JVI Accepts, published online ahead of print on 21 May 2008

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J. Virol. doi:10.1128/JVI.00772-08
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

DIMERIZATION OF THE PAPILLOMAVIRUS E2 PROTEIN IS REQUIRED FOR EFFICIENT MITOTIC CHROMOSOME ASSOCIATION AND BRD4 BINDING

Juan Cardenas-Mora, Jonathan E. Spindler, Moon Kyoo Jang, and Alison A. McBride^*

Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA

^* To whom correspondence should be addressed. Email: amcbride{at}nih.gov.

The E2 protein of several papillomaviruses links the viral genome to mitotic chromosomes to ensure retention and efficient partitioning of genomes to daughter cells following cell division. BPV-1 E2 binds to chromosomes in complex with Brd4, a cellular bromodomain protein. Interaction with Brd4 is also important for E2-mediated transcriptional regulation. The transactivation domain of E2 is crucial for interaction with the Brd4 protein; proteins lacking, or mutated in, this domain do not interact with Brd4. However, we find that the C-terminal DNA binding/dimerization domain of E2 is also required for efficient binding to Brd4. Mutations that eliminate the DNA binding function of E2 have no effect on the ability of E2 to interact with Brd4 but an E2 protein with a mutation that disrupts C-terminal dimerization binds Brd4 with greatly reduced efficiency. Furthermore, E2 proteins in which the C-terminal domain is substituted with the dimeric DNA binding domains of EBNA-1 or Gal4 bind efficiently to the Brd4 protein but substitution with a monomeric Red Fluorescent protein does not rescue efficient Brd4 binding. Thus, E2 binds to Brd4 most efficiently as a dimer. To prove this further, the E2 DNA binding domain was substituted with an FKBP12-derived domain in which dimerization is regulated by a bivalent ligand. This fusion protein binds Brd4 efficiently only in the presence of the ligand, confirming that a dimer of E2 is required. Correspondingly, E2 proteins that can dimerize are able to bind to mitotic chromosomes much more efficiently than monomeric E2 polypeptides.

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