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.

PARP inhibition and immune modulation: scientific rationale and perspectives for the treatment of gynecologic cancers

“…in G1), when a sister chromatid template is not present, thereby preventing completion of HR despite upregulation of HR-related genes 105 , 107 and by impairing the correct localization of RAD51 to DSBs, further hindering HR. There may be additional mechanisms contributing to ineffective HR and/or genomic instability, including decreased levels of RAD52, 108 hypermethylation-related downregulation of RAD51, 109 impaired recruitment of downstream repair factors due to E7-mediated interaction with the E3 ubiquitin ligase RNF168, 110 and suppression of NHEJ and shunting of DSB repair toward the more error-prone microhomology-mediated end joining. 111 Interestingly, in vitro analysis of nine patient-derived cervical-cancer cell lines, eight of which were HPV 16- or HPV 18-positive, found that none met the criteria of HR deficiency, defined using log2-ratios and allele frequencies to generate a loss of heterozygosity score in a method used previously in EOC trials.…”

Epigenetic Regulation of the Human Papillomavirus Life Cycle

“…Additionally, a recent study by Sitz et al demonstrated a critical role for RNF168 in the productive replication of HPV31 [114]. RNF168 protein levels are substantially upregulated in HPV31-positive cells, and transient depletion of RNF168 using small hairpin RNAs blocks productive viral replication upon differentiation, while having minimal effect on episomal maintenance in undifferentiated cells [114]. Importantly, this study also showed that high-risk, but not low-risk, E7 proteins directly interact with RNF168 via E7's CR3 domain, hindering the function of RNF168 at cellular DSBs, resulting in decreased 53BP1 recruitment and an increase in HR repair [114].…”

“…Indeed, Nbs1 and Mre11 of the MRN complex, along with the HR factors, Rad51 and BRCA1, are required for productive replication, indicating that HPV utilizes ATM activity to direct repair to HR on viral chromatin [112,113]. Additionally, a recent study by Sitz et al demonstrated a critical role for RNF168 in the productive replication of HPV31 [114]. RNF168 protein levels are substantially upregulated in HPV31-positive cells, and transient depletion of RNF168 using small hairpin RNAs blocks productive viral replication upon differentiation, while having minimal effect on episomal maintenance in undifferentiated cells [114].…”

“…RNF168 protein levels are substantially upregulated in HPV31-positive cells, and transient depletion of RNF168 using small hairpin RNAs blocks productive viral replication upon differentiation, while having minimal effect on episomal maintenance in undifferentiated cells [114]. Importantly, this study also showed that high-risk, but not low-risk, E7 proteins directly interact with RNF168 via E7's CR3 domain, hindering the function of RNF168 at cellular DSBs, resulting in decreased 53BP1 recruitment and an increase in HR repair [114]. Interestingly, recent studies from the Laimins lab demonstrated that breaks in the HPV31 genome are preferentially repaired at the expense of cellular DNA upon differentiation [115].…”

Regulation of the Human Papillomavirus Life Cycle by DNA Damage Repair Pathways and Epigenetic Factors

“…Alternatively, interactions of viral proteins with cellular replication factors may also contribute. For instance, E7 has been shown to bind to the E3 ubiquitin ligase, RNF168, which is critical for proper DNA repair leading to enhanced viral replication [ 67 ]. In addition to E6 and E7, high-level expression of E1 from heterologous promoters has also been shown to activate the ATM DNA damage response.…”