Analyzing the Human Papillomavirus (HPV) Life Cycle in Primary Keratinocytes with a Quantitative Colony‐Forming Assay

Michael J. Lace1, Lubomir P. Turek1, James R. Anson2, Thomas H. Haugen1

1 The Department of Pathology, University of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, Iowa, 2 Veterans Healthcare System, University of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, Iowa
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 14B.2
DOI:  10.1002/9780471729259.mc14b02s33
Online Posting Date:  May, 2014
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Papillomavirus genomes replicate as extrachromosomal plasmids within infected keratinocytes, requiring the regulated expression of early viral gene products to initially amplify the viral genomes and subvert cell growth checkpoints as part of a complex path to immortalization. Building on contemporary keratinocyte transfection and culture systems, the methods described in this unit form a detailed approach to analyzing critical events in the human papillomavirus (HPV) life cycle, utilizing physiologic levels of viral gene products expressed from their native promoter(s) in the natural host cells for HPV infection. A quantitative colony‐forming assay permits comparison of the capacities of various transfected HPV types and mutant HPV genomes to initially form colonies and immortalize human keratinocytes. In conjunction with additional methods, these protocols enable examination of genomic stability, viral and cellular gene expression, viral integration, and differentiation patterns influenced by HPV persistence in clonal human keratinocytes that effectively mimic early events in HPV infection. Curr. Protoc. Microbiol. 33:14B.2.1‐14B.2.13. © 2014 by John Wiley & Sons, Inc.

Keywords: HPV replication; keratinocyte immortalization; early gene expression; complementation; viral persistence

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Primary Keratinocyte Isolation
  • Basic Protocol 2: Transfection of Primary Human Keratinocytes with Recircularized HPV Plasmid Genomes
  • Basic Protocol 3: The Quantitative Colony‐Forming Assay
  • Support Protocol 1: Preparation of Irradiated J2 Fibroblast Feeder Layers
  • Support Protocol 2: Quality Control Measures
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Primary Keratinocyte Isolation

  Materials
  • Newborn foreskin (harvested directly after circumcision into 10 ml of E medium by clinical staff and stored at 4°C until processed)
  • E medium (see recipe)
  • 25 mg/ml Dispase II (Roche) in PBS ( ), filter through 0.45‐µm filter and store at −20°C
  • Trypsin solution (see recipe)
  • 0.25 mg/ml soybean trypsin inhibitor (Life Technologies)
  • Freeze medium (see recipe)
  • DMEM medium (e.g., Life Technologies) containing 7% (v/v) FBS
  • Liquid N 2
  • 100‐mm and 35‐mm diameter petri plates
  • Surgical instruments:
    • Scissors
    • Forceps and fine forceps
    • Scalpel
  • 75‐cm2 flasks
  • 15‐ml conical tubes (e.g., BD Falcon)
  • Tabletop centrifuge (IEC Centra, model CL2)
  • Cryovials
  • Cell scrapers
  • Cell freezing container (e.g., Mr Frosty from Nalge)

Basic Protocol 2: Transfection of Primary Human Keratinocytes with Recircularized HPV Plasmid Genomes

  Materials
  • Cloned HPV genome (see above)
  • Appropriate restriction enzymes
  • 5 U/µl T4 DNA ligase (Roche)
  • 10× ligase buffer: 660 mM Tris·Cl, pH 7.5 ( ), 50 mM MgCl 2, 50 mM DTT
  • 100 mM rATP, pH 7.5 (Roche)
  • 3× QBT buffer (see recipe)
  • Qiagen plasmid Megakit including:
    • Qiagen‐tip 500 columns
    • QC buffer
    • QF buffer
  • Qiagen Effectene Transfection Kit (cat. no. 301427), including: Effectene, Enhancer, and EC buffer
  • Isopropanol
  • TE buffer ( )
  • Primary human foreskin keratinocytes ( protocol 1)
  • E medium (see recipe)
  • pRSVneo plasmid DNA (Addgene, cat. no. 37198)
  • G418 (Geneticin; Life Technologies, cat. no. 10131)
  • 100‐mm culture dishes
  • Beckman XL‐90 ultracentrifuge with SW28 rotor
  • Additional reagents and equipment for agarose gel electrophoresis (Voytas, ) purification of DNA from agarose gels (Moore et al., ), and colony‐forming assay ( protocol 3)

Basic Protocol 3: The Quantitative Colony‐Forming Assay

  Materials
  • Transfected cells from protocol 2
  • Irradiated J2 fibroblast feeder layers ( protocol 4)
  • E medium (see recipe) containing 100 to 200 µg/ml G418 (Geneticin; Life Technologies, cat. no. 10131)
  • Trypsin solution (see recipe)
  • Qiagen BloodAmp kit
  • Ambion RNAqueous kit
  • Phase‐contrast microscope
  • Cloning cylinders (see Mortensen and Kingston, )
  • Additional reagents and equipment for use of cloning cylinders to subculture cells (Mortensen and Kingston, )

Support Protocol 1: Preparation of Irradiated J2 Fibroblast Feeder Layers

  Materials
  • 3T3‐Swiss albino J2 mouse embryonic fibroblasts (ATCC, cat. no. CCL‐92)
  • DMEM medium (e.g., Invitrogen) containing 7% FBS with 50 U/ml penicillin and 50 µg/ml streptomycin
  • Trypsin solution (see recipe)
  • 150‐mm‐diameter culture plates
  • XRAD‐320 irradiator (Precision X‐ray, Inc.)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Allen‐Hoffmann, B.L. , Schlosser, S.J. , Ivarie, C.A. , Sattler, C.A. , Meisner, L.F. , and O'Connor, S.L. 2000. Normal growth and differentiation in a spontaneously immortalized near‐diploid human keratinocyte cell line, NIKS. J. Invest. Dermatol. 114:444‐455.
   Boshart, M. , Gissmann, L. , Ikenberg, H. , Kleinheinz, A. , Scheurlen, W. , and zur Hausen, H. 1984. A new type of papillomavirus DNA, its presence in genital cancer biopsies and in cell lines derived from cervical cancer. EMBO J. 3:1151‐1157.
   Brown, T. 1993. Hybridization analysis of DNA blots. Curr. Protoc. Mol. Biol. 21:2.10.1‐2.10.16.
   Brown, T. 1999. Southern blotting. Curr. Protoc. Mol. Biol. 00:2.9.1‐2.9.20.
   Doorbar, J. 2005. The papillomavirus life cycle. J. Clin. Virol. 32:7‐15.
   Frattini, M.G. , Lim, H.B. , and Laimins, L.A. 1996. In vitro synthesis of oncogenic human papillomaviruses requires episomal genomes for differentiation‐dependent late expression. Proc. Natl. Acad. Sci. U.S.A. 93:3062‐3067.
   Frattini, M.G. , Lim, H.B. , Doorbar, J. , and Laimins, L.A. 1997. Induction of human papillomavirus type 18 late gene expression and genomic amplification in organotypic cultures from transfected DNA templates. J. Virol. 71:7068‐7072.
   Gillison, M.L. , Alemany, L. , Snijders, P.J. , Chaturvedi, A. , Steinberg, B.M. , Schwartz, S. , and Castellsague, X. 2012. Human papillomavirus and diseases of the upper airway: Head and neck cancer and respiratory papillomatosis. Vaccine 30:F34‐F54.
   Gissmann, L. , Diehl, V. , Schultz Coulon, H.J. , and zur Hausen, H. 1982. Molecular cloning and characterization of human papilloma virus DNA derived from a laryngeal papilloma. J. Virol. 44:393‐400.
   Haugen, T.H. , Lace, M.J. , Ishiji, T. , Sameshima, A. , Anson, J.R. , and Turek, L.P. 2009. Cellular factors are required to activate bovine papillomavirus‐1 early gene transcription and to establish viral plasmid persistence but are not required for cellular transformation. Virology 389:82‐90.
   Hoffmann, R. , Hirt, B. , Bechtold, V. , Beard, P. , and Raj, K. 2006. Different modes of human papillomavirus DNA replication during maintenance. J. Virol. 80:4431‐4439.
   Hubert, W.G. , Kanaya, T. , and Laimins, L.A. 1999. DNA replication of human papillomavirus type 31 is modulated by elements of the upstream regulatory region that lie 5′ of the minimal origin. J. Virol. 73:1835‐1845.
   Hubert, W.G. and Laimins, L.A. 2002. Human papillomavirus type 31 replication modes during the early phases of the viral life cycle depend on transcriptional and posttranscriptional regulation of E1 and E2 expression. J. Virol. 76:2263‐2273.
   Jeon, S. , Allen‐Hoffmann, B.L. , and Lambert, P.F. 1995. Integration of human papillomavirus type 16 into the human genome correlates with a selective growth advantage of cells. J. Virol. 69:2989‐2997.
   Jewers, R.J. , Hildebrandt, P. , Ludlow, J.W. , Kell, B. , and McCance, D.J. 1992. Regions of human papillomavirus type 16 E7 oncoprotein required for immortalization of human keratinocytes. J. Virol. 66:1329‐1335.
   Lace, M.J. , Anson, J.R. , Thomas, G.S. , Turek, L.P. , and Haugen, T.H. 2008a. The HPV16 E8ˆE2 gene product represses early transcription and replication but is dispensable for viral plasmid persistence in keratinocytes. J. Virol. 82:10841‐10853.
   Lace, M.J. , Anson, J.R. , Turek, L.P. , and Haugen, T.H. 2008b. Functional mapping of the human papillomavirus (HPV) Type 16 E1 cistron. J. Virol. 82:10724‐10734.
   Lace, M.J. , Anson, J.R. , Klingelhutz, A.J. , Lee, J.H. , Bossler, A.D. , Haugen, T.H. , and Turek, L.P. 2009a. Human papillomavirus (HPV) type 18 induces extended growth in primary human cervical, tonsillar, or foreskin keratinocytes more effectively than other high‐risk mucosal HPVs. J. Virol. 83:11784‐11794.
   Lace, M.J. , Isacson, C. , Anson, J.R. , Lorincz, A.T. , Wilczynski, S.P. , Haugen, T.H. , and Turek, L.P. 2009b. Upstream regulatory region alterations found in human papillomavirus type 16 (HPV16) isolates from cervical carcinomas increase transcription, ori function, and HPV immortalization capacity in culture. J. Virol. 83:7457‐7466.
   Lace, M.J. , Anson, J.R. , Klussmann, J.P. , Wang, D.H. , Smith, E.M. , Haugen, T.H. , and Turek, L.P. 2011. Human papillomavirus type 16 (HPV16) genomes integrated in head and neck cancers and in HPV16‐immortalized human keratinocyte clones express chimeric virus‐cell mRNAs similar to those found in cervical cancers. J. Virol. 85:1645‐1654.
   Lee, J.H. , Yi, S.M. , Anderson, M.E. , Berger, K.L. , Welsh, M.J. , Klingelhutz, A.J. , and Ozbun, M.A. 2004. Propagation of infectious human papillomavirus type 16 by using an adenovirus and Cre/LoxP mechanism. Proc. Natl. Acad. Sci. U.S.A. 101:2094‐2099.
   Lörincz, A.T. , Quinn, A.P. , Goldsborough, M.D. , Schmidt, B.J. , and Temple, G.F. 1989. Cloning and partial DNA sequencing of two new human papillomavirus types associated with condylomas and low‐grade cervical neoplasia. J. Virol. 63:2829‐2834.
   Meyers, C. , Frattini, M.G. , Hudson, J.B. , and Laimins, L.A. 1992. Biosynthesis of human papillomavirus from a continuous cell line upon epithelial differentiation. Science 257:971‐973.
   Moore, D. , Dowhan, D. , Chory, J. , and Ribaudo, R. K. 2002. Isolation and purification of large DNA restriction fragments from agarose gels. Curr. Protoc. Mol. Biol. 59:2.6.1‐2.6.12.
   Mortensen, R.M. and Kingston, R.E. 2009. Selection of transfected mammalian cells. Curr. Protoc. Mol. Biol. 86:9.5.1‐9.5.13.
   Peehl, D.M. and Ham, R.G. 1980. Clonal growth of human keratinocytes with small amounts of dialyzed serum. In Vitro 16:526‐540.
   Rheinwald, J.G. and Green, H. 1975. Serial cultivation of strains of human epidermal keratinocytes: The formation of keratinizing colonies from single cells. Cell 6:331‐343.
   Sprague, D.L. , Phillips, S.L. , Mitchell, C.J. , Berger, K.L. , Lace, M. , Turek, L.P. , and Klingelhutz, A.J. 2002. Telomerase activation in cervical keratinocytes containing stably replicating human papillomavirus type 16 episomes. Virology 301:247‐254.
   Ushikai, M. , Lace, M.J. , Yamakawa, Y. , Kono, M. , Anson, J. , Ishiji, T. , Parkkinen, S. , Wicker, N. , Valentine, M.E. , Davidson, I. , Turek, L.P. , and Haugen, T.H. 1994. trans activation by the full‐length E2 proteins of human papillomavirus type 16 and bovine papillomavirus type 1 in vitro and in vivo: Cooperation with activation domains of cellular transcription factors. J. Virol. 68:6655‐6666.
   Voytas, D. 2000. Agarose gel electrophoresis. Curr. Protoc. Mol. Biol. 51:2.5A.1‐2.5A.9.
   Wille, J.J. Jr. , Pittelkow, M.R. , Shipley, G.D. , and Scott, R.E. 1984. Integrated control of growth and differentiation of normal human prokeratinocytes cultured in serum‐free medium: clonal analyses, growth kinetics, and cell cycle studies. J. Cell Physiol. 121:31‐44.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library