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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Abstract

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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Materials

Basic Protocol 1: Assessing the Functionality of Papillomavirus Origins of Replication During Transient Transfection of Cultured Cells

  Materials
  • 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

  Materials
  • 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

  Materials
  • 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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Figures

  •   FigureFigure 14.B0.1 Schematic overview of the transient replication assay. The input DNAs for the co‐transfection consist of a papillomavirus origin‐containing test vector (pBOR) and two expression vectors, pE1 and pE2, that provide the viral E1 and E2 proteins, respectively. All the input plasmid DNAs will normally be methylated at specific adenine residues due to the endogenous dam methylation system in E. coli. After cotransfection of the three plasmids into an appropriate cell line, the presence of a functional papillomavirus origin will allow E1‐ and E2‐dependent replication of the test vector. During replication, the adenine methylation will be lost, as metazoan cells lack this activity. Subsequent Hirt extraction isolates the small nucleic acids, including the replicated and unreplicated plasmid DNAs. The input, methylated plasmid DNA is susceptible to DpnI digestion and will be cleaved into small fragments. In contrast, replicated plasmid DNA will be unmethylated and, therefore, resistant to DpnI cleavage. A simultaneous second digestion with a single‐cut restriction enzyme (e.g. HindIII) facilitates subsequent quantitation by converting supercoiled and open circular forms of the plasmid into a single linear species. The replicated and unreplicated DNAs are separated by agarose gel electrophoresis and visualized by Southern blotting with a probe specific for the test vector. Details of this overall process are described in the text.
  •   FigureFigure 14.B0.2 Ethidium bromide–stained agarose gel before and after transfer of the DNA to the GeneScreen membrane. The gel was photographed under UV illumination. Numbers above the lanes indicate the hour post‐transfection at which the DNA samples were harvested.
  •   FigureFigure 14.B0.3 Phosphordensitometer image of a typical replication experiment. Numbers above the lanes indicate the time (in hr) post‐transfection at which each transfected cell culture was harvested. The presence (+) or absence (−) of the pE1 and pE2 DNAs in the original transfection mixture for each sample is as indicated. Samples also received either pUC (nonorigin DNA) or pBOR (bovine papillomavirus origin clone) as shown. Lane M is a marker containing 200 pg of linearized pBOR DNA. The first four transfection lanes are negative controls with the nonorigin DNA or the origin DNA lacking either E1 or E2. Note that there is a minor signal at the position of the marker DNA in the 96‐hr pUC sample, indicating that the DpnI digestion was slightly incomplete for this sample. The remaining transfection lanes show increasing replication of pBOR over time. The bracket indicates the digested input DNA ( DpnI‐sensitive), and the arrow indicates the replicated pBOR DNA ( DpnI‐resistant) that was linearized with HindIII.
  •   FigureFigure 14.B0.4 Diagram of the organization of the gel transfer stacking setup. Details of the assembly and transfer are described in the text.

Videos

Literature Cited

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   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.
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   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.
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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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