Direct interaction with the BRD4 carboxyl-terminal motif (CTM) and TopBP1 is required for human papillomavirus 16 E2 association with mitotic chromatin and plasmid segregation function.

IMPORTANCE: Human papillomavirus 16 (HPV16) is a causative agent in around 3%-4% of all human cancers, and currently, there are no anti-viral therapeutics available for combating this disease burden. In order to identify new therapeutic targets, we must increase our understanding of the HPV16 life cycle. Previously, we demonstrated that an interaction between E2 and the cellular protein TopBP1 mediates the plasmid segregation function of E2, allowing distribution of viral genomes into daughter nuclei following cell division. Here, we demonstrate that E2 interaction with an additional host protein, BRD4, is also essential for E2 segregation function, and that BRD4 exists in a complex with TopBP1. Overall, these results enhance our understanding of a critical part of the HPV16 life cycle and presents several therapeutic targets for disruption of the viral life cycle.

Direct interaction with the BRD4 carboxyl-terminal motif (CTM) and TopBP1 is required for human papillomavirus 16 E2 association with mitotic chromatin and plasmid segregation function.

During the human papillomavirus 16 life cycle, the E2 protein binds simultaneously to the viral genome and host chromatin throughout mitosis, ensuring viral genomes reside in daughter cell nuclei following cell division. Previously, we demonstrated that CK2 phosphorylation of E2 on serine 23 promotes interaction with TopBP1, and that this interaction is required for optimum E2 mitotic chromatin association and plasmid segregation function. Others have implicated BRD4 in mediating the plasmid segregation function of E2 and we have demonstrated that there is a TopBP1-BRD4 complex in the cell. We therefore further investigated the role of the E2-BRD4 interaction in mediating E2 association with mitotic chromatin and plasmid segregation function. Using a combination of immunofluorescence and our novel plasmid segregation assay in U2OS and N/Tert-1 cells stably expressing a variety of E2 mutants, we report that direct interaction with the BRD4 carboxyl-terminal motif (CTM) and TopBP1 is required for E2 association with mitotic chromatin and plasmid segregation. We also identify a novel TopBP1 mediated interaction between E2 and the BRD4 extra-terminal (ET) domain in vivo . Overall, the results demonstrate that direct interaction with TopBP1 and the BRD4 CTM are required for E2 mitotic chromatin association and plasmid segregation function. Disruption of this complex offers therapeutic options for targeting segregation of viral genomes into daughter cells, potentially combatting HPV16 infections, and cancers that retain episomal genomes. Importance: HPV16 is a causative agent in around 3-4% of all human cancers and currently there are no anti-viral therapeutics available for combating this disease burden. In order to identify new therapeutic targets, we must increase our understanding of the HPV16 life cycle. Previously, we demonstrated that an interaction between E2 and the cellular protein TopBP1 mediates the plasmid segregation function of E2, allowing distribution of viral genomes into daughter nuclei following cell division. Here, we demonstrate that E2 interaction with an additional host protein, BRD4, is also essential for E2 segregation function, and that BRD4 exists in a complex with TopBP1. Overall, these results enhance our understanding of a critical part of the HPV16 life cycle and presents several therapeutic targets for disruption of the viral life cycle.

Human Papillomavirus 16 E2 Interaction with TopBP1 Is Required for E2 and Viral Genome Stability during the Viral Life Cycle.

CK2 phosphorylation of HPV16 E2 at serine 23 promotes interaction with TopBP1, and this interaction is important for E2 plasmid segregation function. Here, we demonstrate that the E2-TopBP1 interaction is critical for E2 and viral genome stability during the viral life cycle. Introduction of the S23A mutation into the HPV16 genome results in a loss of E2 expression and viral genome integration during organotypic rafting. Coculture of N/Tert-1+E2-S23A cells with J2 fibroblasts results in E2-S23A degradation via the proteasome; wild-type E2 is not degraded. TopBP1 siRNA treatment of N/Tert-1+E2-WT cells results in E2 degradation only in the presence of J2 cells demonstrating the critical role for TopBP1 in maintaining E2 stability. The CK2 inhibitor CX4945 promotes E2-WT degradation in the presence of fibroblasts as it disrupts E2-TopBP1 interaction. siRNA targeting SIRT1 rescues E2-S23A stability in N/Tert-1 cells treated with J2 fibroblasts, with an increased E2-S23A acetylation. The results demonstrate that the E2-TopBP1 interaction is critical during the viral life cycle as it prevents fibroblast stimulated SIRT1 mediated deacetylation of E2 that promotes protein degradation. This means that the E2-TopBP1 complex maintains E2 and viral genome stability and that disruption of this complex can promote viral genome integration. Finally, we demonstrate that HPV11 E2 also interacts with TopBP1 and that this interaction is critical for HPV11 E2 stability in the presence of J2 cells. Treatment of N/Tert-1 + 11E2-WT cells with CX4945 results in 11E2 degradation. Therefore, CK2 inhibition is a therapeutic strategy for alleviating HPV11 diseases, including juvenile respiratory papillomatosis. IMPORTANCE Human papillomaviruses are pathogens that cause a host of diseases ranging from benign warts to cancers. There are no therapeutics available for combating these diseases that directly target viral proteins or processes; therefore, we must enhance our understanding of HPV life cycles to assist with identifying novel treatments. In this report, we demonstrate that HPV16 and HPV11 E2 protein expression is dependent upon TopBP1 interaction in keratinocytes interacting with fibroblasts, which recapitulate stromal interactions in culture. The degradation of 16E2 promotes HPV16 genome integration; therefore, the E2-TopBP1 interaction is critical during the viral life cycle. We demonstrate that the CK2 inhibitor CX4945 disrupts HPV11 interaction with TopBP1 and destabilizes HPV11 E2 protein in the presence of J2 fibroblasts; we propose that CX4945 could alleviate HPV11 disease burden.

Human papillomavirus 16 E2 interaction with TopBP1 is required for E2 and viral genome stability during the viral life cycle.

CK2 phosphorylation of HPV16 E2 at serine 23 promotes interaction with TopBP1, and this interaction is important for E2 plasmid segregation function. Here we demonstrate that the E2-TopBP1 interaction is critical for E2 and viral genome stability during the viral life cycle. Introduction of the S23A mutation into the HPV16 genome results in a loss of E2 expression and viral genome integration during organotypic rafting. Co-culture of N/Tert-1+E2-S23A cells with J2 fibroblasts results in E2-S23A degradation via the proteasome, wild-type E2 is not degraded. TopBP1 siRNA treatment of N/Tert-1+E2-WT cells results in E2 degradation only in the presence of J2 cells demonstrating the critical role for TopBP1 in maintaining E2 stability. The CK2 inhibitor CX4945 promotes E2-WT degradation in the presence of fibroblasts as it disrupts E2-TopBP1 interaction. siRNA targeting SIRT1 rescues E2-S23A stability in N/Tert-1 cells treated with J2 fibroblasts, with an increased E2-S23A acetylation. The results demonstrate that the E2-TopBP1 interaction is critical during the viral life cycle as it prevents fibroblast stimulated SIRT1 mediated deacetylation of E2 that promotes protein degradation. This means that the E2-TopBP1 complex maintains E2 and viral genome stability and that disruption of this complex can promote viral genome integration. Finally, we demonstrate that HPV11 E2 also interacts with TopBP1 and that this interaction is critical for HPV11 E2 stability in the presence of J2 cells. Treatment of N/Tert-1+11E2-WT cells with CX4945 results in 11E2 degradation. Therefore, CK2 inhibition is a therapeutic strategy for alleviating HPV11 diseases, including juvenile respiratory papillomatosis. Importance: Human papillomaviruses are pathogens that cause a host of diseases ranging from benign warts to cancers. There are no therapeutics available for combating these diseases that directly target viral proteins or processes, therefore we must enhance our understanding of HPV life cycles to assist with identifying novel treatments. In this report, we demonstrate that HPV16 and HPV11 E2 protein expression is dependent upon TopBP1 interaction in keratinocytes interacting with fibroblasts, which recapitulate stromal interactions in culture. The degradation of 16E2 promotes HPV16 genome integration, therefore the E2-TopBP1 interaction is critical during the viral life cycle. We demonstrate that the CK2 inhibitor CX4945 disrupts HPV11 interaction with TopBP1 and destabilizes HPV11 E2 protein in the presence of J2 fibroblasts; we propose that CX4945 could alleviate HPV11 disease burden.

Human papillomavirus 16 E2 blocks cellular senescence in response to activation of the DNA damage response.

Following infection by HPV16, the viral proteins E1 and E2 induce viral genome replication in association with host factors. Here we demonstrate that E2 also plays a role in promoting short-term cellular proliferation in the presence of an active DDR. Cisplatin treatment of E2 expressing cells results in short-term proliferation likely due to a block of cellular senescence and apoptosis. However, long-term growth of E2 expressing cells following cisplatin treatment is attenuated due to an accumulation of DNA damage. We discuss a possible role for this E2 function during the viral life cycle. It is also notable that E2 expressing HPV16 positive cancers have a better clinical outcome than non-E2 expressing tumors. While there are a variety of reasons for the better outcome of patients with E2 expressing tumors, this report suggests that E2 regulation of the DNA damage response may be a contributory factor.

Interaction with TopBP1 Is Required for Human Papillomavirus 16 E2 Plasmid Segregation/Retention Function during Mitosis.

Human papillomavirus 16 (HPV16) E2 is a DNA-binding protein that regulates transcription, replication and potentially, segregation of the HPV16 genome during the viral life cycle. In the segregation model, E2 simultaneously binds to viral and host chromatin, acting as a bridge to ensure that viral genomes reside in daughter nuclei following cell division. The host chromatin receptor for E2 mediating this function is unknown. Recently, we demonstrated that CK2 phosphorylation of E2 on serine 23 (S23) is required for interaction with TopBP1, and that this interaction promotes E2 and TopBP1 recruitment to mitotic chromatin. Here, we demonstrate that in U2OS cells expressing wild-type E2 and a non-TopBP1-binding mutant (S23A, serine 23 mutated to alanine), interaction with TopBP1 is essential for E2 recruitment of plasmids to mitotic chromatin. Using novel quantitative segregation assays, we demonstrate that interaction with TopBP1 is required for E2 plasmid segregation function in U2OS and N/Tert-1 cells. Small interfering RNA (siRNA) knockdown of TopBP1 or CK2 enzyme components disrupts E2 segregation/retention function. The interaction of E2 with TopBP1 promotes increased levels of E2 protein during mitosis in U2OS and N/Tert-1 cells, as well as in human foreskin keratinocytes (HFK) immortalized by the HPV16 genome. Overall, our results demonstrate that E2 has plasmid segregation activity, and that the E2-TopBP1 interaction is essential for this E2 function. IMPORTANCE HPV16 causes 3% to 4% of all human cancers. It is proposed that during the viral life cycle, the viral genome is actively segregated into daughter nuclei, ensuring viral replication in the subsequent S phase. The E2 protein potentially bridges the viral and host genomes during mitosis to mediate segregation of the circular viral plasmid. Here, we demonstrate that E2 has the ability to mediate plasmid segregation, and that this function is dependent upon interaction with the host protein TopBP1. Additionally, we demonstrate that the E2-TopBP1 interaction promotes enhanced E2 expression during mitosis, which likely promotes the plasmid segregation function of E2. Overall, our results present a mechanism of how HPV16 can segregate its viral genome during an active infection, a critical aspect of the viral life cycle.

A Critical Role for p53 during the HPV16 Life Cycle.

Human papillomaviruses (HPV) are causative agents in ano-genital and oral cancers; HPV16 is the most prevalent type detected in human cancers. The HPV16 E6 protein targets p53 for proteasomal degradation to facilitate proliferation of the HPV16 infected cell. However, in HPV16 immortalized cells E6 is predominantly spliced (E6*) and unable to degrade p53. Here, we demonstrate that human foreskin keratinocytes immortalized by HPV16 (HFK+HPV16), and HPV16 positive oropharyngeal cancers, retain significant expression of p53. In addition, p53 levels increase in HPV16+ head and neck cancer cell lines following treatment with cisplatin. Introduction of full-length E6 into HFK+HPV16 resulted in attenuation of cellular growth (in hTERT immortalized HFK, E6 expression promoted enhanced proliferation). An understudied interaction is that between E2 and p53 and we investigated whether this was important for the viral life cycle. We generated mutant genomes with E2 unable to interact with p53 resulting in profound phenotypes in primary HFK. The mutant induced hyper-proliferation, but an ultimate arrest of cell growth; ß-galactosidase staining demonstrated increased senescence, and COMET assays showed increased DNA damage compared with HFK+HPV16 wild-type cells. There was failure of the viral life cycle in organotypic rafts with the mutant HFK resulting in premature differentiation and reduced proliferation. The results demonstrate that p53 expression is critical during the HPV16 life cycle, and that this may be due to a functional interaction between E2 and p53. Disruption of this interaction has antiviral potential. IMPORTANCE Human papillomaviruses are causative agents in around 5% of all cancers. There are currently no antivirals available to combat these infections and cancers, therefore it remains a priority to enhance our understanding of the HPV life cycle. Here, we demonstrate that an interaction between the viral replication/transcription/segregation factor E2 and the tumor suppressor p53 is critical for the HPV16 life cycle. HPV16 immortalized cells retain significant expression of p53, and the critical role for the E2-p53 interaction demonstrates why this is the case. If the E2-p53 interaction is disrupted then HPV16 immortalized cells fail to proliferate, have enhanced DNA damage and senescence, and there is premature differentiation during the viral life cycle. Results suggest that targeting the E2-p53 interaction would have therapeutic benefits, potentially attenuating the spread of HPV16.

CK2 Phosphorylation of Human Papillomavirus 16 E2 on Serine 23 Promotes Interaction with TopBP1 and Is Critical for E2 Interaction with Mitotic Chromatin and the Viral Life Cycle.

During the human papillomavirus 16 (HPV16) life cycle, the E2 protein interacts with host factors to regulate viral transcription, replication, and genome segregation/retention. Our understanding of host partner proteins and their roles in E2 functions remains incomplete. Here we demonstrate that CK2 phosphorylation of E2 on serine 23 promotes interaction with TopBP1 in vitro and in vivo and that E2 is phosphorylated on this residue during the HPV16 life cycle. We investigated the consequences of mutating serine 23 on E2 functions. E2-S23A (E2 with serine 23 mutated to alanine) activates and represses transcription identically to E2-WT (wild-type E2), and E2-S23A is as efficient as E2-WT in transient replication assays. However, E2-S23A has compromised interaction with mitotic chromatin compared with E2-WT. In E2-WT cells, both E2 and TopBP1 levels increase during mitosis compared with vector control cells. In E2-S23A cells, neither E2 nor TopBP1 levels increase during mitosis. Introduction of the S23A mutation into the HPV16 genome resulted in delayed immortalization of human foreskin keratinocytes (HFK) and higher episomal viral genome copy number in resulting established HFK. Remarkably, S23A cells had a disrupted viral life cycle in organotypic raft cultures, with a loss of E2 expression and a failure of viral replication. Overall, our results demonstrate that CK2 phosphorylation of E2 on serine 23 promotes interaction with TopBP1 and that this interaction is critical for the viral life cycle. IMPORTANCE Human papillomaviruses are causative agents in around 5% of all cancers, with no specific antiviral therapeutics available for treating infections or resultant cancers. In this report, we demonstrate that phosphorylation of HPV16 E2 by CK2 promotes formation of a complex with the cellular protein TopBP1 in vitro and in vivo. This complex results in stabilization of E2 during mitosis. We demonstrate that CK2 phosphorylates E2 on serine 23 in vivo and that CK2 inhibitors disrupt the E2-TopBP1 complex. Mutation of E2 serine 23 to alanine disrupts the HPV16 life cycle, hindering immortalization and disrupting the viral life cycle, demonstrating a critical function for this residue.

Human Papillomavirus 16 (HPV16) E2 Repression of TWIST1 Transcription Is a Potential Mediator of HPV16 Cancer Outcomes.

Human papillomaviruses (HPVs) are causative agents in around 5% of all cancers, including cervical and oropharyngeal. A feature of HPV cancers is their better clinical outcome compared with non-HPV anatomical counterparts. In turn, the presence of E2 predicts a better clinical outcome in HPV-positive cancers; the reason(s) for the better outcome of E2-positive patients is not fully understood. Previously, we demonstrated that HPV16 E2 regulates host gene transcription that is relevant to the HPV16 life cycle in N/Tert-1 cells. One of the genes repressed by E2 and the entire HPV16 genome in N/Tert-1 cells is TWIST1. Here, we demonstrate that TWIST1 RNA levels are reduced in HPV-positive versus HPV-negative head and neck cancer and that E2 and HPV16 downregulate both TWIST1 RNA and protein in our N/Tert-1 model; E6/E7 cannot repress TWIST1. E2 represses the TWIST1 promoter in transient assays and is localized to the TWIST1 promoter; E2 also induces repressive epigenetic changes on the TWIST1 promoter. TWIST1 is a master transcriptional regulator of the epithelial to mesenchymal transition (EMT), and a high level of TWIST1 is a prognostic marker indicative of poor cancer outcomes. We demonstrate that TWIST1 target genes are also downregulated in E2-positive N/Tert-1 cells and that E2 promotes a failure in wound healing, a phenotype of low TWIST1 levels. We propose that the presence of E2 in HPV-positive tumors leads to TWIST1 repression and that this plays a role in the better clinical response of E2-positive HPV tumors.IMPORTANCE HPV16-positive cancers have a better clinical outcome that their non-HPV anatomical counterparts. Furthermore, the presence of HPV16 E2 RNA predicts a better outcome for HPV16-positive tumors; the reasons for this are not known. Here, we demonstrate that E2 represses expression of the TWIST1 gene; an elevated level of this gene is a marker of poor prognosis for a variety of cancers. We demonstrate that E2 directly binds to the TWIST1 promoter and actively represses transcription. TWIST1 is a master regulator promoting EMT, and here, we demonstrate that the presence of E2 reduces the ability of N/Tert-1 cells to wound heal. Overall, we propose that the E2 repression of TWIST1 may contribute to the better clinical outcome of E2-positive HPV16-positive tumors.

Estrogen Attenuates the Growth of Human Papillomavirus-Positive Epithelial Cells.

Human papillomaviruses (HPVs) are small, double-stranded DNA viruses that are significant risk factors in the development of cancer, and HPV accounts for approximately 5% of all worldwide cancers. Recent studies using data from The Cancer Genome Atlas (TCGA) have demonstrated that elevated levels of estrogen receptor alpha (ERα) are associated with improved survival in oropharyngeal cancers, and these elevated receptor levels were linked with human papillomavirus-positive cancers (HPV+cancers). There has been a dramatic increase in HPV-related head and neck squamous cell carcinomas (HPV+HNSCCs) over the last 2 decades, and therapeutic options for this ongoing health crisis are a priority; currently, there are no antiviral therapeutics available for combatting HPV+cancers. During our TGCA studies on head and neck cancer, we had also discovered the overexpression of ERα in HPV+cancers. Here, we demonstrate that 17ß-estradiol (estrogen) attenuates the growth/cell viability of HPV+cancers in vitro, but not HPV-negative cancer cells. In addition, N/Tert-1 cells (foreskin keratinocytes immortalized with human telomerase reverse transcriptase [hTERT]) containing human papillomavirus 16 (HPV16) have elevated levels of ERα and growth sensitivity after estrogen treatment compared with parental N/Tert-1 cells. Finally, we demonstrate that there are potentially two mechanisms contributing to the attenuation of HPV+ cell growth following estrogen treatment. First, estrogen represses the viral transcriptional long control region (LCR) downregulating early gene expression, including E6/E7. Second, expression of E6 and E7 by themselves sensitizes cells to estrogen. Overall, our results support the recent proposal that estrogen could be exploited therapeutically for the treatment of HPV-positive oral cancers.IMPORTANCE Human papillomaviruses cause around 5% of all human cancers, yet there are no specific antiviral therapeutic approaches available for combatting these cancers. These cancers are currently treated with standard chemoradiation therapy (CRT). Specific antiviral reagents are desperately required, particularly for HPV+HNSCC whose incidence is increasing and for which there are no diagnostic tools available for combatting this disease. Using data from The Cancer Genome Atlas (TCGA), we and others determined that the estrogen receptor alpha (ERα) is overexpressed in HPV+HNSCC and that elevated levels are associated with an improved disease outcome. This has led to the proposal that estrogen treatment could be a novel therapeutic approach for combatting HPV+cancers. Here, we demonstrate that estrogen attenuates the growth of HPV+epithelial cells using multiple mechanisms, supporting the idea that estrogen has potential as a therapeutic agent for the treatment of HPV+HNSCC.

Human Papillomavirus 16 E6 and E7 Synergistically Repress Innate Immune Gene Transcription.

Human papillomaviruses (HPV) are causative agents in 5% of all cancers, including the majority of anogenital and oropharyngeal cancers. Downregulation of innate immune genes (IIGs) by HPV to promote the viral life cycle is well documented; E6 and E7 are known repressors of these genes. More recently, we demonstrated that E2 could also repress IIGs. These studies have been carried out in cells overexpressing the viral proteins, and to further investigate the role of individual viral proteins in this repression, we introduced stop codons into E6 and/or E7 in the entire HPV16 genome and generated N/Tert-1 cells stably maintaining the HPV16 genomes. We demonstrate that E6 or E7 individually is not sufficient to repress IIG expression in the context of the entire HPV16 genome; both are required for a synergistic repression. The DNA damage response (DDR) is activated by HPV16 irrespective of E6 and E7 expression, presumably due to viral replication; E1 is a known activator of the DDR. In addition, replication stress was apparent in HPV16-positive cells lacking E6 and E7, manifested by attenuated cellular growth and activation of replication stress genes. These studies led us to the following model. Viral replication per se can activate the DDR following infection, and this activation is a known inducer of IIG expression, which may induce cellular senescence. To combat this, E6 and E7 synergistically combine to manipulate the DDR and actively repress innate immune gene expression promoting cellular growth; neither protein by itself is able to do this.IMPORTANCE The role of human papillomavirus 16 (HPV16) in human cancers is well established; however, to date there are no antiviral therapeutics that are available for combatting these cancers. To identify such targets, we must enhance the understanding of the viral life cycle. Innate immune genes (IIGs) are repressed by HPV16, and we have reported that this repression persists through to cancer. Reversal of this repression would boost the immune response to HPV16-positive tumors, an area that is becoming more important given the advances in immunotherapy. This report demonstrates that E6 and E7 synergistically repress IIG expression in the context of the entire HPV16 genome. Removal of either protein activates the expression of IIGs by HPV16. Therefore, gaining a precise understanding of how the viral oncogenes repress IIG expression represents an opportunity to reverse this repression and boost the immune response to HPV16 infections for therapeutic gain.