Elucidating the role of the human papillomavirus type 31 E7 histone deacetylase binding site in the viral life cycle

The E7 oncoprotein of high risk Human Papillomavirus (HPV) facilitates immortalization by altering the action of cell cycle regulatory proteins such as the Rb family of proteins as well as the Histone Deacetylases (HDACs). Expression of the HPV31 E7 protein has also been shown to be necessary for long term stable maintenance of episomes in Human Foreskin Keratinocytes (HFKs). This work describes studies which (1) demonstrate the necessity of the E7 HDAC binding site for episomal maintenance, (2) show evidence that E7 binding to HDACs is necessary for increased expression of E2F2 which is essential for viral replication, and (3) uncover a new localization pattern for the HDAC3 protein in HPV31 positive HFKs. Mutational analyses on the E7 open reading frame in the context of the complete HPV31 genome revealed the E7 HDAC binding site to be necessary for episomal maintenance and for late viral processes. These studies also demonstrated the importance of the zinc finger cysteine residues for stability of the E7 protein and confirmed that E7's binding to Rb is necessary for late viral processes and immortalization of HFKs. In an attempt to uncover a mechanism whereby E7-HDAC binding could facilitate viral replication, the expression of E2F transcription factors was examined. E2F2 was the only family member whose expression was significantly changed in HPV31 positive cells in comparison to untransfected HFKs. Transfection of the E7 HDAC binding mutant genome and chromatin immunoprecipitation analyses demonstrated E2F2 upregulation to be modulated by E7's binding to HDACs which sequesters them away from the E2F2 promoter. Transfection of E2F2 siRNA into HPV31 positive HFKs decreased total viral DNA amounts, without noticeable cell death, suggesting that E2F2 could be a therapeutic target for treatment of HPV infection. While HDAC expression and activity were maintained at higher levels following differentiation of HPV positive cells, the localization of Class I HDACs did not change in comparison to untransfected HFKs. An unexpected result of studying localization of HDAC proteins in HPV positive HFKs, was the discovery of a novel membrane localization pattern for HDAC3.