Skip to main content
NCBI home page
Journal of Virology logoLink to Journal of Virology
. 1996 Jan;70(1):23–29. doi: 10.1128/jvi.70.1.23-29.1996

Amino acids critical for the functions of the bovine papillomavirus type 1 E2 transactivator.

J L Brokaw 1, M Blanco 1, A A McBride 1
PMCID: PMC189783  PMID: 8523530

Abstract

The N-terminal domain of the bovine papillomavirus type 1 E2 protein is important for viral DNA replication, for transcriptional transactivation, and for interaction with the E1 protein. To determine which residues of this 200-amino-acid domain are important for these activities, single conservative amino acid substitutions have been generated in 17 residues that are invariant among all papillomavirus E2 proteins. The resulting mutated E2 proteins were tested for the ability to support viral DNA replication, activate transcription, and cooperatively bind to the origin of replication with the E1 protein. We identified five mutated proteins that were completely defective for transcriptional activation and either were defective or could support viral DNA replication at only low levels. However, several of these proteins could still interact efficiently with the E1 protein. In addition, we identified several mutated proteins that were unable to efficiently cooperatively bind to the origin with the E1 protein. Although a number of the mutated proteins demonstrated wild-type activity in all of the functions tested, only 3 out of 17 mutated viral genomes were able to induce foci in a C127 focus formation assay when the mutations were generated in the background of the entire bovine papillomavirus type 1 genome. This finding suggests that the E2 protein may have additional activities that are important for the viral life cycle.

Full Text

The Full Text of this article is available as a PDF (321.7 KB).

Selected References

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

  1. Chan S. Y., Delius H., Halpern A. L., Bernard H. U. Analysis of genomic sequences of 95 papillomavirus types: uniting typing, phylogeny, and taxonomy. J Virol. 1995 May;69(5):3074–3083. doi: 10.1128/jvi.69.5.3074-3083.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. Deng W. P., Nickoloff J. A. Site-directed mutagenesis of virtually any plasmid by eliminating a unique site. Anal Biochem. 1992 Jan;200(1):81–88. doi: 10.1016/0003-2697(92)90280-k. [DOI] [PubMed] [Google Scholar]
  4. DiMaio D. Nonsense mutation in open reading frame E2 of bovine papillomavirus DNA. J Virol. 1986 Feb;57(2):475–480. doi: 10.1128/jvi.57.2.475-480.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. 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]
  7. Dvoretzky I., Shober R., Chattopadhyay S. K., Lowy D. R. A quantitative in vitro focus assay for bovine papilloma virus. Virology. 1980 Jun;103(2):369–375. doi: 10.1016/0042-6822(80)90195-6. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. 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]
  10. Ham J., Dostatni N., Gauthier J. M., Yaniv M. The papillomavirus E2 protein: a factor with many talents. Trends Biochem Sci. 1991 Nov;16(11):440–444. doi: 10.1016/0968-0004(91)90172-r. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Hermonat P. L., Howley P. M. Mutational analysis of the 3' open reading frames and the splice junction at nucleotide 3225 of bovine papillomavirus type 1. J Virol. 1987 Dec;61(12):3889–3895. doi: 10.1128/jvi.61.12.3889-3895.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hubbert N. L., Schiller J. T., Lowy D. R., Androphy E. J. Bovine papilloma virus-transformed cells contain multiple E2 proteins. Proc Natl Acad Sci U S A. 1988 Aug;85(16):5864–5868. doi: 10.1073/pnas.85.16.5864. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Lambert P. F., Hubbert N. L., Howley P. M., Schiller J. T. Genetic assignment of multiple E2 gene products in bovine papillomavirus-transformed cells. J Virol. 1989 Jul;63(7):3151–3154. doi: 10.1128/jvi.63.7.3151-3154.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lambert P. F., Spalholz B. A., Howley P. M. A transcriptional repressor encoded by BPV-1 shares a common carboxy-terminal domain with the E2 transactivator. Cell. 1987 Jul 3;50(1):69–78. doi: 10.1016/0092-8674(87)90663-5. [DOI] [PubMed] [Google Scholar]
  17. Law M. F., Lowy D. R., Dvoretzky I., Howley P. M. Mouse cells transformed by bovine papillomavirus contain only extrachromosomal viral DNA sequences. Proc Natl Acad Sci U S A. 1981 May;78(5):2727–2731. doi: 10.1073/pnas.78.5.2727. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Le Moal M. A., Yaniv M., Thierry F. The bovine papillomavirus type 1 (BPV1) replication protein E1 modulates transcriptional activation by interacting with BPV1 E2. J Virol. 1994 Feb;68(2):1085–1093. doi: 10.1128/jvi.68.2.1085-1093.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. 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]
  22. McBride A. A., Romanczuk H., Howley P. M. The papillomavirus E2 regulatory proteins. J Biol Chem. 1991 Oct 5;266(28):18411–18414. [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. 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]
  26. Sandler A. B., Vande Pol S. B., Spalholz B. A. Repression of bovine papillomavirus type 1 transcription by the E1 replication protein. J Virol. 1993 Sep;67(9):5079–5087. doi: 10.1128/jvi.67.9.5079-5087.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sarver N., Byrne J. C., Howley P. M. Transformation and replication in mouse cells of a bovine papillomavirus--pML2 plasmid vector that can be rescued in bacteria. Proc Natl Acad Sci U S A. 1982 Dec;79(23):7147–7151. doi: 10.1073/pnas.79.23.7147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. 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]
  30. Spalholz B. A., Yang Y. C., Howley P. M. Transactivation of a bovine papilloma virus transcriptional regulatory element by the E2 gene product. Cell. 1985 Aug;42(1):183–191. doi: 10.1016/s0092-8674(85)80114-8. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. 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]
  33. 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]
  34. 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]
  35. 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]
  36. 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]
  37. 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]
  38. Yang L., Mohr I., Fouts E., Lim D. A., Nohaile M., Botchan M. The E1 protein of bovine papilloma virus 1 is an ATP-dependent DNA helicase. Proc Natl Acad Sci U S A. 1993 Jun 1;90(11):5086–5090. doi: 10.1073/pnas.90.11.5086. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Yang Y. C., Okayama H., Howley P. M. Bovine papillomavirus contains multiple transforming genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1030–1034. doi: 10.1073/pnas.82.4.1030. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES