cultures The mammalian expression vector pSV40, which contains the entire SV40 genome, has been described previously (Clever and Kasamatsu, 1993). BamHI (Roche Molecular Biochemicals) was used to remove the SV40 genome from the pSV40 plasmid. The DNA was electrophoresed on a 1% agarose gel containing 1 mg/ml ethidium bromide in Tris-borate buffer (TBE) (0.09 M Tris base, 0.09 M boric acid, 0.002 M EDTA) (Maniatis et al., 1989) and the SV40 band was excised and gel extracted with a QIAquick gel extraction kit (Qiagen) using the recommended protocol. CV-1 African green monkey kidney epithelial cells (ATCC) were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum (FCS) and 1× penicillin-streptomycinFungizone (PSF) (10 U/ml penicillin, 10 mg/ml streptomycin, and 0.25 mg/ml amphotericin B) (Gibco/BRL).
2.2. Transfections CV-1 cells were seeded onto 35-mm dishes and grown to 50–70% confluency. DNA transfections were performed using Lipofectamine, a cationic lipid, and PLUS reagent (Gibco/BRL). For each dish, 3 mg of linear SV40 DNA was diluted in 200 ml Opti-MEM reduced serum media (Gibco/BRL) and was incubated at room temperature for 20 min with 3 ml PLUS reagent. Concurrently, 12 ml Lipofectamine was diluted in 200 ml Opti-MEM and incubated at room temperature for 20 min. Both mixes were then combined and incubated at room temperature for another 30 min. Cells were rinsed twice with Opti-MEM, overlaid with the DNA-lipid mixture plus an additional 1 ml of
Opti-MEM, and incubated for 5 h at 37°C in 5.5% CO2. After 5 h, 1 ml of DMEM plus 20% FCS was added to the cells and incubation was continued for 2 h more. At hour 7, all media was removed, the cells were rinsed once with 1 × phosphate buffered saline (PBS) (0.137 M NaCl, 2.68 mM KCl, 10.14 mM Na2HPO4, 1.76 mM KH2PO4), and 2 ml of DMEM with 10% FCS and 1 × PSF was added to each dish. Cells were kept at 37°C with 5.5% CO2 until 100% of the cells showed cytopathic effect (CPE).
2.3. Virion and 6irus DNA isolation Transfected cells were scraped and rinsed with an additional 500 ml PBS and were then subjected to four freeze/thaw cycles in a dry ice/ethanol bath. Cellular debris was removed by centrifugation at 11 000 rpm for 10 min. To the supernatant, DNase I (Roche Molecular Biochemicals) was added to a final concentration of 0.5 U/ml in the presence of 42 mM MgCl2 and the sample was incubated on ice for 10 min. The reaction was stopped by the addition of EDTA to a final concentration of 5 mM. In initial isolations, samples were also treated with 0.4 mg/ml RNase for 45 min at 37°C. Solid NaCl was then added to a final concentration of 1 M and incubation was continued on ice for an additional hour. The sample was then centrifuged at 7500 rpm for 10 min and any pellet was discarded. To precipitate virion particles, polyethylene glycol (PEG, MW 4000, BDH Laboratory Supplies) was added (10% w/v) to the supernatant and dissolved by gentle vortexing. The sample was then incubated on ice for 1 h followed by centrifugation at 7500 rpm for 10 min. The supernatant was discarded and the pellet was resuspended in 50 ml 10 mM Tris–HCl pH 8.0. The sample was stored at 4°C as virion stock or was used to isolate viral DNA. To recover viral DNA, proteinase K (Qiagen) was added to a final concentration of 50 mg/ml and the sample was incubated at 56°C for 1 h. Dithiothreitol (DTT) was then added to a final concentration of 25 mM and incubation continued at 56°C for an additional 30 min. Viral DNA was extracted with phenol/chloroform and was ethanol precipitated.
S.J. Orlando et al. / Journal of Virological Methods 90 (2000) 109–114
2.4. Infection and plaque assays CV-1 cells were infected to test the integrity and infectivity of the recovered virions. To 70% confluent cells, 10 − 3 – 10 − 4 dilutions of virion stock in serum free DMEM were added. Plates were rocked every 15 min for 1.5 h at 37°C in 5.5% CO2. Cells were then overlaid with an additional 2 ml of DMEM with 3% FCS and were incubated for 2 days. The media were then removed and the cells were rinsed once with 1× PBS followed by the addition of DMEM containing 0.8% agarose, 10% FCS, and 1× PSF. Upon solidification of the agarose overlay an additional milliliter of liquid media was added. To visualize plaques, agarose was removed gently by inversion and cells were rinsed once with 1 × PBS and stained with crystal violet (Fendrick and Hallick, 1983).
2.5. Restriction and Southern blot analysis of 6iral DNA
111
gen) to a final concentration of 1 mg/ml and 1× reaction buffer. Samples were cycled 30 times in a Promega PCR machine and results were visualized on a 2% agarose gel containing 1 mg/ml ethidium bromide.
3. Results The general procedure for SV40 virion and viral DNA isolation is outlined in Fig. 1. Viral DNA can be harvested in under 6 h. We examined the viability of DNase I treated, PEG precipitated virus. If virions were compromised by DNase I treatment, we would see reduced or lack of plaque formation. Plaque assays from DNase I treated samples showed no reduction in plaque formation compared with non-DNase I treated ones (Fig. 2a). This confirmed the integrity of the viral capsid. Based on the number of plaques formed, the average titer of the isolated virions was calculated to be 75 000 PFUs from one
One half of the viral DNA yield from a 35-mm dish was digested with NdeI, or EcoRV (Roche Molecular Biochemicals) for 2 h and was electrophoresed in a 1% agarose gel containing 1 mg/ml ethidium bromide in TBE. For Southern blot analysis, one fifth of DNase untreated, DNase treated, or DNase and RNase treated viral yield from a single 35-mm dish was subjected to DNA isolation and electrophoresed as mentioned above. Electrophoresed DNA was transferred to nitrocellulose by capillary transfer and baked in a vacuum oven for 1.5 h at 80°C (Maniatis et al., 1989). Blots were probed with two 32P end-labeled SV40 oligonucleotides (Operon Technologies).
2.6. PCR Viral DNA was subjected to PCR using forward and reverse SV40 primers, 5%-TCCTAATGTGCAGTCAGGTG-3% and 5%-GATGTTCATCAGGATTGCCC-3%, with expected amplified DNA of 326 bp (SV40 1366 – 1692). Samples contained 50–500 ng of template DNA, 5 pmol each of forward and reverse primers, dNTPs to a final concentration of 2.4 mM, Taq polymerase (Qia-
Fig. 1. Flow chart of rapid small-scale SV40 and SV40 DNA isolation.
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S.J. Orlando et al. / Journal of Virological Methods 90 (2000) 109–114
Fig. 2. Virions and packaged viral DNA are resistant to DNase I treatment. (a) The infectivity of DNase I treated, PEG precipitated virions was tested. CV-1 cells were infected with recovered virions, overlaid with 0.8% agarose, and monitored for plaque formation. Both DNase I treated and untreated virions produced plaques. (b) Southern blot analysis of extracted DNA from DNase I treated and untreated PEG precipitated virions probed with two 32P end-labeled SV40 oligonucleotides. An equivalent amount of sample (one fifth of a single 35-mm harvest) was loaded into each lane. Lane 1 contains PEG precipitated virions without DNase I and RNase treatment. Lane 2 contains PEG precipitated virions treated with only DNase I. Lane 3 contains PEG precipitated virions treated with both DNase I and RNase.
35-mm dish. Extracted DNA from DNase I treated and untreated samples were subjected to Southern blot analysis using SV40 derived oligonucleotides (Fig. 2b). Results from Southerns, together with DNA profiles seen in ethidium bromide gels (Fig. 3a), confirmed SV40 DNA as the only recovered DNA species. A portion of the recovered DNA is DNase I sensitive (Fig. 2b), yet plaque formation is not (Fig. 2a), suggesting degradation of unpackaged SV40 DNA by DNase I treatment. Up to 1 mg of naked DNA containing SV40 sequences was completely degraded under the same conditions (data not shown). Thus, DNase I was effective in eliminating cellular and/or unpackaged viral DNA but did not compromise virion infectivity. RNase treatment of samples did not result in any differ-
ences in Southern profiles (Fig. 2b), and no evidence of RNA contamination was detected in the ethidium bromide gels (Fig. 3a). Therefore, RNase treatment was not routinely included. DNA recovered from each sample dish ranged from a low of 100 ng to a high of 1.5 mg with most samples containing 500–700 ng DNA. Recovered SV40 DNA was clean enough for restriction digest and for subsequent restriction analysis directly on ethidium bromide gels (as in Fig. 3a). Consistently, there was no cellular DNA contamination in samples. Extracted DNA was also a sufficient template for PCR using SV40 derived primers (Fig. 3b). PCR products were sequenced (CSU Northridge, DNA Sequencing Facility) and further confirmed SV40 sequences.
S.J. Orlando et al. / Journal of Virological Methods 90 (2000) 109–114
4. Discussion A method is described for rapid small-scale isolation of SV40 virus and SV40 DNA. The entire procedure can be carried out in 1.5-ml microcentrifuge tubes. DNA isolation can be completed in under 6 h for a large number of samples, yields an average of 500 ng DNA from a single transfected 35-mm dish, is cost-effective, and does not require the use of radioactivity. The
113
viral isolation method can be used to obtain initial recombinant SV40 virus from recombinant SV40 DNA for further plaque purification. The DNA isolation method can be used to screen SV40 mutants or to harvest packaged viral DNA. Thus, the rapid small-scale procedure is ideal for screening SV40 packaging and/or assembly mutants (Gharakhanian et al., 2000). Although the amount of DNA recovered varies between samples, consistently enough viral DNA, devoid of cellular DNA or RNA contamination, is obtained for direct examination in ethidium bromide gels. This procedure has been repeated several times and found it can be scaled up using a 60-mm dish and can begin with an infection versus a transfection to recover more virus and/or DNA. This procedure may also be adapted for isolation of other DNA viruses with permissive modes of infection. Limiting factors would be the size of the viral genome since transfection efficiency is reduced as the size of the DNA is increased, and the transfection efficiency of the host cells. If beginning from an infection, however, one should find the harvesting procedure very useful for rapid isolation of a large variety of DNA viruses and their genomes from permissive host cells. Acknowledgements
Fig. 3. Restriction and PCR analysis of SV40 DNA minipreparation. (a) One half of viral DNA from a single 35-mm dish was digested with NdeI (lane 3) or EcoRV (lane 4) and electrophoresed on a 1% agarose gel containing 1 mg/ml ethidium bromide. Bacteriophage l DNA digested with HindIII (lane 1) and linearized SV40 (lane 2) were used as size markers. The arrows in lane 3 denote the two expected fragments produced from the digestion with NdeI, (4.2 and 1.0 kb). Lane 4 contains viral DNA restricted once with EcoRV and matches the linearized SV40 DNA (lane 2) at 5.2 kb. (b) PCR analysis to test the quality of extracted viral DNA. Using SV40 derived primers 100 ng viral DNA was amplified with PCR. The size marker used was a 100-bp ladder (lane 1). Lane 2 contains the PCR products at an expected size of 326 bp.
This work was made possible by a grant from the National Science Foundation-Research for Undergraduate Institutions (MCB-9630904) to E.G. S.J.O. was supported by the California State University at Long Beach, College of Natural Sciences and Mathematics. M.N. was partially supported by MCB-9630904. References Clever, J.L., Kasamatsu, H., 1993. Identification of a DNAbinding domain in simian virus 40 capsid proteins Vp2 and Vp3. J. Biol. Chem. 268, 20877 – 20882. Darville, J.M., 1983. A miniaturised and simplified technique for typing and subtyping herpes simplex virus. J. Clin. Pathol. 36, 929 – 934. Fendrick, J.L., Hallick, L.M., 1983. Optimal conditions for titration of SV40 by the plaque assay method. J. Virol. Methods 7, 93 – 102.
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