The E1 and E2 proteins are the only human papillomavirus (HPV) proteins required for transient replication of plasmids containing the viral origin. The E2 gene products play key roles in both viral transcription and replication. In this study we have analysed in further detail the nature of the association between E1 and E2 using a series of E2 proteins mutated in conserved regions of the N- terminal domain. These proteins were tested for their ability to activate transcription and to stimulate viral DNA replication. Several of these mutants revealed that the two functions of E2 can be separated, and that they define three widely spaced regions of the N-terminal domain which are important for DNA replication, two of which retain E1- binding activity. This suggests that E2 may have a role in viral DNA replication other than simply localizing E1 to the origin of replication. Additional important elements for regulating viral gene expression have been shown to be glucocorticoid hormones and epidermal growth factor (EGF). We show here that they may also be involved in regulating viral DNA replication. Our studies show that the addition of glucocorticoid hormone significantly stimulates viral DNA replication. In contrast, addition of EGF results in modest repression of viral DNA replication. These results have important implications for the pathogenesis of HPV infection and suggest that the relative levels of E2, glucocorticoid hormone and EGF may significantly affect the outcome of an HPV infection.
AndrophyE. J., LowyD. R., SchillerJ. T.1987; Bovine papillomavirus E2 trans-activating gene product binds to specific sites in the papillomavirus DNA. Nature 325:70–73
BakerC. C., PhelpsW. C., LindgrenV., BraunM. J., GondaM. A., HowleyP. M.1987; Structural and transcriptional analysis of human papillomavirus type 16 sequences in cervical carcinoma cell lines. Journal of Virology 61:962–971
BauknechtT., AngelP., RoyerH. -D., zur HausenH.1992; Identification of a negative regulatory domain in the human papillomavirus type 18 promoter: interaction with the transcriptional repressor YY1. EMBO Journal 11:4607–4617
BlitzI., LaiminsL. A.1991; The 68-kilodalton E1 protein of bovine papillomavirus is a DNA binding phosphoprotein which associates with the E2 transcription factor in vitro. Journal of Virology 65: 649–656
BouvardV., StoreyA., PimD., BanksL.1994a; Characterization of the human papillomavirus E2 protein: evidence of trans-activation and trans-repression in cervical keratinocytes. EMBO Journal 13:5451–5459
BouvardV., MatlashewskiG., GuZ. -M., StoreyA., BanksL.1994b; The human papillomavirus type 16 E5 gene cooperates with the E7 gene to stimulate proliferation of primary cells and increases viral gene expression. Virology 203:73–80
BreamG. L., OhmstedeC. A., PhelpsW. C.1993; Characterization of the human papillomavirus type 11 E1 and E2 proteins expressed in insect cells. Journal of Virology 67: 2655–2663
ChanW. -K., ChongT., BernardH. -U., KlockG.1990; Transcription of the transforming genes of the oncogenic human papillomavirus-16 is stimulated by tumor promoters through AP-1 binding sites. Nucleic Acids Research 18:763–769
ChiangC. -M., UstavA., StenlundA., HoT. F., BrokerT. R., ChowL. T.1992; Viral E1 and E2 proteins support replication of homologous and heterologous papillomaviral origins. Proceedings of the National Academy of Sciences USA: 89 5799–5803
ChongT., AptD., GlossB., IsaM., BernardH. -U.1991; The enhancer of human papillomavirus type 16 : binding sites for the ubiquitous transcription factors oct-1, NFA, TEF-2, NF1 and AP1 participate in epithelial cell-specific transcription. Journal of Virology 65:5933–5943
CripeT. P., HaugenT. H., TurkJ. P., TabatabaiF., SchmidP. G., DürstM., GissmannL., RomanA., TurekL.1987; Transcriptional regulation of the human papillomavirus-16 E6-E7 promoter by a keratinocyte-dependent enhancer, and by a viral E2 transactivator and repressor gene products: implications for cervical carcinogenesis. EMBO Journal 6:3745–3753
GilbertD. M., CohenS. N.1987; Bovine papilloma virus plasmids replicate randomly in mouse fibroblasts throughout S phase of the cell cycle. Cell 50:59–68
GiriI., YanivM.1988; Structural and mutational analysis of E2 transactivating proteins of papillomaviruses reveals three distinct functional domains. EMBO Journal 7:2823–2829
GlossB., BernardH. -U.1990; The E6/E7 promoter of human papillomavirus type 16 is activated in the absence of E2 proteins by a sequence-aberrant Sp1 distal element. Journal of Virology 64:5577–5584
GlossB., BernardH. -U., KlockG.1987; The upstream regulatory region of the human papillomavirus type 16 contains an E2 protein- independent enhancer which is specific for cervical carcinoma cells and regulated by glucocorticoid hormones. EMBO Journal 6: 3735–3743
HibmaM. H., RajK., ElyS. J., StanleyM., CrawfordL.1995; The interaction between human papillomavirus type 16 E1 and E2 proteins is blocked by an antibody to the N-terminal region of E2. European Journal of Biochemistry 229:517–525
LambertP. F., SpalholzB. A., HowleyP. M.1987; A transcriptional repressor encoded by BPV-1 shares a common carboxy-terminal domain with the E2 transactivator. Cell 50:69–78
LawM. F., LowyD. R., DvoretzkyI., HowleyP. M.1981; Mouse cells transformed by bovine papillomavirus contain only extrachromosomal viral DNA sequences. Proceedings of the National Academy of Sciences USA: 782727–2731
LeesE., OsbornK., BanksL., CrawfordL.1990; Transformation of primary BRK cells by human papillomavirus type 16 and EJ-ras is increased by overexpression of the viral E2 protein. Journal of General Virology 71:183–193
MatsukuraT., KoiS., SugaseM.1989; Both episomal and integrated forms of human papillomavirus type 16 are involved in invasive cervical cancers. Virology 172:63–72
PicciniA., StoreyA., MassimiP., BanksL.1995; Mutations in the human papillomavirus type 16 E2 protein identify multiple regions of the protein involved in binding to E1. Journal of General Virology 76:2909–2913
RemmM., BrainR., JenkinsJ. R.1992; The E2 binding sites determine the efficiency of replication for the origin of human papillomavirus type 18. Nucleic Acids Research 20:6015–6021
SakaiH., ToshiharuY., BensonJ. D., DowhanickJ. J., HowleyP. M.1996; Targeted mutagenesis of the human papillomavirus type 16 E2 transactivation domain reveals separable transcriptional activation and replication functions. Journal of Virology 70:1602–1611
SchwarzE., FreeseU. K., GissmannL., MayerW., RoggenbuckB., StremlauA., zur HausenH.1985; Structure and transcription of human papillomavirus sequences in cervical carcinoma cells. Nature 314:111–114
SeoY. -S., MüllerF., LuskyM., HurwitzJ.1993; Bovine papillomavirus (BPV)-encoded E1 protein contains multiple activities required for DNA replication. Proceedings of the National Academy of Sciences USA: 90702–706
SibbetG. J., CampoM. S.1990; Multiple interactions between cellular factors and the non-coding region of human papillomavirus type 16. Journal of General Virology 71:2699–2707
StoreyA., PicciniA., MassimiP., BouvardV., BanksL.1995; Mutations in the human papillomavirus type 16 E2 protein identify a region of the protein involved in binding to E1 protein. Journal of General Virology 76:819–826
SunS., ThornerL., LentzM., MacPhersonP., BotchanM.1990; Identification of a 68-kilodalton nuclear ATP-binding phosphoprotein encoded by bovine papillomavirus type 1. Journal of Virology 64:5093–5105
UshikaiM., LaceM. J., YamakawaY., KonoM., AnsonJ., IshijiT., ParkkinenS., WickerN., ValentineM. -E., DavidsonI., TurekL. P., HaugenT. 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. Journal of Virology 68: 6655–6666
UstavM., StenlundA.1991; Transient replication ofBPV-1 requires two viral polypeptides encoded by the E1 and E2 open reading frames. EMBO Journal 10:505–509
WilsonV. G., Ludes-MeyersJ.1991; A bovine papillomavirus E1- related protein binds specifically to bovine papillomavirus DNA. Journal of Virology 65: 5314–5322
ZuoF., MertzJ. E.1995; Simian virus 40 late gene expression is regulated by members of the steroid/thyroid hormone receptor superfamily. Proceedings of the National Academy of Sciences USA: 928586–8590
zur HausenH., SchneiderA.1987; The role of papillomaviruses in human anogenital cancer. In The Papovaviridae: The Papillomaviruses pp 245–263HowleyP. M., SalzmanN. Edited by New York: Plenum Press;