Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1996 Jul;70(7):4193–4199. doi: 10.1128/jvi.70.7.4193-4199.1996

Genetic analysis of the activation domain of bovine papillomavirus protein E2: its role in transcription and replication.

M K Ferguson 1, M R Botchan 1
PMCID: PMC190348  PMID: 8676438

Abstract

The bovine papillomavirus protein E2 serves dual functions in viral transcription and in the initiation of viral replication. As a transcription factor, E2 can cooperatively interact with cellular proteins such as SP1 and stimulate transcription of distal promoters. In replication, E2 and the helicase El are the only viral proteins required for accurate replication of templates containing the viral origin. The amino terminus of E2 is a functionally separable domain critical for activation of both replication and transcription; its primary sequence is conserved between many strains of papillomavirus. We targeted conserved residues spanning the activation domain and constructed a series of 30 amino acid substitution mutants. These mutant E2 genes were analyzed for the ability to activate DNA replication and gene expression in cells. The majority of the substitutions affected the ability of E2 to support both viral replication and transcriptional activation, revealing substantial overlap of the functional determinants for these two processes. Replication and transcription activities are genetically separable, however, as mutations at amino acids 73 and 74 retained replication function but failed to activate transcription. Additionally, a mutation at position 39 substantially reduced replication activity but left transcriptional activation intact. Interestingly, over two-thirds of the mutations analyzed reduced function and protein accumulation, many in a temperature-dependent manner. The correspondence between the replication and transcription phenotypes of mutations spanning the activation domain may indicate that the entire region is folded into a single domain required for both functions.

Full Text

The Full Text of this article is available as a PDF (374.2 KB).

Selected References

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

  1. Armentero M. T., Horwitz M., Mermod N. Targeting of DNA polymerase to the adenovirus origin of DNA replication by interaction with nuclear factor I. Proc Natl Acad Sci U S A. 1994 Nov 22;91(24):11537–11541. doi: 10.1073/pnas.91.24.11537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Benson J. D., Howley P. M. Amino-terminal domains of the bovine papillomavirus type 1 E1 and E2 proteins participate in complex formation. J Virol. 1995 Jul;69(7):4364–4372. doi: 10.1128/jvi.69.7.4364-4372.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brokaw J. L., Blanco M., McBride A. A. Amino acids critical for the functions of the bovine papillomavirus type 1 E2 transactivator. J Virol. 1996 Jan;70(1):23–29. doi: 10.1128/jvi.70.1.23-29.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chen M., Mermod N., Horwitz M. S. Protein-protein interactions between adenovirus DNA polymerase and nuclear factor I mediate formation of the DNA replication preinitiation complex. J Biol Chem. 1990 Oct 25;265(30):18634–18642. [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. Church G. M., Gilbert W. Genomic sequencing. Proc Natl Acad Sci U S A. 1984 Apr;81(7):1991–1995. doi: 10.1073/pnas.81.7.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Del Vecchio A. M., Romanczuk H., Howley P. M., Baker C. C. Transient replication of human papillomavirus DNAs. J Virol. 1992 Oct;66(10):5949–5958. doi: 10.1128/jvi.66.10.5949-5958.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. DiMaio D., Settleman J. Bovine papillomavirus mutant temperature sensitive for transformation, replication and transactivation. EMBO J. 1988 Apr;7(4):1197–1204. doi: 10.1002/j.1460-2075.1988.tb02931.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dutta A. Trans-plication factors? Curr Biol. 1993 Oct 1;3(10):709–712. doi: 10.1016/0960-9822(93)90076-z. [DOI] [PubMed] [Google Scholar]
  12. Giri I., Yaniv M. Structural and mutational analysis of E2 trans-activating proteins of papillomaviruses reveals three distinct functional domains. EMBO J. 1988 Sep;7(9):2823–2829. doi: 10.1002/j.1460-2075.1988.tb03138.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gorman C. M., Moffat L. F., Howard B. H. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982 Sep;2(9):1044–1051. doi: 10.1128/mcb.2.9.1044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Ham J., Steger G., Yaniv M. Cooperativity in vivo between the E2 transactivator and the TATA box binding protein depends on core promoter structure. EMBO J. 1994 Jan 1;13(1):147–157. doi: 10.1002/j.1460-2075.1994.tb06244.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hanawalt P. C. Transcription-coupled repair and human disease. Science. 1994 Dec 23;266(5193):1957–1958. doi: 10.1126/science.7801121. [DOI] [PubMed] [Google Scholar]
  16. Haugen T. H., Turek L. P., Mercurio F. M., Cripe T. P., Olson B. J., Anderson R. D., Seidl D., Karin M., Schiller J. Sequence-specific and general transcriptional activation by the bovine papillomavirus-1 E2 trans-activator require an N-terminal amphipathic helix-containing E2 domain. EMBO J. 1988 Dec 20;7(13):4245–4253. doi: 10.1002/j.1460-2075.1988.tb03322.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hegde R. S., Grossman S. R., Laimins L. A., Sigler P. B. Crystal structure at 1.7 A of the bovine papillomavirus-1 E2 DNA-binding domain bound to its DNA target. Nature. 1992 Oct 8;359(6395):505–512. doi: 10.1038/359505a0. [DOI] [PubMed] [Google Scholar]
  18. Heike T., Miyatake S., Yoshida M., Arai K., Arai N. Bovine papilloma virus encoded E2 protein activates lymphokine genes through DNA elements, distinct from the consensus motif, in the long control region of its own genome. EMBO J. 1989 May;8(5):1411–1417. doi: 10.1002/j.1460-2075.1989.tb03522.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Heintz N. H. Transcription factors and the control of DNA replication. Curr Opin Cell Biol. 1992 Jun;4(3):459–467. doi: 10.1016/0955-0674(92)90012-2. [DOI] [PubMed] [Google Scholar]
  20. Lambert P. F., Dostatni N., McBride A. A., Yaniv M., Howley P. M., Arcangioli B. Functional analysis of the papilloma virus E2 trans-activator in Saccharomyces cerevisiae. Genes Dev. 1989 Jan;3(1):38–48. doi: 10.1101/gad.3.1.38. [DOI] [PubMed] [Google Scholar]
  21. Li R., Botchan M. R. Acidic transcription factors alleviate nucleosome-mediated repression of DNA replication of bovine papillomavirus type 1. Proc Natl Acad Sci U S A. 1994 Jul 19;91(15):7051–7055. doi: 10.1073/pnas.91.15.7051. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Li R., Botchan M. R. The acidic transcriptional activation domains of VP16 and p53 bind the cellular replication protein A and stimulate in vitro BPV-1 DNA replication. Cell. 1993 Jun 18;73(6):1207–1221. doi: 10.1016/0092-8674(93)90649-b. [DOI] [PubMed] [Google Scholar]
  23. Li R., Knight J. D., Jackson S. P., Tjian R., Botchan M. R. Direct interaction between Sp1 and the BPV enhancer E2 protein mediates synergistic activation of transcription. Cell. 1991 May 3;65(3):493–505. doi: 10.1016/0092-8674(91)90467-d. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Mohr I. J., Clark R., Sun S., Androphy E. J., MacPherson P., Botchan M. R. Targeting the E1 replication protein to the papillomavirus origin of replication by complex formation with the E2 transactivator. Science. 1990 Dec 21;250(4988):1694–1699. doi: 10.1126/science.2176744. [DOI] [PubMed] [Google Scholar]
  26. Myers R. M., Lerman L. S., Maniatis T. A general method for saturation mutagenesis of cloned DNA fragments. Science. 1985 Jul 19;229(4710):242–247. doi: 10.1126/science.2990046. [DOI] [PubMed] [Google Scholar]
  27. Park P., Copeland W., Yang L., Wang T., Botchan M. R., Mohr I. J. The cellular DNA polymerase alpha-primase is required for papillomavirus DNA replication and associates with the viral E1 helicase. Proc Natl Acad Sci U S A. 1994 Aug 30;91(18):8700–8704. doi: 10.1073/pnas.91.18.8700. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Rank N. M., Lambert P. F. Bovine papillomavirus type 1 E2 transcriptional regulators directly bind two cellular transcription factors, TFIID and TFIIB. J Virol. 1995 Oct;69(10):6323–6334. doi: 10.1128/jvi.69.10.6323-6334.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Russell J., Botchan M. R. cis-Acting components of human papillomavirus (HPV) DNA replication: linker substitution analysis of the HPV type 11 origin. J Virol. 1995 Feb;69(2):651–660. doi: 10.1128/jvi.69.2.651-660.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sakai H., Yasugi T., Benson J. D., Dowhanick J. J., Howley P. M. Targeted mutagenesis of the human papillomavirus type 16 E2 transactivation domain reveals separable transcriptional activation and DNA replication functions. J Virol. 1996 Mar;70(3):1602–1611. doi: 10.1128/jvi.70.3.1602-1611.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Sedman J., Stenlund A. Co-operative interaction between the initiator E1 and the transcriptional activator E2 is required for replicator specific DNA replication of bovine papillomavirus in vivo and in vitro. EMBO J. 1995 Dec 15;14(24):6218–6228. doi: 10.1002/j.1460-2075.1995.tb00312.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Seo Y. S., Müller F., Lusky M., Gibbs E., Kim H. Y., Phillips B., Hurwitz J. Bovine papilloma virus (BPV)-encoded E2 protein enhances binding of E1 protein to the BPV replication origin. Proc Natl Acad Sci U S A. 1993 Apr 1;90(7):2865–2869. doi: 10.1073/pnas.90.7.2865. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Skiadopoulos M. H., McBride A. A. The bovine papillomavirus type 1 E2 transactivator and repressor proteins use different nuclear localization signals. J Virol. 1996 Feb;70(2):1117–1124. doi: 10.1128/jvi.70.2.1117-1124.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Spalholz B. A., McBride A. A., Sarafi T., Quintero J. Binding of bovine papillomavirus E1 to the origin is not sufficient for DNA replication. Virology. 1993 Mar;193(1):201–212. doi: 10.1006/viro.1993.1116. [DOI] [PubMed] [Google Scholar]
  35. Steger G., Ham J., Lefebvre O., Yaniv M. The bovine papillomavirus 1 E2 protein contains two activation domains: one that interacts with TBP and another that functions after TBP binding. EMBO J. 1995 Jan 16;14(2):329–340. doi: 10.1002/j.1460-2075.1995.tb07007.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Stenlund A., Botchan M. R. The E2 trans-activator can act as a repressor by interfering with a cellular transcription factor. Genes Dev. 1990 Jan;4(1):123–136. doi: 10.1101/gad.4.1.123. [DOI] [PubMed] [Google Scholar]
  37. Tanaka M., Herr W. Differential transcriptional activation by Oct-1 and Oct-2: interdependent activation domains induce Oct-2 phosphorylation. Cell. 1990 Feb 9;60(3):375–386. doi: 10.1016/0092-8674(90)90589-7. [DOI] [PubMed] [Google Scholar]
  38. Thorner L. K., Lim D. A., Botchan M. R. DNA-binding domain of bovine papillomavirus type 1 E1 helicase: structural and functional aspects. J Virol. 1993 Oct;67(10):6000–6014. doi: 10.1128/jvi.67.10.6000-6014.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Triezenberg S. J. Structure and function of transcriptional activation domains. Curr Opin Genet Dev. 1995 Apr;5(2):190–196. doi: 10.1016/0959-437x(95)80007-7. [DOI] [PubMed] [Google Scholar]
  40. Ushikai M., Lace M. J., Yamakawa Y., Kono M., Anson J., Ishiji T., Parkkinen S., Wicker N., Valentine M. E., Davidson I. 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. 1994 Oct;68(10):6655–6666. doi: 10.1128/jvi.68.10.6655-6666.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Ustav E., Ustav M., Szymanski P., Stenlund A. The bovine papillomavirus origin of replication requires a binding site for the E2 transcriptional activator. Proc Natl Acad Sci U S A. 1993 Feb 1;90(3):898–902. doi: 10.1073/pnas.90.3.898. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. 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]
  43. Winokur P. L., McBride A. A. Separation of the transcriptional activation and replication functions of the bovine papillomavirus-1 E2 protein. EMBO J. 1992 Nov;11(11):4111–4118. doi: 10.1002/j.1460-2075.1992.tb05504.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. 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]
  45. Yang L., Mohr I., Li R., Nottoli T., Sun S., Botchan M. Transcription factor E2 regulates BPV-1 DNA replication in vitro by direct protein-protein interaction. Cold Spring Harb Symp Quant Biol. 1991;56:335–346. doi: 10.1101/sqb.1991.056.01.040. [DOI] [PubMed] [Google Scholar]

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

RESOURCES