Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1997 Sep;71(9):7068–7072. doi: 10.1128/jvi.71.9.7068-7072.1997

Induction of human papillomavirus type 18 late gene expression and genomic amplification in organotypic cultures from transfected DNA templates.

M G Frattini 1, H B Lim 1, J Doorbar 1, L A Laimins 1
PMCID: PMC191995  PMID: 9261437

Abstract

The genetic analysis of human papillomavirus (HPV) functions during the vegetative viral life cycle is dependent upon the ability to generate human keratinocyte cell lines which maintain episomal copies of transfected viral genomes. We have previously demonstrated that lipofection of normal human foreskin keratinocytes with recircularized cloned HPV-31 genomic sequences resulted in a high frequency of cell lines which maintained viral genomes as extrachromosomal elements (M.G. Frattini, H. Lim, and L.A. Laimins, Proc. Natl. Acad. Sci. USA 93:3062-3067, 1996). Following the growth of these cell lines in organotypic (raft) cultures, the differentiation-dependent expression of viral late genes, the amplification of viral genomes, and virion biosynthesis were observed. In the present study, we demonstrate that these methodologies are not restricted to HPV-31 but are applicable to other HPV types, including the oncogenic HPV-18. HPV-18 genomes were purified from bacterial vector sequences, religated, and transfected into normal human foreskin keratinocytes together with a neomycin-selectable marker. Following drug selection, resistant cells were expanded and examined for the state of the viral DNA. All cell lines examined were found to contain approximately 100 to 200 episomal copies of HPV-18 DNA per cell. Growth of these cell lines in raft cultures resulted in the differentiation-dependent expression of the E1 [symbol: see text] E4 and L1 capsid genes. In addition, viral genome amplification was observed in suprabasal cells following DNA in situ hybridization analysis of differentiated raft cultures. The induction of these late viral functions has previously been shown to be directly associated with differentiation-dependent virion biosynthesis. Our studies indicate the ability to perform a detailed genetic analysis of the various phases of the viral life cycle, including control of the differentiation-dependent late viral functions, using a second oncogenic HPV type.

Full Text

The Full Text of this article is available as a PDF (300.3 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ball R. D., Walker G. K., Bernstein I. A. Histidine-rich proteins as molecular markers of epidermal differentiation. J Biol Chem. 1978 Aug 25;253(16):5861–5868. [PubMed] [Google Scholar]
  2. Bedell M. A., Jones K. H., Laimins L. A. The E6-E7 region of human papillomavirus type 18 is sufficient for transformation of NIH 3T3 and rat-1 cells. J Virol. 1987 Nov;61(11):3635–3640. doi: 10.1128/jvi.61.11.3635-3640.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brune W., Dürst M. Epithelial differentiation fails to support replication of cloned human papillomavirus type 16 DNA in transfected keratinocytes. J Invest Dermatol. 1995 Feb;104(2):277–281. doi: 10.1111/1523-1747.ep12612814. [DOI] [PubMed] [Google Scholar]
  4. Chiang C. M., Ustav M., Stenlund A., Ho T. F., Broker T. R., Chow L. T. Viral E1 and E2 proteins support replication of homologous and heterologous papillomaviral origins. Proc Natl Acad Sci U S A. 1992 Jul 1;89(13):5799–5803. doi: 10.1073/pnas.89.13.5799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chow L. T., Nasseri M., Wolinsky S. M., Broker T. R. Human papillomavirus types 6 and 11 mRNAs from genital condylomata acuminata. J Virol. 1987 Aug;61(8):2581–2588. doi: 10.1128/jvi.61.8.2581-2588.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Christensen N. D., Dillner J., Eklund C., Carter J. J., Wipf G. C., Reed C. A., Cladel N. M., Galloway D. A. Surface conformational and linear epitopes on HPV-16 and HPV-18 L1 virus-like particles as defined by monoclonal antibodies. Virology. 1996 Sep 1;223(1):174–184. doi: 10.1006/viro.1996.0466. [DOI] [PubMed] [Google Scholar]
  7. Dollard S. C., Wilson J. L., Demeter L. M., Bonnez W., Reichman R. C., Broker T. R., Chow L. T. Production of human papillomavirus and modulation of the infectious program in epithelial raft cultures. OFF. Genes Dev. 1992 Jul;6(7):1131–1142. doi: 10.1101/gad.6.7.1131. [DOI] [PubMed] [Google Scholar]
  8. Doorbar J., Coneron I., Gallimore P. H. Sequence divergence yet conserved physical characteristics among the E4 proteins of cutaneous human papillomaviruses. Virology. 1989 Sep;172(1):51–62. doi: 10.1016/0042-6822(89)90106-2. [DOI] [PubMed] [Google Scholar]
  9. Doorbar J., Ely S., Sterling J., McLean C., Crawford L. Specific interaction between HPV-16 E1-E4 and cytokeratins results in collapse of the epithelial cell intermediate filament network. Nature. 1991 Aug 29;352(6338):824–827. doi: 10.1038/352824a0. [DOI] [PubMed] [Google Scholar]
  10. Doorbar J., Parton A., Hartley K., Banks L., Crook T., Stanley M., Crawford L. Detection of novel splicing patterns in a HPV16-containing keratinocyte cell line. Virology. 1990 Sep;178(1):254–262. doi: 10.1016/0042-6822(90)90401-c. [DOI] [PubMed] [Google Scholar]
  11. Frattini M. G., Laimins L. A. Binding of the human papillomavirus E1 origin-recognition protein is regulated through complex formation with the E2 enhancer-binding protein. Proc Natl Acad Sci U S A. 1994 Dec 20;91(26):12398–12402. doi: 10.1073/pnas.91.26.12398. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Frattini M. G., Laimins L. A. The role of the E1 and E2 proteins in the replication of human papillomavirus type 31b. Virology. 1994 Nov 1;204(2):799–804. doi: 10.1006/viro.1994.1596. [DOI] [PubMed] [Google Scholar]
  13. Frattini M. G., Lim H. B., Laimins L. A. In vitro synthesis of oncogenic human papillomaviruses requires episomal genomes for differentiation-dependent late expression. Proc Natl Acad Sci U S A. 1996 Apr 2;93(7):3062–3067. doi: 10.1073/pnas.93.7.3062. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Grassmann K., Rapp B., Maschek H., Petry K. U., Iftner T. Identification of a differentiation-inducible promoter in the E7 open reading frame of human papillomavirus type 16 (HPV-16) in raft cultures of a new cell line containing high copy numbers of episomal HPV-16 DNA. J Virol. 1996 Apr;70(4):2339–2349. doi: 10.1128/jvi.70.4.2339-2349.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Griffiths A. D., Williams S. C., Hartley O., Tomlinson I. M., Waterhouse P., Crosby W. L., Kontermann R. E., Jones P. T., Low N. M., Allison T. J. Isolation of high affinity human antibodies directly from large synthetic repertoires. EMBO J. 1994 Jul 15;13(14):3245–3260. doi: 10.1002/j.1460-2075.1994.tb06626.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hummel M., Hudson J. B., Laimins L. A. Differentiation-induced and constitutive transcription of human papillomavirus type 31b in cell lines containing viral episomes. J Virol. 1992 Oct;66(10):6070–6080. doi: 10.1128/jvi.66.10.6070-6080.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hummel M., Lim H. B., Laimins L. A. Human papillomavirus type 31b late gene expression is regulated through protein kinase C-mediated changes in RNA processing. J Virol. 1995 Jun;69(6):3381–3388. doi: 10.1128/jvi.69.6.3381-3388.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Laimins L. A. The biology of human papillomaviruses: from warts to cancer. Infect Agents Dis. 1993 Apr;2(2):74–86. [PubMed] [Google Scholar]
  19. Meyers C., Frattini M. G., Hudson J. B., Laimins L. A. Biosynthesis of human papillomavirus from a continuous cell line upon epithelial differentiation. Science. 1992 Aug 14;257(5072):971–973. doi: 10.1126/science.1323879. [DOI] [PubMed] [Google Scholar]
  20. Mungal S., Steinberg B. M., Taichman L. B. Replication of plasmid-derived human papillomavirus type 11 DNA in cultured keratinocytes. J Virol. 1992 May;66(5):3220–3224. doi: 10.1128/jvi.66.5.3220-3224.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Pray T. R., Laimins L. A. Differentiation-dependent expression of E1--E4 proteins in cell lines maintaining episomes of human papillomavirus type 31b. Virology. 1995 Jan 10;206(1):679–685. doi: 10.1016/s0042-6822(95)80088-3. [DOI] [PubMed] [Google Scholar]
  22. Roberts S., Ashmole I., Johnson G. D., Kreider J. W., Gallimore P. H. Cutaneous and mucosal human papillomavirus E4 proteins form intermediate filament-like structures in epithelial cells. Virology. 1993 Nov;197(1):176–187. doi: 10.1006/viro.1993.1578. [DOI] [PubMed] [Google Scholar]
  23. Sun S., Thorner L., Lentz M., MacPherson P., Botchan M. Identification of a 68-kilodalton nuclear ATP-binding phosphoprotein encoded by bovine papillomavirus type 1. J Virol. 1990 Oct;64(10):5093–5105. doi: 10.1128/jvi.64.10.5093-5105.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Ustav M., Stenlund A. Transient replication of BPV-1 requires two viral polypeptides encoded by the E1 and E2 open reading frames. EMBO J. 1991 Feb;10(2):449–457. doi: 10.1002/j.1460-2075.1991.tb07967.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES