Human papillomavirus E7 oncoprotein targets RNF168 to hijack the host DNA damage response

Abstract

High-risk human papillomaviruses (HR-HPVs) promote cervical cancer as well as a subset of anogenital and head and neck cancers. Due to their limited coding capacity, HPVs hijack the host cell's DNA replication and repair machineries to replicate their own genomes. How this host-pathogen interaction contributes to genomic instability is unknown. Here, we report that HPV-infected cancer cells express high levels of RNF168, an E3 ubiquitin ligase that is critical for proper DNA repair following DNA double-strand breaks, and accumulate high numbers of 53BP1 nuclear bodies, a marker of genomic instability induced by replication stress. We describe a mechanism by which HPV E7 subverts the function of RNF168 at DNA double-strand breaks, providing a rationale for increased homology-directed recombination in E6/E7-expressing cervical cancer cells. By targeting a new regulatory domain of RNF168, E7 binds directly to the E3 ligase without affecting its enzymatic activity. As RNF168 knockdown impairs viral genome amplification in differentiated keratinocytes, we propose that E7 hijacks the E3 ligase to promote the viral replicative cycle. This study reveals a mechanism by which tumor viruses reshape the cellular response to DNA damage by manipulating RNF168-dependent ubiquitin signaling. Importantly, our findings reveal a pathway by which HPV may promote the genomic instability that drives oncogenesis.

Keywords: 53BP1 nuclear bodies; DNA double-strand break; E7 protein; RNF168; high-risk human papillomavirus.

Fig. 1.

Expression and localization of RNF168 and 53BP1 in HPV⁺ carcinoma. (A) Expression of the indicated genes in cervical cancers (CESC) and head and neck cancers (HNSC) stratified by human papillomavirus (HPV) status. Normalized RNA-seq data extracted from The Cancer Genome Atlas (TCGA) database are presented. For HNSC, normal-adjacent control tissues were used as an extra control (black). Statistical significance was assessed by a Mann–Whitney U test. (B) Whole cell extracts (WCE) were analyzed by immunoblotting with the indicated antibodies. KAP1 and Tubulin were used as loading controls. (C) Representative images of 53BP1 and RNF168 foci observed in the indicated cell lines. Cells were fixed and processed for γ-H2AX, 53BP1, and RNF168 immunofluorescence. (Scale bar, 5 µm.) (D) Quantification of 53BP1 (Upper) and RNF168 (Lower) colocalizing with γ-H2AX. Data are presented as the mean ± SEM (n = 3; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001). ns, not significant. (E) Representative images of GFP-53BP1^FFR and BRCA1 in CaSki cells treated or not with 1 Gy. Cyclin A staining was used to identify cells in S/G2 phase (positive) and G1 phase (negative).

Fig. 2.

E7 protein directly interacts with RNF168. (A) Schematic representation of the tethering LacO/LacR system inserted in U2OS 2-6-5. LacO, Lac operator; LacR, Lac repressor. (B) U2OS 2-6-5 cells transfected with plasmids expressing the indicated mCherry-LacR fusion protein or induced for the expression of ER-mCherry-LacR-FokI-DD were fixed and processed for 53BP1 immunofluorescence. (Scale bars, 5 µm.) (C–H) Quantification of the mCherry-LacR foci colocalizing with 53BP1 (C and G), γ-H2AX (D), and Flag (E and F) staining. In G, cells were depleted for RNF8 prior to transfection with mCherry-LacR constructs. In H, HPV31 E6 or E7 fused to a GFP tag were used. All of the quantifications are represented as the mean ± SD (n = 3; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001). (I) Pull-down of His-RNF168 with GST-HPV16 E7. GST alone and MBP-His were used as negative controls. Inputs and pull-down were analyzed by immunoblotting with the indicated antibodies.

Fig. 3.

E7 from HR-HPVs binds to RNF168 through the CR3 domain. (A) Schematic representation of HPV E7 protein. CR, conserved region; CXXC, zinc-finger motif; LXCXE, pRb-binding sites. An alignment of the CR3 domain from different types of HPV (beta, yellow; alpha low-risk, green; alpha high-risk, pink) is also presented. Conserved residues are indicated in bold, the zinc-binding domains are indicated in dark red, and the residues mutated used in this study are shown in light gray. (B–D) Quantification of the mCherry-LacR-HPV E7 foci colocalizing with Flag-RNF168 were done as described in Fig. 2E. (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.) (E) Immunoprecipitation of GFP-RNF168 from U2OS 2-6-5 cell extracts. The indicated mCherry-LacR fusion proteins were transfected in GFP- or GFP-RNF168–expressing cells. GFP fusion proteins were purified over GFP-binding proteins beads (GFP-Trap) and analyzed by immunoblotting. GAPDH was used as a loading control.

Fig. 4.

RNF168 is required for viral replication. (A) CIN612 9E cells were transiently transduced with lentiviral particles containing a control scrambled shRNA (shScram) or one of the 2 RNF168-specific shRNAs for 72 h. DNA and proteins were harvested at T0 (undifferentiated) or following differentiation in high-calcium medium for 72 h. DNA was digested with BamHI (does not cut the viral genome) and HindIII (linearizes the viral genome) and analyzed by Southern blotting using the HPV31 genome as a probe. WCEs were analyzed by immunoblotting with the indicated antibody. Involucrin and K-10 were used to demonstrate cellular differentiation. KAP1 and GAPDH were used as loading controls. (B) Quantification of the experiment present in A using densitometry of episomal bands with ImageJ software. Shown is the fold change normalized to T0 shScram, which is set to 1. Error bars represent means ± SD (n = 3; *P ≤ 0.05). (C) RNF168 expression in HFK and CIN612 9E cells was analyzed 0, 48, and 96 h after differentiation in high-calcium media by immunoblotting with the indicated antibodies. GAPDH and KAP1 were used as loading controls.

Fig. 5.

E7 hinders the function of RNF168 at DSBs. (A) Representative images of U2OS cells transfected with the indicated mCherry-LacR protein and Flag-RNF168. (B and C) Quantification of γ-H2AX and 53BP1 foci in U2OS cells (B) and hTERT-HFK (C) treated with the indicated amount of Gy. In B, FK2 foci were also quantified. Expression of GFP-HPV31 E7 full-length and CR1/2 were induced 24 h before treatment. Results are represented as the mean ± SEM (n = 3; **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001). (D) Schematic representation of the HDR assay based on gene editing of the LMNA locus. (E) C33-A stable cell lines were transfected with Cas9, sgRNA against LMNA, and mClover donor vectors. Percentage of mClover-positive cells were analyzed by flow cytometry 72 h posttransfection and normalized over percentage of mClover-positive C33-A cells in each replicate. Data are represented as the mean ± SEM (n = 3).

Fig. 6.

E7 interacts with a previously uncharacterized region of RNF168. (A) Schematic representation of RNF168 and its functional domains. LRM, LR-motif; PID, PALB2-interacting domain; R, Ring domain; UDM, Ub-dependent DSB recruitment module; UIM/MIU, motif-interacting with ubiquitin. (B) In vitro ubiquitylation assay (UbRx) on mononucleosomes (MNs) isolated from HeLa cells with or without a gradient of GST or GST-HPV16 E7. A reaction without ubiquitin was used as a negative control. (C) In vitro ubiquitylation assay on GST and GST-HPV16 E7 with MNs. A reaction without ubiquitin was used as a negative control. Asterisk indicates a nonspecific band. (D and E) RNF168-depleted U2OS 2-6-5 cells were transfected with plasmids expressing GFP-E7 and the indicated mCherry-LacR RNF168 construct and processed for 53BP1 immunofluorescence. Accumulation of GFP-HPV31 E7 (Left) and 53BP1 (Right) at the mCherry-LacR RNF168 foci are represented as the mean ± SD (n = 3) in D (*P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001). Representative images obtained with the indicated mutation/truncation of RNF168 are presented in E. (Scale bars, 5 µm.) (F) Proposed models for the inhibition of RNF168 function at DSBs by HR-HPV E7. P, phosphorylation; Ub, ubiquitin.