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. 1994 Nov 15;13(22):5451–5459. doi: 10.1002/j.1460-2075.1994.tb06880.x

Characterization of the human papillomavirus E2 protein: evidence of trans-activation and trans-repression in cervical keratinocytes.

V Bouvard 1, A Storey 1, D Pim 1, L Banks 1
PMCID: PMC395503  PMID: 7957111

Abstract

The major regulator of papillomavirus transcription is encoded by the viral E2 gene. The E2 gene has been well characterized in bovine papillomavirus (BPV) where it encodes at least three different polypeptides which differentially affect viral gene expression. In human papillomaviruses (HPVs) the E2 gene product is much less well characterized. In this study we have analysed the mechanism of action of the HPV-16, HPV-18 and BPV-1 E2 proteins in cervical keratinocytes. We show that the full length HPV E2 protein acts as a potent transcriptional activator of viral gene expression in both normal and immortalized keratinocytes. In contrast, the BPV-1 E2 protein produces transcriptional repression under identical conditions. A cDNA encoding the C-terminal half of the HPV-16 E2 protein in these assays weakly repressed viral gene expression. Further, co-transfection of this cDNA with the full length clone progressively abolishes the activation in trans by the full length HPV E2 protein. Gel retardation assays have defined a number of protein complexes between the long and short forms of E2 but with no evidence for preferential DNA binding. These results define two distinct activities for the HPV-16 E2 protein, indicate functional differences with the BPV E2 protein and suggest that splicing of the HPV E2 mRNA is a critical mechanism for controlling viral gene expression.

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Selected References

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

  1. Androphy E. J., Lowy D. R., Schiller J. T. Bovine papillomavirus E2 trans-activating gene product binds to specific sites in papillomavirus DNA. Nature. 1987 Jan 1;325(6099):70–73. doi: 10.1038/325070a0. [DOI] [PubMed] [Google Scholar]
  2. Baker C. C., Phelps W. C., Lindgren V., Braun M. J., Gonda M. A., Howley P. M. Structural and transcriptional analysis of human papillomavirus type 16 sequences in cervical carcinoma cell lines. J Virol. 1987 Apr;61(4):962–971. doi: 10.1128/jvi.61.4.962-971.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barbosa M. S., Schlegel R. The E6 and E7 genes of HPV-18 are sufficient for inducing two-stage in vitro transformation of human keratinocytes. Oncogene. 1989 Dec;4(12):1529–1532. [PubMed] [Google Scholar]
  4. Bernard B. A., Bailly C., Lenoir M. C., Darmon M., Thierry F., Yaniv M. The human papillomavirus type 18 (HPV18) E2 gene product is a repressor of the HPV18 regulatory region in human keratinocytes. J Virol. 1989 Oct;63(10):4317–4324. doi: 10.1128/jvi.63.10.4317-4324.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chiang C. M., Dong G., Broker T. R., Chow L. T. Control of human papillomavirus type 11 origin of replication by the E2 family of transcription regulatory proteins. J Virol. 1992 Sep;66(9):5224–5231. doi: 10.1128/jvi.66.9.5224-5231.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Choe J., Vaillancourt P., Stenlund A., Botchan M. Bovine papillomavirus type 1 encodes two forms of a transcriptional repressor: structural and functional analysis of new viral cDNAs. J Virol. 1989 Apr;63(4):1743–1755. doi: 10.1128/jvi.63.4.1743-1755.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cripe T. P., Haugen T. H., Turk J. P., Tabatabai F., Schmid P. G., 3rd, Dürst M., Gissmann L., Roman A., Turek L. P. Transcriptional regulation of the human papillomavirus-16 E6-E7 promoter by a keratinocyte-dependent enhancer, and by viral E2 trans-activator and repressor gene products: implications for cervical carcinogenesis. EMBO J. 1987 Dec 1;6(12):3745–3753. doi: 10.1002/j.1460-2075.1987.tb02709.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Dürst M., Dzarlieva-Petrusevska R. T., Boukamp P., Fusenig N. E., Gissmann L. Molecular and cytogenetic analysis of immortalized human primary keratinocytes obtained after transfection with human papillomavirus type 16 DNA. Oncogene. 1987;1(3):251–256. [PubMed] [Google Scholar]
  11. Dürst M., Glitz D., Schneider A., zur Hausen H. Human papillomavirus type 16 (HPV 16) gene expression and DNA replication in cervical neoplasia: analysis by in situ hybridization. Virology. 1992 Jul;189(1):132–140. doi: 10.1016/0042-6822(92)90688-l. [DOI] [PubMed] [Google Scholar]
  12. Galehouse D., Jenison E., DeLucia A. Differences in the integration pattern and episomal forms of human papillomavirus type 16 DNA found within an invasive cervical neoplasm and its metastasis. Virology. 1992 Jan;186(1):339–341. doi: 10.1016/0042-6822(92)90093-5. [DOI] [PubMed] [Google Scholar]
  13. Hawley-Nelson P., Vousden K. H., Hubbert N. L., Lowy D. R., Schiller J. T. HPV16 E6 and E7 proteins cooperate to immortalize human foreskin keratinocytes. EMBO J. 1989 Dec 1;8(12):3905–3910. doi: 10.1002/j.1460-2075.1989.tb08570.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Laug W. E., DeClerck Y. A., Jones P. A. Degradation of the subendothelial matrix by tumor cells. Cancer Res. 1983 Apr;43(4):1827–1834. [PubMed] [Google Scholar]
  15. Lees E., Osborn K., Banks L., Crawford L. Transformation of primary BRK cells by human papillomavirus type 16 and EJ-ras is increased by overexpression of the viral E2 protein. J Gen Virol. 1990 Jan;71(Pt 1):183–193. doi: 10.1099/0022-1317-71-1-183. [DOI] [PubMed] [Google Scholar]
  16. Luckow B., Schütz G. CAT constructions with multiple unique restriction sites for the functional analysis of eukaryotic promoters and regulatory elements. Nucleic Acids Res. 1987 Jul 10;15(13):5490–5490. doi: 10.1093/nar/15.13.5490. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Matlashewski G., Schneider J., Banks L., Jones N., Murray A., Crawford L. Human papillomavirus type 16 DNA cooperates with activated ras in transforming primary cells. EMBO J. 1987 Jun;6(6):1741–1746. doi: 10.1002/j.1460-2075.1987.tb02426.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Matsukura T., Koi S., Sugase M. Both episomal and integrated forms of human papillomavirus type 16 are involved in invasive cervical cancers. Virology. 1989 Sep;172(1):63–72. doi: 10.1016/0042-6822(89)90107-4. [DOI] [PubMed] [Google Scholar]
  19. McBride A. A., Byrne J. C., Howley P. M. E2 polypeptides encoded by bovine papillomavirus type 1 form dimers through the common carboxyl-terminal domain: transactivation is mediated by the conserved amino-terminal domain. Proc Natl Acad Sci U S A. 1989 Jan;86(2):510–514. doi: 10.1073/pnas.86.2.510. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. McBride A. A., Schlegel R., Howley P. M. The carboxy-terminal domain shared by the bovine papillomavirus E2 transactivator and repressor proteins contains a specific DNA binding activity. EMBO J. 1988 Feb;7(2):533–539. doi: 10.1002/j.1460-2075.1988.tb02842.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Moskaluk C. A., Bastia D. Interaction of the bovine papillomavirus type 1 E2 transcriptional control protein with the viral enhancer: purification of the DNA-binding domain and analysis of its contact points with DNA. J Virol. 1988 Jun;62(6):1925–1931. doi: 10.1128/jvi.62.6.1925-1931.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Münger K., Phelps W. C., Bubb V., Howley P. M., Schlegel R. The E6 and E7 genes of the human papillomavirus type 16 together are necessary and sufficient for transformation of primary human keratinocytes. J Virol. 1989 Oct;63(10):4417–4421. doi: 10.1128/jvi.63.10.4417-4421.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Nasseri M., Gage J. R., Lorincz A., Wettstein F. O. Human papillomavirus type 16 immortalized cervical keratinocytes contain transcripts encoding E6, E7, and E2 initiated at the P97 promoter and express high levels of E7. Virology. 1991 Sep;184(1):131–140. doi: 10.1016/0042-6822(91)90829-z. [DOI] [PubMed] [Google Scholar]
  24. Phelps W. C., Howley P. M. Transcriptional trans-activation by the human papillomavirus type 16 E2 gene product. J Virol. 1987 May;61(5):1630–1638. doi: 10.1128/jvi.61.5.1630-1638.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Pirisi L., Yasumoto S., Feller M., Doniger J., DiPaolo J. A. Transformation of human fibroblasts and keratinocytes with human papillomavirus type 16 DNA. J Virol. 1987 Apr;61(4):1061–1066. doi: 10.1128/jvi.61.4.1061-1066.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ptashne M. How eukaryotic transcriptional activators work. Nature. 1988 Oct 20;335(6192):683–689. doi: 10.1038/335683a0. [DOI] [PubMed] [Google Scholar]
  27. Rohlfs M., Winkenbach S., Meyer S., Rupp T., Dürst M. Viral transcription in human keratinocyte cell lines immortalized by human papillomavirus type-16. Virology. 1991 Jul;183(1):331–342. doi: 10.1016/0042-6822(91)90146-3. [DOI] [PubMed] [Google Scholar]
  28. Romanczuk H., Howley P. M. Disruption of either the E1 or the E2 regulatory gene of human papillomavirus type 16 increases viral immortalization capacity. Proc Natl Acad Sci U S A. 1992 Apr 1;89(7):3159–3163. doi: 10.1073/pnas.89.7.3159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Schwarz E., Freese U. K., Gissmann L., Mayer W., Roggenbuck B., Stremlau A., zur Hausen H. Structure and transcription of human papillomavirus sequences in cervical carcinoma cells. Nature. 1985 Mar 7;314(6006):111–114. doi: 10.1038/314111a0. [DOI] [PubMed] [Google Scholar]
  30. Sherman L., Alloul N. Human papillomavirus type 16 expresses a variety of alternatively spliced mRNAs putatively encoding the E2 protein. Virology. 1992 Dec;191(2):953–959. doi: 10.1016/0042-6822(92)90271-p. [DOI] [PubMed] [Google Scholar]
  31. Smotkin D., Wettstein F. O. Transcription of human papillomavirus type 16 early genes in a cervical cancer and a cancer-derived cell line and identification of the E7 protein. Proc Natl Acad Sci U S A. 1986 Jul;83(13):4680–4684. doi: 10.1073/pnas.83.13.4680. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Stanley M. A., Browne H. M., Appleby M., Minson A. C. Properties of a non-tumorigenic human cervical keratinocyte cell line. Int J Cancer. 1989 Apr 15;43(4):672–676. doi: 10.1002/ijc.2910430422. [DOI] [PubMed] [Google Scholar]
  33. Storey A., Greenfield I., Banks L., Pim D., Crook T., Crawford L., Stanley M. Lack of immortalizing activity of a human papillomavirus type 16 variant DNA with a mutation in the E2 gene isolated from normal human cervical keratinocytes. Oncogene. 1992 Mar;7(3):459–465. [PubMed] [Google Scholar]
  34. Storey A., Pim D., Murray A., Osborn K., Banks L., Crawford L. Comparison of the in vitro transforming activities of human papillomavirus types. EMBO J. 1988 Jun;7(6):1815–1820. doi: 10.1002/j.1460-2075.1988.tb03013.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Thierry F., Dostatni N., Arnos F., Yaniv M. Cooperative activation of transcription by bovine papillomavirus type 1 E2 can occur over a large distance. Mol Cell Biol. 1990 Aug;10(8):4431–4437. doi: 10.1128/mcb.10.8.4431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Thierry F., Yaniv M. The BPV1-E2 trans-acting protein can be either an activator or a repressor of the HPV18 regulatory region. EMBO J. 1987 Nov;6(11):3391–3397. doi: 10.1002/j.1460-2075.1987.tb02662.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. 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]
  38. Vaillancourt P., Nottoli T., Choe J., Botchan M. R. The E2 transactivator of bovine papillomavirus type 1 is expressed from multiple promoters. J Virol. 1990 Aug;64(8):3927–3937. doi: 10.1128/jvi.64.8.3927-3937.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Wigler M., Pellicer A., Silverstein S., Axel R., Urlaub G., Chasin L. DNA-mediated transfer of the adenine phosphoribosyltransferase locus into mammalian cells. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1373–1376. doi: 10.1073/pnas.76.3.1373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Yang L., Li R., Mohr I. J., Clark R., Botchan M. R. Activation of BPV-1 replication in vitro by the transcription factor E2. Nature. 1991 Oct 17;353(6345):628–632. doi: 10.1038/353628a0. [DOI] [PubMed] [Google Scholar]

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