A human papillomavirus 16 E2-TopBP1 dependent SIRT1-p300 acetylation switch regulates mitotic viral and human protein levels and activates the DNA damage response.

An interaction between human papillomavirus 16 (HPV16) E2 and the cellular proteins TopBP1 and BRD4 is required for E2 plasmid segregation function. The E2-TopBP1 interaction promotes increased mitotic E2 protein levels in U2OS and N/Tert-1 cells, as well as in human foreskin keratinocytes immortalized by HPV16 (HFK + HPV16). SIRT1 deacetylation reduces E2 protein stability and here we demonstrate that increased E2 acetylation occurs during mitosis in a TopBP1 interacting-dependent manner, promoting E2 mitotic stabilization. p300 mediates E2 acetylation and acetylation is increased due to E2 switching off SIRT1 function during mitosis in a TopBP1 interacting-dependent manner, confirmed by increased p53 stability and acetylation on lysine 382, a known target for SIRT1 deacetylation. SIRT1 can complex with E2 in growing cells but is unable to do so during mitosis due to the E2-TopBP1 interaction; SIRT1 is also unable to complex with p53 in mitotic E2 wild-type cells but can complex with p53 outside of mitosis. E2 lysines 111 and 112 are highly conserved residues across all E2 proteins and we demonstrate that K111 hyper-acetylation occurs during mitosis, promoting E2 interaction with Topoisomerase 1 (Top1). We demonstrate that K112 ubiquitination promotes E2 proteasomal degradation during mitosis. E2-TopBP1 interaction promotes mitotic acetylation of CHK2, promoting phosphorylation and activation of the DNA damage response (DDR). The results present a new model in which the E2-TopBP1 complex inactivates SIRT1 during mitosis, and activates the DDR. This is a novel mechanism of HPV16 activation of the DDR, a requirement for the viral life cycle. IMPORTANCE: Human papillomaviruses (HPVs) are causative agents in around 5% of all human cancers. While there are prophylactic vaccines that will significantly alleviate HPV disease burden on future generations, there are currently no anti-viral strategies available for the treatment of HPV cancers. To generate such reagents, we must understand more about the HPV life cycle, and in particular about viral-host interactions. Here, we describe a novel mitotic complex generated by the HPV16 E2 protein interacting with the host protein TopBP1 that controls the function of the deacetylase SIRT1. The E2-TopBP1 interaction disrupts SIRT1 function during mitosis in order to enhance acetylation and stability of viral and host proteins. We also demonstrate that the E2-TopBP1 interaction activates the DDR. This novel complex is essential for the HPV16 life cycle and represents a novel anti-viral therapeutic target.

A human papillomavirus 16 E2-TopBP1 dependent SIRT1-p300 acetylation switch regulates mitotic viral and human protein levels.

An interaction between human papillomavirus 16 (HPV16) E2 and the cellular proteins TopBP1 and BRD4 is required for E2 plasmid segregation function. The E2-TopBP1 interaction promotes increased mitotic E2 protein levels in U2OS and N/Tert-1 cells, as well as in human foreskin keratinocytes immortalized by HPV16 (HFK+HPV16). SIRT1 deacetylation reduces E2 protein stability and here we demonstrate that increased E2 acetylation occurs during mitosis in a TopBP1 interacting dependent manner, promoting E2 mitotic stabilization. p300 mediates E2 acetylation and acetylation is increased due to E2 switching off SIRT1 function during mitosis in a TopBP1 interacting dependent manner, confirmed by increased p53 stability and acetylation on lysine 382, a known target for SIRT1 deacetylation. SIRT1 can complex with E2 in growing cells but is unable to do so during mitosis due to the E2-TopBP1 interaction; SIRT1 is also unable to complex with p53 in mitotic E2 wild type cells but can complex with p53 outside of mitosis. E2 lysines 111 and 112 are highly conserved residues across all E2 proteins and we demonstrate that K111 hyper-acetylation occurs during mitosis, promoting E2 interaction with Topoisomerase 1 (Top1). We also demonstrate that K112 ubiquitination promotes E2 proteasomal degradation during mitosis. The results present a model in which the E2-TopBP1 complex inactivates SIRT1 during mitosis and E2 acetylation on K111 by p300 increases, promoting interaction with Top1 that protects K112 from ubiquitination and therefore E2 proteasomal degradation. Importance: Human papillomaviruses are causative agents in around 5% of all human cancers. While there are prophylactic vaccines that will significantly alleviate HPV disease burden on future generations, there are currently no anti-viral strategies available for the treatment of HPV cancers. To generate such reagents, we must understand more about the HPV life cycle, and in particular about viral-host interactions. Here we describe a novel mitotic complex generated by the HPV16 E2 protein interacting with the host protein TopBP1 that controls the function of the deacetylase SIRT1. The E2-TopBP1 interaction disrupts SIRT1 function during mitosis in order to enhance acetylation and stability of viral and host proteins. This novel complex is essential for the HPV16 life cycle and represents a novel anti-viral therapeutic target.

Human Papillomavirus 16 replication converts SAMHD1 into a homologous recombination factor and promotes its recruitment to replicating viral DNA.

We have demonstrated that SAMHD1 (sterile alpha motif and histidine-aspartic domain HD-containing protein 1) is a restriction factor for the HPV16 life cycle. Here we demonstrate that in HPV negative cervical cancer C33a cells and human foreskin keratinocytes immortalized by HPV16 (HFK+HPV16), SAMHD1 is recruited to E1-E2 replicating DNA. Homologous recombination (HR) factors are required for HPV16 replication and viral replication promotes phosphorylation of SAMHD1, which converts it from a dNTPase to an HR factor independent from E6/E7 expression. A SAMHD1 phosphor-mimic (SAMHD1 T592D) reduces E1-E2 mediated DNA replication in C33a cells and has enhanced recruitment to the replicating DNA. In HFK+HPV16 cells SAMHD1 T592D is recruited to the viral DNA and attenuates cellular growth, but does not attenuate growth in isogenic HFK cells immortalized by E6/E7 alone. SAMHD1 T592D also attenuates the development of viral replication foci following keratinocyte differentiation. The results indicated that enhanced SAMHD1 phosphorylation could be therapeutically beneficial in cells with HPV16 replicating genomes. Protein phosphatase 2A (PP2A) can dephosphorylate SAMHD1 and PP2A function can be inhibited by endothall. We demonstrate that endothall reduces E1-E2 replication and promotes SAMHD1 recruitment to E1-E2 replicating DNA, mimicking the SAMHD1 T592D phenotypes. Finally, we demonstrate that in head and neck cancer cell lines with HPV16 episomal genomes endothall attenuates their growth and promotes recruitment of SAMHD1 to the viral genome. The results suggest that targeting cellular phosphatases has therapeutic potential for the treatment of HPV infections and cancers. Importance: Human papillomaviruses are causative agents in around 5% of all human cancers. The development of anti-viral therapeutics depends upon an increased understanding of the viral life cycle. Here we demonstrate that HPV16 replication converts SAMHD1 into an HR factor via phosphorylation. This phosphorylation promotes recruitment of SAMHD1 to viral DNA to assist with replication. A SAMHD1 mutant that mimics phosphorylation is hyper-recruited to viral DNA and attenuates viral replication. Expression of this mutant in HPV16 immortalized cells attenuates the growth of these cells, but not cells immortalized by the viral oncogenes E6/E7 alone. Finally, we demonstrate that the phosphatase inhibitor endothall promotes hyper-recruitment of endogenous SAMHD1 to HPV16 replicating DNA and can attenuate the growth of both HPV16 immortalized human foreskin keratinocytes and HPV16 positive head and neck cancer cell lines. We propose that phosphatase inhibitors represent a novel tool for combating HPV infections and disease.

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 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.

SAMHD1 Regulates Human Papillomavirus 16-Induced Cell Proliferation and Viral Replication during Differentiation of Keratinocytes.

Human papillomaviruses induce a host of anogenital cancers, as well as oropharyngeal cancer (HPV+OPC); human papillomavirus 16 (HPV16) is causative in around 90% of HPV+OPC cases. Using telomerase reverse transcriptase (TERT) immortalized foreskin keratinocytes (N/Tert-1), we have identified significant host gene reprogramming by HPV16 (N/Tert-1+HPV16) and demonstrated that N/Tert-1+HPV16 support late stages of the viral life cycle. Expression of the cellular dNTPase and homologous recombination factor sterile alpha motif and histidine-aspartic domain HD-containing protein 1 (SAMHD1) is transcriptionally regulated by HPV16 in N/Tert-1. CRISPR/Cas9 removal of SAMHD1 from N/Tert-1 and N/Tert-1+HPV16 demonstrates that SAMHD1 controls cell proliferation of N/Tert-1 only in the presence of HPV16; the deletion of SAMHD1 promotes hyperproliferation of N/Tert-1+HPV16 cells in organotypic raft cultures but has no effect on N/Tert-1. Viral replication is also elevated in the absence of SAMHD1. This new system has allowed us to identify a specific interaction between SAMHD1 and HPV16 that regulates host cell proliferation and viral replication; such studies are problematic in nonimmortalized primary keratinocytes due to their limited life span. To confirm the relevance of our results, we repeated the analysis with human tonsil keratinocytes (HTK) immortalized by HPV16 (HTK+HPV16) and observed the same hyperproliferative phenotype following CRISPR/Cas9 editing of SAMHD1. Identical results were obtained with three independent CRISPR/Cas9 guide RNAs. The isogenic pairing of N/Tert-1 with N/Tert-1+HPV16, combined with HTK+HPV16, presents a unique system to identify host genes whose products functionally interact with HPV16 to regulate host cellular growth in keratinocytes.IMPORTANCE HPVs are causative agents in human cancers and are responsible for around of 5% of all cancers. A better understanding of the viral life cycle in keratinocytes will facilitate the development of novel therapeutics to combat HPV-positive cancers. Here, we present a unique keratinocyte model to identify host proteins that specifically interact with HPV16. Using this system, we report that a cellular gene, SAMHD1, is regulated by HPV16 at the RNA and protein levels in keratinocytes. Elimination of SAMHD1 from these cells using CRISPR/Cas9 editing promotes enhanced cellular proliferation by HPV16 in keratinocytes and elevated viral replication but not in keratinocytes that do not have HPV16. Our study demonstrates a specific intricate interplay between HPV16 and SAMHD1 during the viral life cycle and establishes a unique model system to assist exploring host factors critical for HPV pathogenesis.

Structural and functional probing of PorZ, an essential bacterial surface component of the type-IX secretion system of human oral-microbiomic Porphyromonas gingivalis.

Porphyromonas gingivalis is a member of the human oral microbiome abundant in dysbiosis and implicated in the pathogenesis of periodontal (gum) disease. It employs a newly described type-IX secretion system (T9SS) for secretion of virulence factors. Cargo proteins destined for secretion through T9SS carry a recognition signal in the conserved C-terminal domain (CTD), which is removed by sortase PorU during translocation. Here, we identified a novel component of T9SS, PorZ, which is essential for surface exposure of PorU and posttranslational modification of T9SS cargo proteins. These include maturation of enzyme precursors, CTD removal and attachment of anionic lipopolysaccharide for anchorage in the outer membrane. The crystal structure of PorZ revealed two ß-propeller domains and a C-terminal ß-sandwich domain, which conforms to the canonical CTD architecture. We further documented that PorZ is itself transported to the cell surface via T9SS as a full-length protein with its CTD intact, independently of the presence or activity of PorU. Taken together, our results shed light on the architecture and possible function of a novel component of the T9SS. Knowledge of how T9SS operates will contribute to our understanding of protein secretion as part of host-microbiome interactions by dysbiotic members of the human oral cavity.