HPV Binding Assay to Laminin-332/Integrin α6β4 on Human Keratinocytes

Part of the Methods in Molecular Biology book series (MIMB, volume 1249)


Human papillomaviruses (HPVs) have been shown to bind to Laminin-332 (Ln-332) on the extracellular matrix (ECM) secreted by human keratinocytes. The assay described here is an important tool to study HPV receptor binding to the ECM. The assay can also be modified to study the receptors required for HPV infection and for binding to tissues. We previously showed that Ln-332 is essential for the binding of HPV11 to human keratinocytes and that infectious entry of HPV11 requires α6β4 integrin for the transfer of HPV11 from ECM to host cells (Culp et al., J Virol 80:8940–8950, 2006). We also demonstrated that several of the high-risk HPV types (16, 18, 31 and 45) bind to Ln-332 and/or other components of the ECM in vitro (Broutian et al., J Gen Virol 91:531–540, 2010). The exact binding and internalization mechanism(s) for HPV are still under investigation. A better understanding of these mechanisms will aid in the design of therapeutics against HPVs and ultimately help prevent many cancers. In this chapter, we describe the HPV binding assay to Ln-332/integrin α6β4 on human keratinocytes (ECM). We also present data and suggestions for modifying the assay for testing the specificity of HPV for receptors (by blocking receptors) and binding to human tissues (basement membrane, BM) in order to study binding mechanisms.

Key words

Human papillomavirus (HPV) Laminin 332 (Ln-332) Integrin α6β4 Extracellular matrix (ECM) Basement membrane (BM) Immunocytochemistry 


  1. 1.
    Parkin DM, Bray F (2006) The burden of HPV-related cancers. Vaccine 24:11–25CrossRefGoogle Scholar
  2. 2.
    Schiffman M, Wentzensen N, Wacholder S, Kinney W, Gage JC, Castle PE (2011) Human papillomavirus testing in the prevention of cervical cancer. J Natl Cancer Inst 103:368–383PubMedCrossRefPubMedCentralGoogle Scholar
  3. 3.
    Zandberg DP, Bhargava R, Badin S, Cullen KJ (2013) The role of human papillomavirus in nongenital cancers. CA Cancer J Clin 63: 57–81PubMedCrossRefGoogle Scholar
  4. 4.
    Culp TD, Budgeon LR, Marinkovich MP, Meneguzzi G, Christensen ND (2006) Keratinocyte-secreted laminin 5 can function as a transient receptor for human papillomaviruses by binding virions and transferring them to adjacent cells. J Virol 80:8940–8950PubMedCrossRefPubMedCentralGoogle Scholar
  5. 5.
    Richards RM, Lowy DR, Schiller JT, Day PM (2006) Cleavage of the papillomavirus minor capsid protein, L2, at a furin consensus site is necessary for infection. Proc Natl Acad Sci U S A 103:1522–1527PubMedCrossRefPubMedCentralGoogle Scholar
  6. 6.
    Sapp M, Bienkowska-Haba M (2009) Viral entry mechanisms: human papillomavirus and a long journey from extracellular matrix to the nucleus. FEBS J 276:7206–7216PubMedCrossRefPubMedCentralGoogle Scholar
  7. 7.
    Schiller JT, Day PM, Kines RC (2010) Current understanding of the mechanism of HPV infection. Gynecol Oncol 118:S12–S17PubMedCrossRefPubMedCentralGoogle Scholar
  8. 8.
    Culp TD, Budgeon LR, Christensen ND (2006) Human papillomaviruses bind a basal extracellular matrix component secreted by keratinocytes which is distinct from a membrane-associated receptor. Virology 347:147–159PubMedCrossRefGoogle Scholar
  9. 9.
    Broutian TR, Brendle SA, Christensen ND (2010) Differential binding patterns to host cells associated with particles of several human alphapapillomavirus types. J Gen Virol 91: 531–540PubMedCrossRefPubMedCentralGoogle Scholar
  10. 10.
    Boukamp P, Petrussevska RT, Breitkreutz D, Hornung J, Markham A, Fusenig NE (1988) Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. J Cell Biol 106:761–771PubMedCrossRefGoogle Scholar
  11. 11.
    Kreider JW, Howett MK, Leuredupree AE, Zaino RJ, Weber JA (1987) Laboratory production in vivo of infectious human papillomavirus type-11. J Virol 61:590–593PubMedPubMedCentralGoogle Scholar
  12. 12.
    Christensen ND, Kirnbauer R, Schiller JT et al (1994) Human papillomavirus type-6 and type-11 have antigenically distinct strongly immunogenic conformationally dependent neutralizing epitopes. Virology 205:329–335PubMedCrossRefGoogle Scholar
  13. 13.
    Meyers C, Frattini MG, Hudson JB, Laimins LA (1992) Biosynthesis of human papillomavirus from a continuous cell-line upon epithelial differentiation. Science 257:971–973PubMedCrossRefGoogle Scholar
  14. 14.
    Brendle SA, Culp TD, Broutian TR, Christensen ND (2010) Binding and neutralization characteristics of a panel of monoclonal antibodies to Human Papillomavirus 58. J Gen Virol 91(Pt 7):1834–1839PubMedCrossRefPubMedCentralGoogle Scholar
  15. 15.
    Christensen ND, Dillner J, Eklund C et al (1996) Surface conformational and linear epitopes on HPV-16 and HPV-18L1 virus-like particles as defined by monoclonal antibodies. Virology 223:174–184PubMedCrossRefGoogle Scholar
  16. 16.
    Christensen ND, Cladel NM, Reed CA (1995) Postattachment neutralization of papillomaviruses by monoclonal and polyclonal antibodies. Virology 207:136–142PubMedCrossRefGoogle Scholar
  17. 17.
    Christensen ND, Reed CA, Cladel NM, Hall K, Leiserowitz GS (1996) Monoclonal antibodies to HPV-6L1 virus-like particles identify conformational and linear neutralizing epitopes on HPV-11 in addition to type-specific epitopes on HPV-6. Virology 224:477–486PubMedCrossRefGoogle Scholar
  18. 18.
    Mejia AF, Culp TD, Cladel NM et al (2006) Preclinical model to test human papillomavirus virus (HPV) capsid vaccines in vivo using infectious HPV/cottontail rabbit papillomavirus chimeric papillomavirus particles. J Virol 80: 12393–12397PubMedCrossRefPubMedCentralGoogle Scholar
  19. 19.
    Buck CB, Pastrana DV, Lowy DR, Schiller JT (2004) Efficient intracellular assembly of papillomaviral vectors. J Virol 78:751–757PubMedCrossRefPubMedCentralGoogle Scholar
  20. 20.
    Buck CB, Thompson CD, Pang YYS, Lowy DR, Schiller JT (2005) Maturation of papillomavirus capsids. J Virol 79:2839–2846PubMedCrossRefPubMedCentralGoogle Scholar
  21. 21.
    Christensen ND, Cladel NM, Reed CA et al (2001) Hybrid papillomavirus L1 molecules assemble into virus-like particles that reconstitute conformational epitopes and induce neutralizing antibodies to distinct HPV types. Virology 291:324–334PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of Pathology, The Jake Gittlen Laboratories for Cancer ResearchPennsylvania State University College of MedicineHersheyUSA

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