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Journal of Virology, May 2009, p. 4127-4139, Vol. 83, No. 9
0022-538X/09/$08.00+0     doi:10.1128/JVI.02468-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Proteasomal Degradation of the Papillomavirus E2 Protein Is Inhibited by Overexpression of Bromodomain-Containing Protein 4{triangledown} ,{ddagger}

David Gagnon,{dagger} Simon Joubert,{dagger} Hélène Sénéchal, Amélie Fradet-Turcotte, Sabrina Torre, and Jacques Archambault*

Laboratory of Molecular Virology, Institut de Recherches Cliniques de Montréal, and Department of Biochemistry, University of Montreal, Montreal, Quebec H2W 1R7, Canada

Received 1 December 2008/ Accepted 3 February 2009

The E2 protein of human papillomavirus (HPV) binds to specific sites in the viral genome to regulate its transcription, replication, and maintenance in infected cells. Like most regulatory proteins, E2 is rapidly turned over. A high-throughput assay was developed to quantify the expression and stability of E2 in vivo, based on its fusion to Renilla luciferase (RLuc). The steady-state levels of Rluc-E2 were quantified by measuring the amounts of associated luciferase activity, and its degradation was measured by monitoring the decrease in enzymatic activity occurring after a block of translation with cycloheximide. Using this assay, the E2 proteins from a low-risk (HPV11) and a high-risk (HPV31) human papillomavirus (HPV) type were found to have short half-lives of 60 min in C33A cervical carcinoma cells and to be ubiquitinated and degraded by the proteasome. Analysis of mutant proteins showed that the instability of E2 is independent of its DNA-binding and transcriptional activities but is encoded within its transactivation domain, the region that binds to the cellular chromatin factor bromodomain-containing protein 4 (Brd4) to regulate viral gene transcription. Overexpression of Brd4, or of its C-terminal E2-interaction domain, was found to increase the steady-state levels and stability of wild-type E2 but not of E2 mutants defective for binding Brd4. These results indicate that the stability of E2 is increased upon complex formation with Brd4 and highlight the value of the luciferase assay for the study of E2 degradation.

* Corresponding author. Mailing address: Laboratory of Molecular Virology, Institut de Recherches Cliniques de Montréal, 110 Pine Avenue West, Montreal, Quebec H2W 1R7, Canada. Phone: (514) 987-5739. Fax: (514) 987-5741. E-mail: jacques.archambault{at}ircm.qc.ca

{triangledown} Published ahead of print on 11 February 2009.

{ddagger} Supplemental material for this article may be found at http://jvi.asm.org/.

{dagger} D.G. and S.J. contributed equally to this study.

Journal of Virology, May 2009, p. 4127-4139, Vol. 83, No. 9
0022-538X/09/$08.00+0     doi:10.1128/JVI.02468-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Zheng, G., Schweiger, M.-R., Martinez-Noel, G., Zheng, L., Smith, J. A., Harper, J. W., Howley, P. M. (2009). Brd4 Regulation of Papillomavirus Protein E2 Stability. J. Virol. 83: 8683-8692 [Abstract] [Full Text]  


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