Fig 1.
The biotinylated peptides used in this study; the peptides correspond to the 10 C-terminal amino acid residues from the E6 protein of each virus type.
The scrambled peptide has the same amino acid composition as the HPV-18 peptide. Group 1 HPV types are defined as definitely carcinogenic in humans; Group 2B as possibly carcinogenic in humans, and Group 3 as no risk (IARC Monograph 100B).
Fig 2.
Peptide pulldown assays analysed by mass spectroscopy.
Mass spectroscopy results of the pulldown assays from HaCat cells. Each histogram shows the numbers of peptides from each cellular PDZ domain–containing protein that was pulled down from HaCat extracts; n = total number of PDZ proteins pulled down by the E6 C-terminal peptide from HPV types 16, 18, 31, 33, 35, 51, 56 (Group 1); HPV types 26, 66, 70 (Group 2B); HPV type 40 (Group 3). The histograms were drawn using the Prism program, error bars show Standard error of the Mean. The scrambled peptide pulled down no PDZ domain-containing proteins in any assay.
Fig 3.
The mean numbers of PDZ domain-containing proteins pulled down by peptides from the Group 1 HPV types was significantly greater than the number pulled down by Group 2B (P = 0.0029) or Group 3 (P = 0.0015) types.
Numbers were analysed using the Prism software and standard deviations are shown. The scrambled peptide pulled down no PDZ domain-containing proteins in any assay.
Fig 4.
The number of cellular PDZ proteins targeted by the E6 PBMs increases with the cancer-causing potential of the virus type.
The histograms show the number of PDZ domain-containing protein peptides pulled down by each E6 PBM, adjusted for the molecular weight of the protein concerned. The histograms are arranged by HPV phylogenetic group (boxes) and by the percentage of total invasive cervical cancers associated with each HPV type (highest on the left); the percentages are shown.
Fig 5.
Mass spectroscopy results of the pulldown assays from Normal Immortal Keratinocytes (NIKS), using E6 C-terminal peptides from HPV types representing Group 1 (HPV-16, HPV-18), Group 2 (HPV-66) and Group 3 (HPV-40).
The histograms show the numbers of peptides from each cellular PDZ domain–containing protein that was pulled down from NIKS cell extracts; n = total number of PDZ proteins pulled down by each E6 C-terminal peptide. The histograms were analysed using the Prism program and standard deviations are shown. The scrambled peptide pulled down no PDZ domain-containing proteins in any assay.
Fig 6.
DLG1 is a common target of the E6 proteins, regardless of oncogenic potential.
A. DLG1 binds to the E6 PBMs of both oncogenic and non-oncogenic HPV types; the histogram shows mean peptide numbers from the mass spectroscopy analysis of all pulldowns, analysed using Prism software, error bars show Standard Error of the Mean. B. The non-oncogenic HPV-40 E6 PBM binds DLG1 to a level similar to the HPV-16 E6 PBM. A peptide pulldown from HaCat cell extract, using the control (scr), HPV-16 and HPV-40 peptides, was analysed by western blot and probed for DLG1, CASK, hScrib and ZO-2, as indicated. C. DLG1 binds to the E6 proteins of both oncogenic and non-oncogenic HPV types. In vitro translated radiolabelled DLG1 was incubated with the full-length HPV-66 and HPV-40 E6 proteins expressed as GST fusion proteins. GST alone, GST-HPV-16E6 and HPV-18-E6 were included as controls. The upper panel shows the autoradiograph of bound DLG1 and the lower panel shows the Coomassie stained gel. Arrows indicate the GST-E6 proteins. The histogram shows the quantitation of at least three binding assays, analysed by the ImageJ and Prism programmes. Standard deviations are shown. D. The non-oncogenic HPV-40 E6 protein binds to DLG1 via its PDZ domains. A GST-pulldown assay, using in vitro translated radiolabelled HPV-18 and HPV-40 E6 proteins, together with GST fusion proteins with full-length DLG1(FL), the N-terminus of DLG1, lacking any PDZ domain (NT), and the first 401 amino acid residues of DLG1, containing the PDZ domains 1 and 2 (1/2). The upper panel shows the autoradiograph of the bound E6 proteins, the lower panel shows the Coomassie stained gel. Arrows indicate the GST fusion proteins.
Fig 7.
hScrib is preferentially bound by cancer-causing HPV E6 proteins.
A. The histogram shows the mean numbers of hScrib peptides pulled down by each E6 C-terminus peptide, detected by mass spectroscopy. Standard Errors of the Mean are shown. B. Comparison of the numbers of hScrib peptides pulled down by the Group 1 and Group 2B peptides shows a statistically significant (P = 0.0083) difference. Numbers were analysed by Prism software and standard deviations are shown. C. hScrib is bound by the E6 proteins of oncogenic HPV types. In vitro translated radiolabelled hScrib was incubated with the full-length GST-HPV-16E6, HPV-18-E6, HPV-66-E6 and HPV-40-E6 proteins. The upper panel shows the autoradiograph of bound hScrib and the lower panel shows the Coomassie stained gel. Arrows indicate the GST-E6 proteins; the GST alone is not visible on a gel of low enough percentage acrylamide to allow hScrib visualisation, but was equivalent to that in Fig 2C and 2D. D. The histogram shows the quantitation of at least three binding assays, analysed by the Image J and Prism programmes. Standard deviations are shown.
Fig 8.
Novel interacting partners of HPV E6 proteins.
ZO-2 binds to HPV E6 proteins in a PBM-dependent manner. (A). Extract of 293 cells transfected with ZO-2-expressing plasmid was incubated with GST-E6 proteins from HPV types 16, 18, 31, 51, 66, 70, 40 and the HPV18 T156E mutant, which ablates PDZ binding. Above: bound proteins detected by western blot probed with anti-HA antibody. Below: Ponceau staining of the nitrocellulose membranes, showing the GST-E6 proteins (arrowed). (B). The histogram shows the collated results of at least 3 assays. Standard deviations are shown. (C). No binding is seen with HA-ZO-1. Extract of 293 cells transfected with ZO-1-expressing plasmid was incubated with GST-E6 proteins from HPV types 16, 18, 31, 51, 66, 70, 40 and the HPV18 T156E mutant, which ablates PDZ binding. Above: bound proteins detected by western blot probed with anti-HA antibody. Below: Ponceau staining of the nitrocellulose membranes, showing the GST-E6 proteins (arrowed).
Fig 9.
Novel interacting partners of HPV E6 proteins.
Beta-2-syntrophin (SNTB2) binds to HPV E6 proteins in a PBM-dependent manner. (A). Autoradiograph of a GST pulldown assay using in vitro translated, radiolabelled SNTB2, together with GST-E6 fusion proteins from HPV types 16, 18, 31, 51, 66, 70, 40 and the 18E6T156E mutant. The Coomassie stained gel shows the GST fusion protein input (arrowed). (B). The histogram shows the collated results of at least 3 assays, standard deviations are shown.
Fig 10.
ZO-2 levels are increased upon HPV E6 expression.
Western blot analysis of (A). ZO-2 and (B). SNTB2 levels in HeLa cells. si-ablation of E6/E7 expression results in a significant (p = 0.0063) reduction in ZO-2 levels. HeLa cells were transfected with siRNA to Luciferase (Luci) as control, with si-HPV-18 E6/E7 to ablate E6 expression, with si-ZO-2 or si-SNTB2. ZO-2 and SNTB2 levels were determined by Western Blot after 48h. The histograms show the results of at least three independent assays, analysed by Image J and Prism programmes and standard deviations are shown. (C). A representative western blot assay.
Fig 11.
Increased ZO-2 levels promote cell migration.
Ablation of ZO-2 expression reduces wound healing ability in HeLa cells, to a similar degree as ablation of E6 expression. (A). Confluent HeLa cells were scratched with a plastic pipette tip 48h after being transfected with si-RNA to luciferase, HPV 18 E6/E7, ZO-2 or SNTB2. The cells were photographed immediately post-scratch (0h) and after 24h. (B). The histogram shows the percentage area of the scratch remaining at 24h, from three independent experiments and standard deviations are shown.
Fig 12.
The molecular basis for substrate selection.
Histograms representing the mass spectroscopy results of peptide pulldowns from HaCat cell extract, using peptides representing the C-termini of HPV-16 and HPV-33, and peptides in which the penultimate amino acid residue was swapped: HPV-16Q150A and HPV-33A148Q. The most marked effects of the change are arrowed. The histograms were drawn using the Prism software, the peptide sequences are shown on each histogram.