Cell Culture Assay for Transient Replication of Human and Animal Papillomaviruses

Van G. Wilson1

1 Texas A&M University Health Science Center, Bryan, Texas
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 14B.1
DOI:  10.1002/9780471729259.mc14b01s24
Online Posting Date:  February, 2012
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This unit contains protocols for evaluation of replication functionality of papillomavirus genomes or subgenomic fragments. Replication is measured after transient cotransfection of the genome (or subgenomic fragment) with expression vectors encoding the viral E1 and E2 proteins. Input DNA is methylated at the adenine of GATC sequences by propagation in E. coli. DNA that replicates in mammalian cells will lose the adenine methylation and become DpnI‐resistant, while residual methylated input DNA will remain DpnI‐sensitive. After transfection, DNA extraction, and DpnI digestion, replicated DNA can be detected by Southern blotting as a full‐length plasmid, since it is resistant to digestion. This assay can be used to map the genomic location of a functional origin or to evaluate replication activity of mutations in either the origin DNA sequences or the E1 or E2 proteins. Curr. Protoc. Microbiol. 24:14B.1.1‐14B.1.18. © 2012 by John Wiley & Sons, Inc.

Keywords: replication; transfection; HIRT extraction; Southern blot; papillomaviruses

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Table of Contents

  • Introduction
  • Basic Protocol 1: Assessing the Functionality of Papillomavirus Origins of Replication During Transient Transfection of Cultured Cells
  • Support Protocol 1: Hirt Precipitation
  • Support Protocol 2: Southern Blotting
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Assessing the Functionality of Papillomavirus Origins of Replication During Transient Transfection of Cultured Cells

  • Chinese hamster ovary (CHO) cells (American Type Culture Collection)
  • Ham's F12 medium (e.g., Life Technologies) with and without 10% fetal bovine serum (FBS)
  • Trypsin, tissue culture grade
  • DNA stocks for transfection (store at −20°C), prepared in TE buffer, pH 7.0 ( appendix 2A):
    • 500 ng/µl pE1 (E1 expression vector)
    • 500 ng/µl pE2 (E2 expression vector)
    • 100 ng/µl pBOR (origin positive vector)
    • 100 ng/µl pUC (origin negative; parental vector for pBOR; New England Biolabs)
    • 500 ng/µl pcDNA3.1 (parental vector for pE1 and pE2; Invitrogen)
  • Lipofectamine 2000 (Invitrogen)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 70% (v/v) ethanol
  • TE buffer, pH 7.0 ( appendix 2A)
  • 500 µg/ml RNase, DNase‐free (Roche Applied Science)
  • 10× restriction enzyme buffer compatible with DpnI and HindIII
  • ≥20 U/µl DpnI restriction endonuclease
  • ≥20 U/µl HindIII restriction endonuclease
  • Molecular‐biology‐grade H 2O
  • 10× agarose sample buffer (see recipe)
  • Molecular‐biology‐grade agarose
  • DNA quantitation standards (see recipe)
  • 1% (w/v) ethidium bromide
  • 60‐mm and 100‐mm tissue culture plates
  • 68°C water bath or heating block
  • Ultraviolet lamp
  • Plastic sheet protector (i.e., folder for papers available from office supply store)
  • Additional reagents and equipment for counting cells using a hemacytometer (Strober, ), Hirt precipitation (see protocol 2), agarose gel electrophoresis (Voytas, ), Southern blotting (see protocol 3), and detection of radiolabeled DNA in blots by autoradiography or phosphor imaging (Voytas and Ning, )
NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified. Maintain cells at subclonfluency.

Support Protocol 1: Hirt Precipitation

  • Plates containing cells to be lysed (see protocol 1, step )
  • Hirt lysis buffer (see recipe)
  • 5.0 M NaCl (sterilized by autoclaving)
  • 20 mg/ml proteinase K stock in sterile H 2O (store up to 1 year at −20°C)
  • 25:24:1 phenol/chloroform/isoamyl alcohol (Ambion)
  • Chloroform
  • TE buffer, pH 7.0 ( appendix 2A)
  • Isopropanol
  • 50°C water bath or heating block

Support Protocol 2: Southern Blotting

  • Agarose gel containing separated proteins (see protocol 1, step )
  • 0.25 M HCl
  • 0.4 M NaOH
  • 20× SSC ( appendix 2A)
  • 0.4 mg/ml ethidium bromide
  • Probe DNA stock: prepare linearized pBOR as described for DNA quantitation standards (see recipe), but resuspend at 1 µg/ml in TE buffer, pH 7.0
  • Prime‐A‐Gene kit (Promega) containing:
    • dATP, dGTP, and dTTP stock solutions
    • 5× buffer
    • Nuclease‐free H 2O
    • Nuclease‐free BSA stock solution
    • DNA polymerase I large (Klenow) fragment stock solution
  • 10 mCi/ml [32P]dCTP (3000 Ci/mmol)
  • Rapid‐Hyb buffer (GE Healthcare)
  • 0.1× SSC ( appendix 2A)/0.1% (w/v) SDS
  • Gel staining trays
  • GeneScreen Plus membrane (PerkinElmer Life Sciences)
  • Whatman 3MM filter paper
  • Platform rocker
  • Stratalinker UV cross‐linker (Stratagene)
  • 65° or 95°C water bath or heating block
  • Hybridization oven
  • Hybridization bottles
  • 15‐ml conical tubes
CAUTION: Radioactive materials require special handling. See unit 1.4.
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Literature Cited

   Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K. (eds.) 2005. Current Protocols in Molecular Biology. John Wiley & Sons, Hoboken, N.J.
   Brown, T. 1999. Southern blotting. Curr. Protoc. Mol. Biol. 68:2.9.1‐2.9.20.
   Chiang, C.M., Ustav, M., Stenlund, A., Ho, T.F., Broker, T.R., and Chow, L.T. 1992. Viral E1 and E2 proteins support replication of homologous and heterologous papillomaviral origins. Proc. Natl. Acad. Sci. U.S.A. 89:5799‐5803.
   Fradet‐Turcotte, A., Morin, G., Lehoux, M., Bullock, P.A., and Archambault, J. 2010. Development of quantitative and high‐throughput assays of polymovavirus and papillomavirus DNA replication. J. Virol. 399:65‐76.
   Gopalakrishnan, V. and Khan, S.A. 1994. E1 protein of human papillomavirus type 1a is sufficient for initiation of viral DNA replication. Proc. Natl. Acad. Sci. U.S.A. 91:9597‐9601.
   Holt, S.E. and Wilson, V.G. 1995. Mutational analysis of the 18‐base‐pair inverted repeat element at the bovine papillomavirus origin of replication: Identification of critical sequences for E1 binding and in vivo replication. J. Virol. 69:6525‐6532.
   Lin, B.Y., Ma, T.L., Liu, J.S., Kuo, S.R., Jin, G., Broker, T.R., Harper, J.W., and Chow, L.T. 2000. HeLa cells are phenotypically limiting in cyclin E/CDK2 for efficient human papillomavirus DNA replication. J. Biol. Chem. 275:6167‐6174.
   Lu, J.Z., Sun, Y.N., Rose, R.C., Bonnez, W., and McCance, D.J. 1993. Two E2 binding sites (E2BS) alone or one E2BS plus an A/T‐rich region are minimal requirements for the replication of the human papillomavirus type 11 origin. J. Virol. 67:7131‐7139.
   Lusky, M. and Botchan, M.R. 1984. Characterization of the bovine papilloma virus plasmid maintenance sequences. Cell 36:391‐401.
   Lusky, M. and Botchan, M.R. 1986. Transient replication of bovine papilloma virus type 1 plasmids: cis and trans requirements. Proc. Natl. Acad. Sci. U.S.A. 83:3609‐3613.
   McShan, G.D. and Wilson, V.G. 1997. Reconstitution of a functional bovine Papillomavirus Type 1 origin of replication reveals a modular tripartite replicon with an essential AT‐rich element. Virology 237:198‐208.
   Mino, T., Hatono, T., Matsumoto, N., Mori, T., Mineta, Y., Aoyama, Y., and Sera, T. 2006. Inhibition of DNA replication of human papillomavirus by artificial zinc finger proteins. J. Virol. 80:5405‐5412.
   Peden, K.W.C., Pipas, Y.M., Pearson‐White, S., and Nathans, D. 1980. Isolation of mutants of an animal virus in bacteria. Science 203:1392.
   Piirsoo, M., Ustav, E., Mandel, T., Stenlund, A., and Ustav, M. 1996. Cis and trans requirements for stable episomal maintenance of the BPV‐1 replicator. EMBO. J. 15:1‐11.
   Strober, W. 1997. Monitoring cell growth. Curr. Protoc. Immunol. 21:A.3A.1‐A.3A.2.
   Sverdrup, F. and Khan, S.A. 1994. Replication of human papillomavirus (HPV) DNAs supported by the HPV type 18 E1 and E2 proteins. J. Virol. 68:505‐509.
   Taylor, E.R. and Morgan, I.M. 2003. A novel technique with enhanced detection and quantitation of HPV‐16 E1‐ and E2‐mediated DNA replication. Virology 315:103‐109.
   Titolo, S., Pelletier, A., Sauve, F., Brault, K., Wardrop, E., White, P.W., Amin, A., Cordingley, M.G., and Archambault, J. 1999. Role of the ATP‐binding domain of the human papillomavirus type 11 E1 helicase in E2‐dependent binding to the origin. J. Virol. 73:5282‐5293.
   Ustav, M. and Stenlund, A. 1991. Transient replication of BPV‐1 requires two viral polypeptides encoded by the E1 and E2 open reading frames. EMBO. J. 10:449‐457.
   Ustav, M., Ustav, E., Szymanski, P., and Stenlund, A. 1991. Identification of the origin of replication of bovine papillomavirus and characterization of the viral origin recognition factor E1. EMBO. J. 10:4321‐4329.
   Voytas, D. 2000. Agarose gel electrophoresis. Curr. Protoc. Mol. Biol. 51:2.5A.1‐2.5A.9.
   Voytas, D. and Ning, K. 2000. Detection and quantitation of radiolabeled proteins and DNA in gels and blots. Curr. Protoc. Immunol. 50:A.3J.1‐A.3J.10.
   Wang, X.M., Jansen, K.U., and McClements, W.L. 2003. DNA replicative functions of highly‐expressed, codon‐optimized human papillomavirus proteins E1 and E2. J Virol Methods 108:83‐90.
   West, M., Flanery, D., Woytek, K., Rangasamy, D., and Wilson, V.G. 2001. Functional mapping of the DNA binding domain of bovine papillomavirus E1 protein. J. Virol. 75:11948‐11960.
   White, P.W., Titolo, S., Brault, K., Thauvette, L., Pelletier, A., Welchner, E., Bourgon, L., Doyon, L., Ogilvie, W.W., Yoakim, C., Cordingley, M.G., and Archambault, J. 2003. Inhibition of human papillomavirus DNA replication by small molecule antagonists of the E1‐E2 protein interaction. J. Biol. Chem. 278:26765‐26772.
Key References
   Ustav and Stenlund, 1991. See above.
  This is the first study to document that the viral E1 and E2 proteins were necessary and sufficient to support replication of a papillomavirus origin in a transient transfection assay.
   Ustav et al., 1991. See above.
  This study correctly identified and mapped the bovine papillomavirus origin of replication. This was the first authentic definition of a functional papillomavirus origin, and documented that a DpnI assay could be used to successfully detect transient replication of papillomavirus origin–containing DNA.
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