title: Amino acid substitutions that specifically impair the transcriptional activity of papillomavirus E2 affect binding to the long isoform of Brd4.
abstract: The E2 protein of papillomaviruses binds to specific sites in the viral genome to regulate its transcription, replication and segregation in mitosis. Amino acid substitutions in the transactivation domain (TAD) of E2, of Arg37 and Ile73, have been shown previously to impair the transcriptional activity of the protein but not its ability to support viral DNA replication. To understand the biochemical basis of this defect, we have used the TADs of a low-risk (HPV11) and a high-risk (HPV31) human papillomavirus (HPV) as affinity ligands to capture proteins from whole cell extracts that can associate with these domains. The major TAD-binding protein was identified by mass spectrometry and western blotting as the long isoform of Brd4. Binding to Brd4 was also demonstrated for the E2 TADs of other papillomaviruses including cutaneous and animal types. For HPV11, HPV31 and CRPV E2, we found that binding to Brd4 is significantly reduced by substitutions of Arg37 and Ile73. Since these amino acids are located near each other in the 3-dimensional structure of the TAD, we suggest that they define a conserved surface involved in binding Brd4 to regulate viral gene transcription.
authors.affiliation: Laboratory of Molecular Virology, Institut de recherches cliniques de Montréal, 110 Pine Avenue West, Montreal, Quebec, Canada H2W 1R7.
authors.forename: Hélène
authors.initials: H
authors.lastname: Sénéchal
authors.forename: Guy G
authors.initials: GG
authors.lastname: Poirier
authors.forename: Benoit
authors.initials: B
authors.lastname: Coulombe
authors.forename: Laimonis A
authors.initials: LA
authors.lastname: Laimins
authors.forename: Jacques
authors.initials: J
authors.lastname: Archambault
mesh_terms: D019943:Amino Acid Substitution; D015153:Blotting, Western; D018797:Cell Cycle Proteins; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry; D008958:Models, Molecular; D009687:Nuclear Proteins; D015514:Oncogene Proteins, Fusion / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000032:analysis / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000092468:Bromodomain Containing Proteins
chemical_list: D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D015514:Oncogene Proteins, Fusion; D011506:Proteins; D014157:Transcription Factors; D014764:Viral Proteins; C441289:BRD4 protein, human; D000092468:Bromodomain Containing Proteins; C422983:E2 protein, Cottontail rabbit papillomavirus; C117355:E2 protein, Human papillomavirus type 11; C098597:E2 protein, Human papillomavirus type 31