The DNA binding domain of a papillomavirus E2 programs a chimeric nuclease to cleave human papillomavirus DNA

Viral DNA binding proteins that direct nuclease domains or other functional protein domains to viral DNA in lytically or latently infected cells may provide a novel approach to modulate viral gene expression or replication. Cervical carcinogenesis is initiated by high-risk human papillomavirus (HPV) infection, and viral DNA persists in the cancer cells. To test if a DNA binding domain of a papillomavirus protein can direct a nuclease domain to cleave HPV DNA in cervical cancer cells, we fused the site-specific DNA binding domain of either the bovine papillomavirus type 1 (BPV1) or HPV16 E2 protein to the catalytic domain of the modular FokI restriction endonuclease, generating either a BPV1 E2-FokI (BEF) chimeric nuclease or an HPV16 E2-FokI chimeric nuclease. These chimeric nucleases cleaved DNA substrates containing E2 binding sites in vitro, and only a single E2 binding site was required for cleavage. BEF introduced DNA double-strand breaks on both sides of an E2 binding site in vitro to generate 5' overhangs of 4 nucleotides. Both the DNA binding and catalytic activities of BEF were required for efficient DNA cleavage in vitro. After expression of BEF in HeLa cervical carcinoma cells, we detected cleavage at E2 binding sites 1 and 4 in the integrated HPV18 DNA in these cells. Cleavage was also detected at an E2 binding site in cellular DNA. Expression of BEF in HeLa cells led to repression of the HPV E6 and E7 oncogenes and activation of the p53 and Rb tumor suppressor pathways, resulting in senescence, as measured by growth arrest, altered cellular morphology, increased autofluorescence, and senescence-associated beta-galactosidase activity. This induction of senescence required the DNA binding activity of BEF, but not its nuclease activity. These results demonstrate that DNA binding domains of viral proteins can target effector molecules to cognate binding sites in virally infected cells.