Comparative analysis of transforming properties of E6 and E7 from different beta human papillomavirus types

Abstract

The cutaneous beta human papillomavirus (beta HPV) types appear to be involved in skin carcinogenesis. However, only a few beta HPVs have been investigated so far. Here, we compared the properties of E6 and E7 oncoproteins from six uncharacterized beta HPVs (14, 22, 23, 24, 36, 49). Only HPV49 E6 and E7 immortalized primary human keratinocytes and efficiently deregulated the p53 and pRb pathways. Furthermore, HPV49 E6, similarly to E6 from the oncogenic HPV16, promoted p53 degradation.

Fig 1

HPV49 E6 and E7 efficiently immortalize primary HFK. (A) Total RNA was extracted from HFK transduced with the indicated recombinant retroviruses and subjected to reverse transcription. PCR was performed using the different cDNAs as templates with HPV type-specific primers. (B) Morphology of primary HFK transduced with the indicated recombinant retroviruses 20 days postransduction. The same magnification was used (×10) for all photomicrographs. (C) Total RNA was extracted from the indicated cells and subjected to reverse transcription. PCR analysis was performed using specific primers for hTERT and GAPDH. The graph represents the quantification of the results from three independent experiments.

Fig 2

HPV49 E7 alters the pRB-regulated pathway. (A) HaCaT cell extracts were incubated with different GST fusion proteins, and the bound proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The amount of pRb associated with the different GST E7 proteins was determined by immunoblotting, using a specific anti-pRb (554136; BD Pharmingen) antibody. One-tenth of the total cellular extract (60 μg) used in the GST pulldown assay was loaded on the SDS-PAGE gel as a control (input). (B) NIH 3T3 cells were transduced with the indicated recombinant retroviruses. After completion of the drug selection, cells were cultured in the presence of cycloheximide (CHX) for the indicated times. At each time point, cells were collected. Protein extracts were prepared and analyzed by immunoblotting with the indicated antibodies (top). The levels of pRb were quantified by Quantity one (Bio-Rad) and normalized to the levels of β-actin (bottom). (C) Protein extracts of HFK transduced with empty retrovirus vector (pLXSN HFK) or expressing the different beta HPV E6 and E7 genes were analyzed by immunoblotting using anti-pRb (554136; BD Pharmingen) and β-actin (clone C4; MP Biomedicals) antibodies. β-Actin staining was included as a loading control. (D) Protein extracts from HFK transduced with empty (pLXSN) or beta HPV38 or 49 E6/E7 retrovirus were incubated in the presence and absence of lambda phosphatase (λ-pp) and analyzed by immunoblotting using antibodies that recognize pRb phosphorylated at serine 795 (9301S; Cellsignalling), all forms of pRb (554136; BD Pharmingen), or β-actin (clone c4; MP-Biomedicals). (E) HFK transduced with the indicated retroviruses were treated with a CDK inhibitor (219476; Calbiochem) at 520 nM for 48 h. pRb and β-actin levels were determined by SDS-PAGE and immunoblotting. (F) Total RNA was extracted from HFK transduced with the indicated recombinant retroviruses and subjected to reverse transcription. Quantitative PCR analysis was then performed with specific primers for the Cdc2, cyclin A, and GAPDH genes. The data are means from three independent experiments performed in duplicate. (G) Protein extracts from HFK transduced with empty vector (pLXSN) or the indicated beta HPV E6/E7 retrovirus were analyzed by immunoblotting using the following antibodies: anti-c-Myc (1667149; Roche), anti-cyclin A (H-432; Santa Cruz), anti-Cdc2 (sc-54; Santa Cruz), and anti-β-actin (clone C4; MP-Biomedicals).

Fig 3

ΔNp73α is upregulated in HPV24 and 36 E6/E7 HFK but not in HPV49 E6/E7 HFK. (A) Protein extracts of primary keratinocytes transduced with empty retrovirus vector (pLXSN HFK) or the indicated recombinant retroviruses were analyzed by immunoblotting using anti-p73α (OP108; Calbiochem) and anti-β-actin (clone C4; MP-Biomedicals) antibodies. (B) HPV24, -38, or -49 E6/E7 HFK were transiently transfected either with scrambled (Scr) small interfering RNA (siRNA) or with siRNA directed against p53. After preparation of total protein extracts, ΔNp73α, p53, and β-actin protein levels were determined by immunoblotting using specific antibodies.

Fig 4

HPV49 E6 deregulates the p53 pathway, promoting its degradation. (A) HFK transduced with the indicated retroviruses were UV irradiated (0.08 J/cm²). After 8 h, total RNA was extracted and subjected to reverse transcription. Then qPCR analysis was performed using specific primers for the p21^WAF1 and GAPDH genes. The data are means from three independent experiments performed in duplicate. (B) HFK transduced with the indicated retroviruses were cultured, and protein extracts were prepared and analyzed by immunoblotting with the indicated antibodies. (C) HFK transduced with the indicated retroviruses were cultured in the presence of cycloheximide (CHX) for the indicated times. At each time point, cells were collected. Protein extracts were prepared and analyzed by immunoblotting with the indicated antibodies. The levels of p53 were quantified by Quantity one (Bio-Rad) and normalized to the levels of β-actin. The data are means from three independent experiments. (D) In vitro-translated p53 was incubated at 30°C for 0, 30, or 60 min, either alone or with in vitro-translated HPV16, -38, or -49 E6. The remaining p53 was visualized by SDS-PAGE and autoradiography (left). p53 levels were measured after normalization to E6 levels in three independent experiments (right). (E) C33A cell extracts were incubated with GST fusion proteins, and bound proteins were analyzed by SDS-PAGE. The amounts of p53 and E6AP associated with the different GST E6 proteins were determined by immunoblotting, using specific anti-E6AP (clone E6AP-330; Sigma) or anti-p53 antibodies (DO-7; Dako). One-tenth of the total cellular extract (60 μg) used in the GST pulldown assay (input) was loaded on the SDS-PAGE gel as a control. (F) HPV49 E6/E7 HFK were transiently transfected with siRNAs specifically directed against either luciferase (Luc) or E6AP. After preparation of total protein extracts, levels of p53, E6AP, and β-actin were determined by immunoblotting using specific antibodies.