Binding of human papillomavirus type 16 E6 to E6AP is not required for activation of hTERT

Abstract

The human papillomavirus (HPV) type 16 (HPV16) E6 protein stimulates transcription of the catalytic subunit of telomerase, hTERT, in epithelial cells. It has been reported that binding to the ubiquitin ligase E6AP is required for this E6 activity, with E6 directing E6AP to the hTERT promoter. We previously reported two E6AP binding-defective HPV16 E6 mutations that induced immortalization of human mammary epithelial cells. Because activation of hTERT is proposed to be necessary for epithelial cell immortalization, we sought to further characterize the relationship between E6/E6AP association and telomerase induction. We demonstrate that while these E6 mutants do not bind E6AP, they retain the capability to stimulate the expression of hTERT. Chromatin immunoprecipitation assays confirmed the presence of Myc, wild-type E6, and the E6AP binding-defective E6 mutants, but not E6AP itself, at the endogenous hTERT promoter. Interestingly, an immortalization-defective E6 mutant localized to the hTERT promoter but failed to increase transcription. We conclude that binding to E6AP is not necessary for E6 localization to or activation of the hTERT promoter and that another activity of E6 is involved in hTERT activation.

FIG. 1.

Mutational analysis of E6 binding to E6AP. GST-E6AP was incubated with in vitro-translated wild-type HPV16 E6 (16E6) and the G130V and L50G (top panel) and F2V, L37S, and L110Q (bottom panel) mutants. Background is shown in the last lane of each panel, in which GST alone (GST C) was used as a negative control.

FIG. 2.

Quantitative binding assay. Five hundred nanograms of each GST-E6 fusion protein was incubated with increasing concentrations of portions of E6AP or hDLG fused to BAP (ligand) in a final volume of 50 μl. After a wash, bound ligand was determined as relative light units (RLU). (A) Binding of GST-E6 proteins to increasing concentrations (0 to 400 nM) of E6AP-BAP. E6 and the F2V mutant bound E6AP and reached saturation; the L37S, L50G, L110Q, and G130V mutants did not bind to the ligand. (B) Binding of GST-E6 proteins to increasing concentrations (0 to 200 nM) of hDLG-BAP. All E6 proteins bound this ligand and reached saturation.

FIG. 3.

The HPV16 E6 L37S and L110Q mutants do not associate with E6AP in vivo. H1299 cells were transfected with Flag-pcDNA3-16 E6, -L37S, or -L110Q and Flag-pcDNA3-E6AP. Cells were treated with MG132 and harvested 24 h after transfection. To demonstrate the association between E6 and E6AP, cell lysates were immunoprecipitated with an anti-E6AP antibody, and bound complexes were immunoblotted with an anti-E6AP or anti-Flag antibody. E6AP immunoprecipitated only with wild-type E6, not with E6 L37S or L110Q.

FIG. 4.

E6AP binding is not required for hTERT activation. Primary HKs were infected with HPV16 E6-expressing retroviruses. Real-time RT-PCR was performed on extracted RNA using TaqMan oligonucleotides specific for hTERT and SYBR green for E6 in parallel with GAPDH for normalization. (A) Ratio of hTERT to GAPDH amplification. The L37S and L110Q E6AP binding-defective mutants activated hTERT to levels similar to those for wild-type HPV16 E6 and the F2V mutant. The L50G and G130V mutants did not increase hTERT transcript levels more than the control (C) retrovirus. (B) Ratio of E6 expression to GAPDH expression.

FIG. 5.

ChIP assays for E6 and Myc at the hTERT promoter. HKs were infected with HPV16 E6-expressing retroviruses and treated with formaldehyde. Extracts were immunoprecipitated, the cross-linking reversed, and PCR performed with primers to amplify hTERT DNA. The input is the extract prior to immunoprecipitation that was subject to PCR. (A) ChIP assay with antiserum to E6 or preimmune serum (PI). E6AP binding-defective HPV16 E6 mutants associate with the endogenous hTERT promoter. E6 mutants were detected at the hTERT promoter by using an anti-E6 peptide antibody. No E6 signal was detected in cells expressing retroviral backbone DNA (lanes C). (B) A ChIP assay using an anti-Myc antibody detects Myc at the hTERT promoter in HKs infected with control (lanes C) and E6-expressing retroviruses. An anti-actin antibody was used as a control.