The Mdm2 ubiquitin ligase enhances transcriptional activity of human papillomavirus E2

Abstract

The regulation of human papillomavirus (HPV) gene expression by the E2 protein is a critical feature of the viral life cycle. Previous studies have shown an important role in transcription for the ubiquitin-proteasome pathway, but its role in HPV gene expression has not been addressed. We now show that HPV E2 requires an active proteasome for its optimal transcriptional activator function. This involves an interaction with the Mdm2 ubiquitin ligase, which together with E2 acts synergistically to activate the HPV type 16 promoter. We also show that HPV E2 recruits Mdm2 onto HPV promoter sequences, providing an explanation for this cooperative activity.

FIG. 1.

The role of the proteasome machinery in the transcriptional activity of E2. To assess E2 transcriptional activity, luciferase assays were conducted in U2OS cells transfected with a reporter construct containing E2 binding sites upstream of the luciferase gene (E2-Luc), plus the Renilla luciferase gene as a transfection control and an untagged HPV-16 E2 expression plasmid. Representative results of three experiments are shown together with standard deviations. RLA, relative luciferase activity. (a, left) E2 transcriptional activity in the presence or absence of proteasome inhibitors (CBZ). (a, right) The effects of CBZ on Dex-induced GR transactivation. U2OS cells were transfected with GR and MMTV-Luc plasmids and treated with 10 nM Dex (Sigma) for 16 h, followed by treatment with CBZ for a further 5 h. (b) The effects of various ubiquitin ligases on E2 transcriptional activity. Cells were transfected with the reporter plasmids and E2 in the presence of expression plasmids of the indicated ubiquitin ligases.

FIG. 2.

Mdm2 enhances the transcriptional activity of E2. (a) The ubiquitin ligase activity of Mdm2 is required to enhance E2 transcriptional activity. A luciferase assay was conducted in the presence of Mdm2C462A (ligase-dead mutant of Mdm2) and CBZ. RLA, relative luciferase activity. (b) Mdm2 enhances E2-mediated transcriptional activity in SAOS-2 (p53^−/− pRb^−/−) (left panel) and H1299 (p53^−/− pRb^+/+) (right panel) cells. Cells were transfected with the reporter construct and an E2 expression vector with or without the expression of Mdm2 as shown in panel a. (c) The transcriptional activity of E2 is reduced in the presence of Mdm2 inhibitors. SAOS-2 cells were transfected as shown in panel b. Five hours prior to harvest, cells were treated with Nutlin-3 (10 μM; Sigma) or Mdm2 E3 ligase inhibitor (10 μM; Calbiochem). (d) The effect of Mdm2 expression on E2 levels was tested by expressing both proteins in U2OS cells. At 24 h after transfection, protein levels were analyzed by Western blotting using antibodies against 16E2 or β-Gal as a transfection control.

FIG. 3.

Mdm2 interacts with 16E2 in vitro and in vivo. (a) In vitro-translated and radiolabeled Mdm2 was incubated with bacterially purified GST-tagged E2. GST alone and GST-p53 were included as negative and positive controls, respectively. Bound proteins were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The Coomassie blue stains of the GST inputs are also included in the top panel. An asterisk indicates the full-length GST fusion proteins. (b) Mdm2 binds to the C-terminal region of E2. GST-16E2 and a number of GST-tagged fragments of E2 (right) were incubated with in vitro-translated and radiolabeled Mdm2, and bound proteins were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Fifty percent of the input is included, and the GST inputs are shown in the bottom panel and are stained with Coomassie blue. An asterisk indicates the full-length GSTs. NE2, N-terminal half of E2 protein; CE2, C-terminal half of E2 protein. (c) E2 and Mdm2 bind in vivo. 293 cells were transfected using either Mdm2 alone or Mdm2 with GFP-tagged E2. Cell extracts were immunoprecipitated (IP) using polyclonal anti-GFP antibodies, followed by Western blot analysis using antibodies against Mdm2 or GFP. An asterisk indicates nonspecific bands. -ve, untransfected cells.

FIG. 4.

Mdm2 binds to E2 on DNA. (a) FLAG-tagged Mdm2 immunoprecipitates with GFP-tagged E2. 293 cell extracts expressing GFP-E2 with or without the coexpression of FLAG-Mdm2 were immunoprecipitated (IP) using anti-FLAG antibodies, and immunoprecipitated proteins were detected by Western blotting (W.B) using antibodies against E2. -ve, untransfected cells. (b) Mdm2 immunoprecipitates with DNA-bound E2. 293 cells were transfected with a E2-Luc construct along with different combinations of FLAG-Mdm2 and GFP-E2 expression plasmids, and cell extracts were immunoprecipitated using the indicated antibodies. The coprecipitated DNA was analyzed by PCR using primers that are complementary to the luciferase gene.