Papillomavirus E6 PDZ interactions can be replaced by repression of p53 to promote episomal human papillomavirus genome maintenance

Abstract

Cancer-associated human papillomaviruses (HPVs) express E6 oncoproteins that target the degradation of p53 and have a carboxy-terminal PDZ ligand that is required for stable episomal maintenance of the HPV genome. We find that the E6 PDZ ligand can be deleted and the HPV genome stably maintained if cellular p53 is inactivated. This indicates that the E6-PDZ interaction promotes HPV genome maintenance at least in part by neutralization of an activity that can arise from residual undegraded p53.

FIG 1

Episomal replication of HPV-16_E6ΔPDZ. (A) The three panels are Southern blot exposures using a HPV-16 genomic probe. Three variants of NIKS keratinocytes are indicated to the left of each panel: NIKS cells, NIKS cells expressing an shRNA directed against p53, and NIKS cells transduced with a retrovirus expressing a dominant-negative p53 (GSE56 [35]). Each of the three NIKS cell lines were transfected with the indicated HPV-16 genomes as indicated above the blots: wild-type (WT) HPV-16, HPV-16 with a premature stop codon at amino acid 12 of E6 (E6_X12), and HPV-16 with a premature stop codon at amino acid 150 of E6 that deletes the last two amino acids of E6, thereby ablating the PDZ ligand (E6ΔPDZ). The bacterial sequences were excised, the HPV genomes were recircularized and transfected into the three indicated NIKS cell lines together with a plasmid conferring resistance to G418, and the cells were selected for drug resistance for 4 days. Surviving pooled drug-resistant cell colonies were then passaged 1 or 2 times per week without further drug coselection for 8 weeks before isolation of total cellular DNA. Ten micrograms of uncut (U) DNA or DNA cut with NcoI that cuts HPV-16 once (N) or DNA cut with HindIII (H) that does not cut HPV-16 were loaded per lane. Lane 1 contains 5 pg of unit-length linear plasmid genomic DNA as a marker. Lanes 14 to 16 contain Hirt lysate samples derived from NIKS cells transfected with WT HPV-16 to enrich for episomal supercoiled HPV-16. (B) Expression levels of p53 in NIKS cell lines. The same NIKS cell lines described above for panel A were treated (+) with mitomycin C (MTC) (10 μg/ml) or the proteasome inhibitor MG132 (10 μM) for 6 h, lysed in 1% SDS, and analyzed by Western blotting with the indicated antibodies directed against p53 (p53 AB8, Oncogene Science, Cambridge, MA.), p21Cip (Abcam, Cambridge, MA.), actin (Thermo Scientific), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Cell Signaling Technology, Boston, MA.).