Papillomavirus E2 is a multifunctional viral protein that regulates many aspects of the viral life cycle including viral episome maintenance, transcriptional activation, and repression. E2 is degraded by the ubiquitin-proteasome pathway. Cellular bromodomain protein Brd4 has been implicated in the stabilization of the E2 protein. E2 normally shuttles between the cytoplasm and the nucleus. In this study, we demonstrate that E2 ubiquitylation mostly occurs in the cytoplasm. We also find that the interaction with Brd4 promotes nuclear retention of papillomavirus E2 proteins and contributes to their stabilization in the nucleus. Compared to wild type E2 proteins, nuclear-localization-defective mutants are rapidly degraded by the ubiquitin-proteasome pathway; however, co-expression of Brd4 redirects these mutants into the nucleus and significantly increases their stability. We further demonstrate that tethering E2 proteins to chromatin as either double-bromodomain fusion proteins or histone 2B (H2B) fusion proteins significantly stabilizes the E2 proteins. Our studies suggest that chromatin recruitment of the E2 protein via interaction with Brd4 prevents E2 ubiquitylation and proteasomal degradation in the cytoplasm, leading to its stabilization in the nucleus. These studies bring new insights for understanding Brd4-mediated E2 stabilization, and provide an additional mechanism by which the chromatin-associated Brd4 regulates E2 functions.
References
[1]
Howley, P.M.; Lowy, D.R. Papillomaviruses and their replication. In Filelds Virology, 4th ed.; Knipe, D.M., Howley, P.M., Eds.; Lippincott Williams & Wilkins: Philadelphia, PA, USA, 2001; Volume 2, p. 2197.
[2]
Zur Hausen, H. Papillomaviruses and cancer: From basic studies to clinical application. Nat. Rev. Cancer 2002, 2, 342–350, doi:10.1038/nrc798.
[3]
Hebner, C.M.; Laimins, L.A. Human papillomaviruses: Basic mechanisms of pathogenesis and oncogenicity. Rev. Med. Virol. 2006, 16, 83–97, doi:10.1002/rmv.488.
[4]
McBride, A.A.; Romanczuk, H.; Howley, P.M. The papillomavirus E2 regulatory proteins. J. Biol. Chem. 1991, 266, 18411–18414.
[5]
Penrose, K.J.; McBride, A.A. Proteasome-mediated degradation of the papillomavirus E2-TA protein is regulated by phosphorylation and can modulate viral genome copy number. J. Virol. 2000, 74, 6031–6038, doi:10.1128/JVI.74.13.6031-6038.2000.
[6]
Bellanger, S.; Demeret, C.; Goyat, S.; Thierry, F. Stability of the human papillomavirus type 18 E2 protein is regulated by a proteasome degradation pathway through its amino-terminal transactivation domain. J. Virol. 2001, 75, 7244–7251, doi:10.1128/JVI.75.16.7244-7251.2001.
[7]
Penrose, K.J.; Garcia-Alai, M.; de Prat-Gay, G.; McBride, A.A. Casein Kinase II phosphorylation-induced conformational switch triggers degradation of the papillomavirus E2 protein. J. Biol. Chem. 2004, 279, 22430–22439.
Zheng, G.; Schweiger, M.R.; Martinez-Noel, G.; Zheng, L.; Smith, J.A.; Harper, J.W.; Howley, P.M. Brd4 regulation of papillomavirus protein E2 stability. J. Virol. 2009, 83, 8683–8692, doi:10.1128/JVI.00674-09.
[10]
Bellanger, S.; Tan, C.L.; Nei, W.; He, P.P.; Thierry, F. The human papillomavirus type 18 E2 protein is a cell cycle-dependent target of the SCFSkp2 ubiquitin ligase. J. Virol. 2010, 84, 437–444, doi:10.1128/JVI.01162-09.
[11]
You, J.; Croyle, J.L.; Nishimura, A.; Ozato, K.; Howley, P.M. Interaction of the bovine papillomavirus E2 protein with Brd4 tethers the viral DNA to host mitotic chromosomes. Cell 2004, 117, 349–360, doi:10.1016/S0092-8674(04)00402-7.
[12]
Dey, A.; Ellenberg, J.; Farina, A.; Coleman, A.E.; Maruyama, T.; Sciortino, S.; Lippincott-Schwartz, J.; Ozato, K. A bromodomain protein, MCAP, associates with mitotic chromosomes and affects G(2)-to-M transition. Mol. Cell. Biol. 2000, 20, 6537–6549, doi:10.1128/MCB.20.17.6537-6549.2000.
[13]
Dey, A.; Chitsaz, F.; Abbasi, A.; Misteli, T.; Ozato, K. The double bromodomain protein Brd4 binds to acetylated chromatin during interphase and mitosis. Proc. Natl. Acad. Sci. USA 2003, 100, 8758–8763, doi:10.1073/pnas.1433065100.
[14]
Abbate, E.A.; Voitenleitner, C.; Botchan, M.R. Structure of the papillomavirus DNA-tethering complex E2:Brd4 and a peptide that ablates HPV chromosomal association. Mol. Cell 2006, 24, 877–889, doi:10.1016/j.molcel.2006.11.002.
[15]
Baxter, M.K.; McPhillips, M.G.; Ozato, K.; McBride, A.A. The mitotic chromosome binding activity of the papillomavirus E2 protein correlates with interaction with the cellular chromosomal protein, Brd4. J. Virol. 2005, 79, 4806–4818, doi:10.1128/JVI.79.8.4806-4818.2005.
[16]
Brannon, A.R.; Maresca, J.A.; Boeke, J.D.; Basrai, M.A.; McBride, A.A. Reconstitution of papillomavirus E2-mediated plasmid maintenance in Saccharomyces cerevisiae by the Brd4 bromodomain protein. Proc. Natl. Acad. Sci. USA 2005, 102, 2998–3003, doi:10.1073/pnas.0407818102.
[17]
Ilves, I.; Maemets, K.; Silla, T.; Janikson, K.; Ustav, M. Brd4 is involved in multiple processes of the bovine papillomavirus type 1 life cycle. J. Virol. 2006, 80, 3660–3665, doi:10.1128/JVI.80.7.3660-3665.2006.
[18]
McPhillips, M.G.; Ozato, K.; McBride, A.A. Interaction of bovine papillomavirus E2 protein with Brd4 stabilizes its association with chromatin. J. Virol. 2005, 79, 8920–8932, doi:10.1128/JVI.79.14.8920-8932.2005.
[19]
McPhillips, M.G.; Oliveira, J.G.; Spindler, J.E.; Mitra, R.; McBride, A.A. Brd4 is required for e2-mediated transcriptional activation but not genome partitioning of all papillomaviruses. J. Virol. 2006, 80, 9530–9543, doi:10.1128/JVI.01105-06.
[20]
Schweiger, M.R.; You, J.; Howley, P.M. Bromodomain protein 4 mediates the papillomavirus E2 transcriptional activation function. J. Virol. 2006, 80, 4276–4285, doi:10.1128/JVI.80.9.4276-4285.2006.
[21]
Wu, S.Y.; Chiang, C.M. The double bromodomain-containing chromatin adaptor Brd4 and transcriptional regulation. J. Biol. Chem. 2007, 282, 13141–13145, doi:10.1074/jbc.R700001200.
Yan, J.; Li, Q.; Lievens, S.; Tavernier, J.; You, J. Abrogation of the Brd4-positive transcription elongation factor B complex by papillomavirus E2 protein contributes to viral oncogene repression. J. Virol. 2010, 84, 76–87, doi:10.1128/JVI.01647-09.
[24]
You, J.; Schweiger, M.R.; Howley, P.M. Inhibition of E2 binding to Brd4 enhances viral genome loss and phenotypic reversion of bovine papillomavirus-transformed cells. J. Virol. 2005, 79, 14956–14961, doi:10.1128/JVI.79.23.14956-14961.2005.
[25]
Lee, A.Y.; Chiang, C.M. Chromatin adaptor Brd4 modulates E2 transcription activity and protein stability. J. Biol. Chem. 2009, 284, 2778–2786.
[26]
Gagnon, D.; Joubert, S.; Senechal, H.; Fradet-Turcotte, A.; Torre, S.; Archambault, J. Proteasomal degradation of the papillomavirus E2 protein is inhibited by overexpression of bromodomain-containing protein 4. J. Virol. 2009, 83, 4127–4139, doi:10.1128/JVI.02468-08.
[27]
Blachon, S.; Bellanger, S.; Demeret, C.; Thierry, F. Nucleo-cytoplasmic shuttling of high risk human Papillomavirus E2 proteins induces apoptosis. J. Biol. Chem. 2005, 280, 36088–36098, doi:10.1074/jbc.M505138200.
[28]
Klucevsek, K.; Wertz, M.; Lucchi, J.; Leszczynski, A.; Moroianu, J. Characterization of the nuclear localization signal of high risk HPV16 E2 protein. Virology 2007, 360, 191–198, doi:10.1016/j.virol.2006.10.018.
[29]
Mochizuki, K.; Nishiyama, A.; Jang, M.K.; Dey, A.; Ghosh, A.; Tamura, T.; Natsume, H.; Yao, H.; Ozato, K. The bromodomain protein Brd4 stimulates G1 gene transcription and promotes progression to S phase. J. Biol. Chem. 2008, 283, 9040–9048, doi:10.1074/jbc.M707603200.
[30]
Yang, Z.; He, N.; Zhou, Q. Brd4 recruits P-TEFb to chromosomes at late mitosis to promote G1 gene expression and cell cycle progression. Mol. Cell. Biol. 2008, 28, 967–976, doi:10.1128/MCB.01020-07.
[31]
Johansson, C.; Graham, S.V.; Dornan, E.S.; Morgan, I.M. The human papillomavirus 16 E2 protein is stabilised in S phase. Virology 2009, 394, 194–199, doi:10.1016/j.virol.2009.08.046.
[32]
Wang, X.; Naidu, S.R.; Sverdrup, F.; Androphy, E.J. Tax1BP1 interacts with papillomavirus E2 and regulates E2-dependent transcription and stability. J. Virol. 2009, 83, 2274–2284, doi:10.1128/JVI.01791-08.
[33]
Chin, K.T.; Chun, A.C.; Ching, Y.P.; Jeang, K.T.; Jin, D.Y. Human T-cell leukemia virus oncoprotein tax represses nuclear receptor-dependent transcription by targeting coactivator TAX1BP1. Cancer Res. 2007, 67, 1072–1081, doi:10.1158/0008-5472.CAN-06-3053.
[34]
Longworth, M.S.; Laimins, L.A. Pathogenesis of human papillomaviruses in differentiating epithelia. MicroBiol. Mol. Biol. Rev. 2004, 68, 362–372, doi:10.1128/MMBR.68.2.362-372.2004.
[35]
Schreiber, E.; Matthias, P.; Muller, M.M.; Schaffner, W. Rapid detection of octamer binding proteins with ‘mini-extracts’, prepared from a small number of cells. Nucleic Acids Res. 1989, 17, 6419, doi:10.1093/nar/17.15.6419.
