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. 1996 Sep;70(9):6169–6179. doi: 10.1128/jvi.70.9.6169-6179.1996

Transcriptional and replicational activation functions in the bovine papillomavirus type 1 E2 protein are encoded by different structural determinants.

A Abroi 1, R Kurg 1, M Ustav 1
PMCID: PMC190641  PMID: 8709243

Abstract

A set of E2 proteins with mutations in the amino-terminal transactivation domain was made by a scheme called clustered charged-to-alanine scan. These mutant E2 proteins were tested for expression, stability, and compartmentalization in cells and for sequence-specific DNA binding, as well as in functional assays for transcriptional and replicational activation. We identified four groups of mutants. First, mutants K111A, K112A, and E176A were unable to activate replication and transcription because of oligomerization-induced retention of oligomers in the cytoplasm. Second, although fractions of the mutant proteins E74A and D143A/ R172C existed in the oligomeric form, they were localized in the nucleus. Certain fractions of these proteins existed as a dimer able to form a specific complex and activate replication; however, these proteins were inactive in transcriptional activation. Third, mutants R37A and D122A were localized in the nucleus, existed in the dimeric form, supported replication efficiently, and were severely crippled in transcriptional activation. The fourth group of mutants did not differ considerably from the wild-type protein. The activation of transcription by the wild type as well as mutant E2 proteins was dependent on the concentration of input E2 expression vector DNA and had a bell-like shape. We suggest that the reduction of transcriptional activation at higher E2 concentrations, the self-squelching activity, is caused by oligomerization of the E2 transactivator and is one of the mechanisms for the regulation of E2 activity. Our results also show that transcriptional and replicational activation activities are encoded by different determinants in the E2 protein.

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Selected References

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  1. Bass S. H., Mulkerrin M. G., Wells J. A. A systematic mutational analysis of hormone-binding determinants in the human growth hormone receptor. Proc Natl Acad Sci U S A. 1991 May 15;88(10):4498–4502. doi: 10.1073/pnas.88.10.4498. [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. Blitz I. L., Laimins L. A. The 68-kilodalton E1 protein of bovine papillomavirus is a DNA binding phosphoprotein which associates with the E2 transcriptional activator in vitro. J Virol. 1991 Feb;65(2):649–656. doi: 10.1128/jvi.65.2.649-656.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bochkarev A., Barwell J. A., Pfuetzner R. A., Furey W., Jr, Edwards A. M., Frappier L. Crystal structure of the DNA-binding domain of the Epstein-Barr virus origin-binding protein EBNA 1. Cell. 1995 Oct 6;83(1):39–46. doi: 10.1016/0092-8674(95)90232-5. [DOI] [PubMed] [Google Scholar]
  5. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Burnett S., Ström A. C., Jareborg N., Alderborn A., Dillner J., Moreno-Lopez J., Pettersson U., Kiessling U. Induction of bovine papillomavirus E2 gene expression and early region transcription by cell growth arrest: correlation with viral DNA amplification and evidence for differential promoter induction. J Virol. 1990 Nov;64(11):5529–5541. doi: 10.1128/jvi.64.11.5529-5541.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Corina K., Grossman S. R., Barsoum J., Prakash S. S., Androphy E. J., Pepinsky R. B. The tryptophan bridge is a critical feature of the papillomavirus E2 DNA binding domain. Virology. 1993 Nov;197(1):391–396. doi: 10.1006/viro.1993.1600. [DOI] [PubMed] [Google Scholar]
  10. Cunningham B. C., Wells J. A. High-resolution epitope mapping of hGH-receptor interactions by alanine-scanning mutagenesis. Science. 1989 Jun 2;244(4908):1081–1085. doi: 10.1126/science.2471267. [DOI] [PubMed] [Google Scholar]
  11. DePamphili M. L. How transcription factors regulate origins of DNA replication in eukaryotic cells. Trends Cell Biol. 1993 May;3(5):161–167. doi: 10.1016/0962-8924(93)90137-p. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Dostatni N., Thierry F., Yaniv M. A dimer of BPV-1 E2 containing a protease resistant core interacts with its DNA target. EMBO J. 1988 Dec 1;7(12):3807–3816. doi: 10.1002/j.1460-2075.1988.tb03265.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Hahn S. Structure(?) and function of acidic transcription activators. Cell. 1993 Feb 26;72(4):481–483. doi: 10.1016/0092-8674(93)90064-w. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  18. 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]
  19. Hermonat P. L., Spalholz B. A., Howley P. M. The bovine papillomavirus P2443 promoter is E2 trans-responsive: evidence for E2 autoregulation. EMBO J. 1988 Sep;7(9):2815–2822. doi: 10.1002/j.1460-2075.1988.tb03137.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Knight J. D., Li R., Botchan M. The activation domain of the bovine papillomavirus E2 protein mediates association of DNA-bound dimers to form DNA loops. Proc Natl Acad Sci U S A. 1991 Apr 15;88(8):3204–3208. doi: 10.1073/pnas.88.8.3204. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Leuther K. K., Salmeron J. M., Johnston S. A. Genetic evidence that an activation domain of GAL4 does not require acidity and may form a beta sheet. Cell. 1993 Feb 26;72(4):575–585. doi: 10.1016/0092-8674(93)90076-3. [DOI] [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. Lusky M., Botchan M. R. Transient replication of bovine papilloma virus type 1 plasmids: cis and trans requirements. Proc Natl Acad Sci U S A. 1986 Jun;83(11):3609–3613. doi: 10.1073/pnas.83.11.3609. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lusky M., Fontane E. Formation of the complex of bovine papillomavirus E1 and E2 proteins is modulated by E2 phosphorylation and depends upon sequences within the carboxyl terminus of E1. Proc Natl Acad Sci U S A. 1991 Jul 15;88(14):6363–6367. doi: 10.1073/pnas.88.14.6363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lusky M., Hurwitz J., Seo Y. S. The bovine papillomavirus E2 protein modulates the assembly of but is not stably maintained in a replication-competent multimeric E1-replication origin complex. Proc Natl Acad Sci U S A. 1994 Sep 13;91(19):8895–8899. doi: 10.1073/pnas.91.19.8895. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Maldonado E., Reinberg D. News on initiation and elongation of transcription by RNA polymerase II. Curr Opin Cell Biol. 1995 Jun;7(3):352–361. doi: 10.1016/0955-0674(95)80090-5. [DOI] [PubMed] [Google Scholar]
  28. McBride A. A., Bolen J. B., Howley P. M. Phosphorylation sites of the E2 transcriptional regulatory proteins of bovine papillomavirus type 1. J Virol. 1989 Dec;63(12):5076–5085. doi: 10.1128/jvi.63.12.5076-5085.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Mikaelian I., Sergeant A. A general and fast method to generate multiple site directed mutations. Nucleic Acids Res. 1992 Jan 25;20(2):376–376. doi: 10.1093/nar/20.2.376. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. Monini P., Grossman S. R., Pepinsky B., Androphy E. J., Laimins L. A. Cooperative binding of the E2 protein of bovine papillomavirus to adjacent E2-responsive sequences. J Virol. 1991 Apr;65(4):2124–2130. doi: 10.1128/jvi.65.4.2124-2130.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Morrissey L. C., Barsoum J., Androphy E. J. Trans activation by the bovine papillomavirus E2 protein in Saccharomyces cerevisiae. J Virol. 1989 Oct;63(10):4422–4425. doi: 10.1128/jvi.63.10.4422-4425.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Piirsoo M., Ustav E., Mandel T., Stenlund A., Ustav M. Cis and trans requirements for stable episomal maintenance of the BPV-1 replicator. EMBO J. 1996 Jan 2;15(1):1–11. [PMC free article] [PubMed] [Google Scholar]
  35. Rabson M. S., Yee C., Yang Y. C., Howley P. M. Bovine papillomavirus type 1 3' early region transformation and plasmid maintenance functions. J Virol. 1986 Nov;60(2):626–634. doi: 10.1128/jvi.60.2.626-634.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. Remm M., Brain R., Jenkins J. R. The E2 binding sites determine the efficiency of replication for the origin of human papillomavirus type 18. Nucleic Acids Res. 1992 Nov 25;20(22):6015–6021. doi: 10.1093/nar/20.22.6015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Rost B., Sander C. Prediction of protein secondary structure at better than 70% accuracy. J Mol Biol. 1993 Jul 20;232(2):584–599. doi: 10.1006/jmbi.1993.1413. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. 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]
  41. 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]
  42. 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]
  43. Spalholz B. A., Lambert P. F., Yee C. L., Howley P. M. Bovine papillomavirus transcriptional regulation: localization of the E2-responsive elements of the long control region. J Virol. 1987 Jul;61(7):2128–2137. doi: 10.1128/jvi.61.7.2128-2137.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. 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]
  45. Stanway C. A., Sowden M. P., Wilson L. E., Kingsman A. J., Kingsman S. M. Efficient activation of transcription in yeast by the BPV1 E2 protein. Nucleic Acids Res. 1989 Mar 25;17(6):2187–2196. doi: 10.1093/nar/17.6.2187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. 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]
  47. Stenlund A., Bream G. L., Botchan M. R. A promoter with an internal regulatory domain is part of the origin of replication in BPV-1. Science. 1987 Jun 26;236(4809):1666–1671. doi: 10.1126/science.3037693. [DOI] [PubMed] [Google Scholar]
  48. 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]
  49. 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]
  50. 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]
  51. 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]
  52. 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]
  53. Ustav M., Ustav E., Szymanski P., Stenlund A. Identification of the origin of replication of bovine papillomavirus and characterization of the viral origin recognition factor E1. EMBO J. 1991 Dec;10(13):4321–4329. doi: 10.1002/j.1460-2075.1991.tb05010.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. 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]
  55. 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]
  56. Yang Y. C., Spalholz B. A., Rabson M. S., Howley P. M. Dissociation of transforming and trans-activation functions for bovine papillomavirus type 1. Nature. 1985 Dec 12;318(6046):575–577. doi: 10.1038/318575a0. [DOI] [PubMed] [Google Scholar]
  57. Zawel L., Reinberg D. Common themes in assembly and function of eukaryotic transcription complexes. Annu Rev Biochem. 1995;64:533–561. doi: 10.1146/annurev.bi.64.070195.002533. [DOI] [PubMed] [Google Scholar]

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