Human papillomavirus oncoprotein E7 targets the promyelocytic leukemia protein and circumvents cellular senescence via the Rb and p53 tumor suppressor pathways

Abstract

Cellular senescence can be triggered by a variety of signals, including loss of telomeric integrity or intense oncogenic signaling, and is considered a potent, natural tumor suppressor mechanism. Previously, it was shown that the promyelocytic leukemia protein (PML) induces cellular senescence when overexpressed in primary human fibroblasts. The mechanism by which the PML IV isoform elicits this irreversible growth arrest is believed to involve activation of the tumor suppressor pathways p21/p53 and p16/Rb; however, a requirement for either pathway has not been demonstrated unequivocally. To investigate the individual contributions of p53 and Rb to PML-induced senescence, we used oncoproteins E6 and E7 from human papillomaviruses (HPVs), which predominantly target p53 and Rb. We show that E7, but not E6, circumvents PML-induced senescence. Using different E7 mutant proteins, dominant negative cyclin-dependent kinase 4, and p16 RNA interference, we demonstrate that Rb-related and Rb-independent mechanisms of E7 are necessary for subversion of PML-induced senescence and we identify PML as a novel target for E7. Interaction between E7 and a functional prosenescence complex composed of PML, p53, and CBP perturbs transcriptional activation of p53, thus highlighting a significant effect also on the p53 tumor suppressor pathway. Given the importance of HPV in the pathogenesis of cervical cancer, our results warrant a more detailed analyses of PML in HPV infections.

FIG. 1.

Effects of E6 and E7 on cell proliferation, DNA synthesis, and induction of cellular senescence by PML IV and Ras. (A) Growth curves of normal WI38 human fibroblasts expressing combinations of PML IV, oncogenic RasV12, or empty vector (B0) plus E6, E7, or empty vector (LX). After drug selection, the number of PDs over the indicated period of time was determined. Day 0 is the first day after selection. PDs foreach time point are the mean values from triplicates. (B) DNA synthesis and (C) SA-β-Gal expression in normal human cells. WI38 cells were infected and selected as described in (A). After selection, [³H]thymidine was added at the indicated time points and left for 3 days (B), and cells were subsequently histochemically stained for SA-β-Gal expression (C) followed by autoradiography as described in Materials and Methods. A minimum of 200 cells were counted to determine the percentages of positive SA-β-Gal expression and radiolabeled nuclei (LN). A cell was considered SA-β-Gal positive only when it was not radiolabeled. (D) Morphology of cells expressing E7/PML IV or LX/PML IV. Cells were photographed under phase-contrast optics at day 10 postselection. Note the similar alterations in cell morphology in LX/PML IV- and E7/PML IV-expressing cells. (E) Inhibition of the Rb tumor suppressor pathway does not rescue PML-induced senescence. Growth curves of WI38 fibroblasts expressing Cdk 4^R24C or siRNA-p16 plus empty vector B0 or PML IV are shown. After drug selection, the number of PDs was determined as in (A). Shown are also the protein levels for Cdk 4 and p16 in the respective cell populations.

FIG. 2.

Rb-related and Rb-independent mechanisms of E7 are necessary to block PML IV-induced senescence and extend life span. (A) Diagram of E7, showing the locations of substitution mutations. (B) Growth curves of WI38 fibroblasts expressing either empty vector LX or its derivatives expressing E7 or mutant E7 genes superinfected with empty vector pBABE (B0) or its derivative expressing PML IV. After drug selection, the number of PDs over the indicated period of time was determined (upper panel). Also shown is the overall level and phosphorylation status of Rb in these cell populations as determined by Western blotting (lower panel). (C) Protein levels of p16, p21, and p53 in cells infected with the indicated viruses at 10 days postselection. Tub, tubulin.

FIG.3.

E7 perturbs a functional complex between PML, p53, and CBP. (A) Nuclear lysates were prepared from WI38 fibroblasts infected with either PML III/LX, PML IV/LX, or PML IV/E7 and immunoprecipitated (IP) with either anti-CBP or anti-p53 antibody. Immunoprecipitated complexes were collected on protein A/G beads and analyzed by Western blotting (WB) with antibodies specific for CBP, p53, or acetylated lysine. IgG-H+L, immunoglobulin G heavy plus light chains. (B) Nuclear lysates were prepared from WI38 fibroblasts infected with either PML IV/E7, PML IV/E7C24G, or PML IV/L82.83R and immunoprecipitated with anti-p53 antibody. Immune complexes were collected on protein A/G beads and analyzed by Western blotting with an antibody specific for p53 or acetylated lysine. (C) Nuclear lysates were prepared from WI38 cells infected either with PML IV/LX, PML IV/E7, PML IV/E7C24G, or PML IV/L82.83R and immunoprecipitated with anti-PML antibody. Immune complexes were collected on protein A/G beads and analyzed by Western blotting with an antibody specific for PML, p53, or CBP. Expression of PML, p53, and E7 was evaluated by immunoblotting of total nuclear lysates. (D) Radiolabeled, in vitro-translated PML IV was incubated with GST-p53 and increasing amounts of recombinant wild-type E7 or E7 L82.83R mutant (mt) proteins (1, 10, and 100 ng). (E) WI38 normal human fibroblasts were infected with the indicated retroviral combinations, selected, and transiently cotransfected when growth arrested with a p21-Luc reporter vector together with a pCMV-β-galactosidase normalization vector. Cells were assayed for β-galactosidase and luciferase reporters. Normalized p21 reporter activity is luciferase/β-galactosidase activity, with the activity of p21-Luc in proliferating LX/B0 cells set at 1. Shown are the averages and standard deviations from three independent experiments. Within each experiment, transfections were done in triplicate. (F) WI38 normal human fibroblasts were infected with the indicated retroviral combinations and selected, and the number of PDs over the indicated period of time was determined.

FIG. 4.

E7 and PML colocalize. (A) U2-OS cells were cotransfected with pcDNA3.1-PML IV and pSG5-E7-FLAG and costained with anti-PML (green) and anti-E7 (red) antibodies. (B) WI38 fibroblasts were retrovirally infected with PML IV and untagged E7 and costained with anti-PML (green) and anti-E7 (red) antibodies. (C) Caski cells were costained with anti-PML (red) and anti-E7 (green) antibodies. (D) Caski cells were transfected with pcDNA3.1-PML IV and costained with anti-PML (green) and anti-E7 (red) antibodies. All samples were analyzed by laser scanning microscopy.

FIG. 5.

E7 and PML physically interact. (A) Nuclear lysates were prepared from WI38 fibroblasts infected with either LX-E7-FLAG/pBABE-PML IV or LX-FLAG-GFP/pBABE-PML IV and immunoprecipitated (IP) with anti-FLAG antibody. Immunoprecipitated complexes were collected on protein A/G beads and were analyzed by Western blotting (WB) with antibodies specific for FLAG and PML. (B) In vivo GST-pull down assay. Nuclear lysates were prepared from WI38 fibroblasts expressing PML IV and affinity purified on GST-E7 or GST beads. Bound protein complexes were analyzed by Western blotting with using an anti-PML antibody. (C) Nuclear lysates were prepared from Caski cells and immunoprecipitated with either anti-E7, anti-PML, or control immunoglobulin G (IgG) antibodies. Immunoprecipitated complexes were analyzed by Western blotting with antibodies specific for E7 and PML. Asterisks mark IgG chains. (D) Radiolabeled, in vitro-translated PML IV or PML IVΔCC was incubated with either recombinant GST-E7 or -E7 mutant fusion protein products. Bound complexes were analyzed by autoradiography. (E) Growth curves of WI38 fibroblasts expressing the indicated retroviral constructs. After drug selection, the number of PDs over the indicated period of time was determined. Also shown is the in vitro interaction between GST-HPV-16 E7, GST-HPV-11 E7, and GST-HPV-6 E7 fusion proteins and radiolabeled, in vitro-translated PML IV.

FIG. 6.

Schematic representation of the inhibitory mechanism of E7. E7 circumvents PML IV-induced cellular senescence by directly targeting Rb for degradation and simultaneously perturbing a prosenescence PML/p53/CBP complex, resulting in the loss of transcriptional activation of p53 and induction of p53 response genes necessary for the execution of senescence. By directly targeting PML, E7 may also compromise the host's antiviral defense response against HPV infection, thereby securing virus survival. Double arrows indicate protein-protein interactions, single arrows indicate processing, and blunt lines indicate inhibitory events.