The membrane-associated ubiquitin ligase MARCHF8 stabilizes the human papillomavirus oncoprotein E7 by degrading CUL1 and UBE2L3 in head and neck cancer.

The human papillomavirus (HPV) oncoprotein E7 is a relatively short-lived protein required for HPV-driven cancer development and maintenance. E7 is degraded through ubiquitination mediated by cullin 1 (CUL1) and the ubiquitin-conjugating enzyme E2 L3 (UBE2L3). However, E7 proteins are maintained at high levels in most HPV-positive cancer cells. A previous proteomics study has shown that UBE2L3 and CUL1 protein levels are increased by the knockdown of the E3 ubiquitin ligase membrane-associated ring-CH-type finger 8 (MARCHF8). We have recently demonstrated that HPV16 upregulates MARCHF8 expression in HPV-positive keratinocytes and head and neck cancer (HPV+ HNC) cells. Here, we report that MARCHF8 stabilizes the HPV16 E7 protein by degrading the components of the S-phase kinase-associated protein 1-CUL1-F-box ubiquitin ligase complex in HPV+ HNC cells. We found that MARCHF8 knockdown in HPV+ HNC cells drastically decreases the HPV16 E7 protein level while increasing the CUL1 and UBE2L3 protein levels. We further revealed that the MARCHF8 protein binds to and ubiquitinates CUL1 and UBE2L3 proteins and that MARCHF8 knockdown enhances the ubiquitination of the HPV16 E7 protein. Conversely, the overexpression of CUL1 and UBE2L3 in HPV+ HNC cells decreases HPV16 E7 protein levels and suppresses tumor growth in vivo. Our findings suggest that HPV-induced MARCHF8 prevents the degradation of the HPV16 E7 protein in HPV+ HNC cells by ubiquitinating and degrading CUL1 and UBE2L3 proteins.IMPORTANCESince human papillomavirus (HPV) oncoprotein E7 is essential for virus replication; HPV has to maintain high levels of E7 expression in HPV-infected cells. However, HPV E7 can be efficiently ubiquitinated by a ubiquitin ligase and degraded by proteasomes in the host cell. Mechanistically, the E3 ubiquitin ligase complex cullin 1 (CUL1) and ubiquitin-conjugating enzyme E2 L3 (UBE2L3) components play an essential role in E7 ubiquitination and degradation. Here, we show that the membrane ubiquitin ligase membrane-associated ring-CH-type finger 8 (MARCHF8) induced by HPV16 E6 stabilizes the E7 protein by degrading CUL1 and UBE2L3 and blocking E7 degradation through proteasomes. MARCHF8 knockout restores CUL1 and UBE2L3 expression, decreasing E7 protein levels and inhibiting the proliferation of HPV-positive cancer cells. Additionally, overexpression of CUL1 or UBE2L3 decreases E7 protein levels and suppresses in vivo tumor growth. Our results suggest that HPV16 maintains high E7 protein levels in the host cell by inducing MARCHF8, which may be critical for cell proliferation and tumorigenesis.

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 human papillomavirus 16 (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 phospho-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 (HPVs) 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 sterile alpha motif and histidine-aspartic domain HD-containing protein 1 (SAMHD1) into a homologous recombination (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 (HFKs) and HPV16-positive head and neck cancer cell lines. We propose that phosphatase inhibitors represent a novel tool for combating HPV infections and disease.

HPV upregulates MARCHF8 ubiquitin ligase and inhibits apoptosis by degrading the death receptors in head and neck cancer.

The membrane-associated RING-CH-type finger ubiquitin ligase MARCHF8 is a human homolog of the viral ubiquitin ligases Kaposi's sarcoma herpesvirus K3 and K5 that promote host immune evasion. Previous studies have shown that MARCHF8 ubiquitinates several immune receptors, such as the major histocompatibility complex II and CD86. While human papillomavirus (HPV) does not encode any ubiquitin ligase, the viral oncoproteins E6 and E7 are known to regulate host ubiquitin ligases. Here, we report that MARCHF8 expression is upregulated in HPV-positive head and neck cancer (HNC) patients but not in HPV-negative HNC patients compared to normal individuals. The MARCHF8 promoter is highly activated by HPV oncoprotein E6-induced MYC/MAX transcriptional activation. The knockdown of MARCHF8 expression in human HPV-positive HNC cells restores cell surface expression of the tumor necrosis factor receptor superfamily (TNFRSF) death receptors, FAS, TRAIL-R1, and TRAIL-R2, and enhances apoptosis. MARCHF8 protein directly interacts with and ubiquitinates the TNFRSF death receptors. Further, MARCHF8 knockout in mouse oral cancer cells expressing HPV16 E6 and E7 augments cancer cell apoptosis and suppresses tumor growth in vivo. Our findings suggest that HPV inhibits host cell apoptosis by upregulating MARCHF8 and degrading TNFRSF death receptors in HPV-positive HNC cells.

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.

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.

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.

PTPN14 degradation by high-risk human papillomavirus E7 limits keratinocyte differentiation and contributes to HPV-mediated oncogenesis.

High-risk human papillomavirus (HPV) E7 proteins enable oncogenic transformation of HPV-infected cells by inactivating host cellular proteins. High-risk but not low-risk HPV E7 target PTPN14 for proteolytic degradation, suggesting that PTPN14 degradation may be related to their oncogenic activity. HPV infects human keratinocytes but the role of PTPN14 in keratinocytes and the consequences of PTPN14 degradation are unknown. Using an HPV16 E7 variant that can inactivate retinoblastoma tumor suppressor (RB1) but cannot degrade PTPN14, we found that high-risk HPV E7-mediated PTPN14 degradation impairs keratinocyte differentiation. Deletion of PTPN14 from primary human keratinocytes decreased keratinocyte differentiation gene expression. Related to oncogenic transformation, both HPV16 E7-mediated PTPN14 degradation and PTPN14 deletion promoted keratinocyte survival following detachment from a substrate. PTPN14 degradation contributed to high-risk HPV E6/E7-mediated immortalization of primary keratinocytes and HPV+ but not HPV- cancers exhibit a gene-expression signature consistent with PTPN14 inactivation. We find that PTPN14 degradation impairs keratinocyte differentiation and propose that this contributes to high-risk HPV E7-mediated oncogenic activity independent of RB1 inactivation.

Identifying predictors of HPV-related head and neck squamous cell carcinoma progression and survival through patient-derived models.

Therapeutic innovation for human papilloma virus-related (HPV+) head and neck squamous cell carcinomas (HNSCCs) is impaired by inadequate preclinical models and the absence of accurate biomarkers. Our study establishes the first well-characterized panel of patient-derived xenografts (PDXs) and organoids from HPV+ HNSCCs while determining fidelity of the models to the distinguishing genetic features of this cancer type. Despite low engraftment rates, whole exome sequencing showed that PDXs retain multiple distinguishing features of HPV+ HNSCC lost in existing cell lines, including PIK3CA mutations, TRAF3 deletion and the absence of EGFR amplifications. Engrafted HPV+ tumors frequently contained NOTCH1 mutations, thus providing new models for a negatively prognostic alteration in this disease. Genotype-phenotype associations in the models were then tested for prediction of tumor progression and survival in published clinical cohorts. Observation of high tumor mutational burdens (TMBs) in the faster-growing models facilitated identification of a novel association between TMB and local progression in both HPV+ and HPV- patients that was prognostic in HPV- cases. In addition, reduced E7 and p16INK4A levels found in a PDX from an outlier case with lethal outcome led to detection of similar profiles among recurrent HPV+ HNSCCs. Transcriptional data from the Cancer Genome Atlas was used to demonstrate that the lower E2F target gene expression predicted by reduced E7 levels has potential as a biomarker of disease recurrence risk. Our findings bridge a critical gap in preclinical models for HPV+ HNSCCs and simultaneously reveal novel potential applications of quantifying mutational burden and viral oncogene functions for biomarker development.

Human Papillomavirus 16 E2 Regulates Keratinocyte Gene Expression Relevant to Cancer and the Viral Life Cycle.

Human papillomaviruses (HPVs) are causative agents in ano-genital and oropharyngeal cancers. The virus must reprogram host gene expression to promote infection, and E6 and E7 contribute to this via the targeting of cellular transcription factors, including p53 and pRb, respectively. The HPV16 E2 protein regulates host gene expression in U2OS cells, and in this study, we extend these observations into telomerase reverse transcriptase (TERT) immortalized oral keratinocytes (NOKs) that are capable of supporting late stages of the HPV16 life cycle. We observed repression of innate immune genes by E2 that are also repressed by the intact HPV16 genome in NOKs. Transcriptome sequencing (RNA-seq) data identified 167 up- and 395 downregulated genes by E2; there was a highly significant overlap of the E2-regulated genes with those regulated by the intact HPV16 genome in the same cell type. Small interfering RNA (siRNA) targeting of E2 reversed the repression of E2-targeted genes. The ability of E2 to repress innate immune genes was confirmed in an ano-genital immortalized keratinocyte cell line, N/Tert-1. We present the analysis of data from The Cancer Genome Atlas (TCGA) for HPV16-positive and -negative head and neck cancers (HNC) suggesting that E2 plays a role in the regulation of the host genome in cancers. Patients with HPV16-positive HNC with a loss of E2 expression exhibited a worse clinical outcome, and we discuss how this could, at least partially, be related to the loss of E2 host gene regulation.IMPORTANCE Human papillomavirus 16 (HPV16)-positive tumors that retain expression of E2 have a better clinical outcome than those that have lost E2 expression. It has been suggested that this is due to a loss of E2 repression of E6 and E7 expression, but this is not supported by data from tumors where there is not more E6 and E7 expression in the absence of E2. Here we report that E2 regulates host gene expression and place this regulation in the context of the HPV16 life cycle and HPV16-positive head and neck cancers (the majority of which retain E2 expression). We propose that this E2 function may play an important part in the increased response of HPV16-positive cancers to radiation therapy. Therefore, host gene regulation by E2 may be important for promotion of the HPV16 life cycle and also for the response of HPV16-positive tumors to radiation therapy.

Using a Human Papillomavirus Model to Study DNA Replication and Repair of Wild Type and Damaged DNA Templates in Mammalian Cells.

Human papillomaviruses have 8kbp DNA episomal genomes that replicate autonomously from host DNA. During initial infection, the virus increases its copy number to 20-50 copies per cell, causing torsional stress on the replicating DNA. This activates the DNA damage response (DDR) and HPV replicates its genome, at least in part, using homologous recombination. An active DDR is on throughout the HPV life cycle. Two viral proteins are required for replication of the viral genome; E2 binds to 12bp palindromic sequences around the A/T rich origin of replication and recruits the viral helicase E1 via a protein-protein interaction. E1 forms a di-hexameric complex that replicates the viral genome in association with host factors. Transient replication assays following transfection with E1-E2 expression plasmids, along with an origin containing plasmid, allow monitoring of E1-E2 replication activity. Incorporating a bacterial lacZ gene into the origin plasmid allows for the determination of replication fidelity. Here we describe how we exploited this system to investigate replication and repair in mammalian cells, including using damaged DNA templates. We propose that this system has the potential to enhance the understanding of cellular components involved in DNA replication and repair.

The Deacetylase SIRT1 Regulates the Replication Properties of Human Papillomavirus 16 E1 and E2.

Human papillomaviruses (HPV) replicate their genomes in differentiating epithelium using the viral proteins E1 and E2 in association with host proteins. While the roles of E1 and E2 in this process are understood, the host factors involved and how they interact with and regulate E1-E2 are not. Our previous work identified the host replication and repair factor TopBP1 as an E2 partner protein essential for optimal E1-E2 replication and for the viral life cycle. The role of TopBP1 in host DNA replication is regulated by the class III deacetylase SIRT1; activation of the DNA damage response prevents SIRT1 deacetylation of TopBP1, resulting in a switch from DNA replication to repair functions for this protein and cell cycle arrest. Others have demonstrated an essential role for SIRT1 in regulation of the HPV31 life cycle; here, we report that SIRT1 can directly regulate HPV16 E1-E2-mediated DNA replication. SIRT1 is part of the E1-E2 DNA replication complex and is recruited to the viral origin of replication in an E1-E2-dependent manner. CRISPR/Cas9 was used to generate C33a clones with undetectable SIRT1 expression and lack of SIRT1 elevated E1-E2 DNA replication, in part due to increased acetylation and stabilization of the E2 protein in the absence of SIRT1. The results demonstrate that SIRT1 is a member of, and can regulate, the HPV16 replication complex. We discuss the potential role of this protein in the viral life cycle.IMPORTANCE HPV are causative agents in a number of human diseases, and currently only the symptoms of these diseases are treated. To identify novel therapeutic approaches for combating these diseases, the viral life cycle must be understood in more detail. This report demonstrates that a cellular enzyme, SIRT1, is part of the HPV16 DNA replication complex and is brought to the viral genome by the viral proteins E1 and E2. Using gene editing technology (CRISPR/Cas9), the SIRT1 gene was removed from cervical cancer cells. The consequence of this was that viral replication was elevated, probably due to a stabilization of the viral replication factor E2. The overall results demonstrate that an enzyme with known inhibitors, SIRT1, plays an important role in controlling how HPV16 makes copies of itself. Targeting this enzyme could be a new therapeutic approach for combating HPV spread and disease.

Evidence supporting a role for TopBP1 and Brd4 in the initiation but not continuation of human papillomavirus 16 E1/E2-mediated DNA replication.

UNLABELLED: To replicate the double-stranded human papillomavirus 16 (HPV16) DNA genome, viral proteins E1 and E2 associate with the viral origin of replication, and E2 can also regulate transcription from adjacent promoters. E2 interacts with host proteins in order to regulate both transcription and replication; TopBP1 and Brd4 are cellular proteins that interact with HPV16 E2. Previous work with E2 mutants demonstrated the Brd4 requirement for the transactivation properties of E2, while TopBP1 is required for DNA replication induced by E2 from the viral origin of replication in association with E1. More-recent studies have also implicated Brd4 in the regulation of DNA replication by E2 and E1. Here, we demonstrate that both TopBP1 and Brd4 are present at the viral origin of replication and that interaction with E2 is required for optimal initiation of DNA replication. Both cellular proteins are present in E1-E2-containing nuclear foci, and the viral origin of replication is required for the efficient formation of these foci. Short hairpin RNA (shRNA) against either TopBP1 or Brd4 destroys the E1-E2 nuclear bodies but has no effect on E1-E2-mediated levels of DNA replication. An E2 mutation in the context of the complete HPV16 genome that compromises Brd4 interaction fails to efficiently establish episomes in primary human keratinocytes. Overall, the results suggest that interactions between TopBP1 and E2 and between Brd4 and E2 are required to correctly initiate DNA replication but are not required for continuing DNA replication, which may be mediated by alternative processes such as rolling circle amplification and/or homologous recombination. IMPORTANCE: Human papillomavirus 16 (HPV16) is causative in many human cancers, including cervical and head and neck cancers, and is responsible for the annual deaths of hundreds of thousands of people worldwide. The current vaccine will save lives in future generations, but antivirals targeting HPV16 are required for the alleviation of disease burden on the current, and future, generations. Targeting viral DNA replication that is mediated by two viral proteins, E1 and E2, in association with cellular proteins such as TopBP1 and Brd4 would have therapeutic benefits. This report suggests a role for these cellular proteins in the initiation of viral DNA replication by HPV16 E1-E2 but not for continuing replication. This is important if viral replication is to be effectively targeted; we need to understand the viral and cellular proteins required at each phase of viral DNA replication so that it can be effectively disrupted.

A new role for human papillomavirus 16 E2: Mitotic activation of the DNA damage response to promote viral genome segregation.

Human papillomaviruses (HPV) are causative agents in around 5% of all human cancers. To identify and develop new targeted HPV therapeutics we must enhance our understanding of the viral life cycle and how it interacts with the host. The HPV E2 protein dimerizes and binds to 12bp target sequences in the viral genome and segregates the viral genome during mitosis. In this function, E2 binds to the viral genome and the host chromatin simultaneously, ensuring viral genomes reside in daughter nuclei following cell division. We have demonstrated that a mitotic interaction between E2 and the DNA damage response (DDR) protein TOPBP1 is required for E2 segregation function. In non-infected cells, following DNA damage, TOPBP1 is recruited to the mitotic host genome via interaction with MDC1 and this interaction protects DNA integrity during mitosis. Recently we demonstrated that the E2-TOPBP1 interaction activates the DNA damage response (DDR) during mitosis independently from external stimuli, promoting TOPBP1 interaction with mitotic chromatin and therefore segregation of the viral genome. Therefore, the virus has hijacked an existing host mechanism in order to segregate the viral genome. This intricate E2 function will be described and discussed.

Inhibition of human cytomegalovirus entry into mucosal epithelial cells.

Human cytomegalovirus (CMV) causes serious developmental disabilities in newborns infected in utero following oral acquisition by the mother. Thus, neutralizing antibodies in maternal saliva have potential to prevent maternal infection and, consequently, fetal transmission and disease. Based on standard cell culture models, CMV entry mediators (and hence neutralizing targets) are cell type-dependent: entry into fibroblasts requires glycoprotein B (gB) and a trimeric complex (TC) of glycoproteins H, L, and O, whereas endothelial and epithelial cell entry additionally requires a pentameric complex (PC) of glycoproteins H and L with UL128, UL130, and UL131A. However, as the mediators of mucosal cell entry and the potential impact of cellular differentiation remained unclear, the present studies utilized mutant viruses, neutralizing antibodies, and soluble TC-receptor to determine the entry mediators required for infection of mucocutaneus cell lines and primary tonsil epithelial cells. Entry into undifferentiated cells was largely PC-dependent, but PC-independent entry could be induced by differentiation. TC-independent entry was also observed and varied by cell line and differentiation. Infection of primary tonsil cells from some donors was entirely TC-independent. In contrast, an antibody to gB or disruption of virion attachment using heparin blocked entry into all cells. These findings indicate that CMV entry into the spectrum of cell types encountered in vivo is likely to be more complex than has been suggested by standard cell culture models and may be influenced by the relative abundance of virion envelope glycoprotein complexes as well as by cell type, tissue of origin, and state of differentiation.

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.

HPV16 genome structure analysis in oropharyngeal cancer PDXs identifies tumors with integrated and episomal genomes.

HPV + oropharyngeal squamous cell carcinoma (OPC) incidence recently surpassed cervical cancer and is the most common HPV-related cancer in the developed world. HPV16 is in ∼90 % of HPV + OPCs, with episomal genomes in the majority of cases. Most existing HPV16+ cancer cell lines derive from outside the oropharynx and harbor integrated HPV genomes. Thus, there is need for OPC preclinical models to evaluate standard and experimental therapeutics in the presence of episomal HPV16 oncogenic drivers. Here we characterize HPV genome structures in eight HPV16+ OPC patient-derived xenografts (PDXs), and evaluate their responses to standard chemotherapy. HPV genome state was investigated by combining Southern blot, T5 exonuclease assay, whole genome sequencing, and RNAseq data. This analysis revealed complexity and variation in integrated vs. episomal HPV forms across PDXs and demonstrated that four PDXs predominantly contain episomal HPV16. Episomal status did not ensure favorable in vivo responses to cisplatin therapy, despite the more favorable prognosis previously attributed to episomal HPV + tumors; this could be due to the small number present in the dataset. Our analysis establishes PDX models as test platforms for novel therapies designed to target maintenance of the episomal forms of HPV16 that commonly appear in OPC.

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.

E7-mediated repression of miR-203 promotes LASP1-dependent proliferation in HPV-positive cervical cancer.

Human papillomaviruses (HPV) are a major cause of malignancy, contributing to ~5% of all human cancers worldwide, including most cervical cancer cases and a growing number of anogenital and oral cancers. The major HPV viral oncogenes, E6 and E7, manipulate many host cellular pathways that promote cell proliferation and survival, predisposing infected cells to malignant transformation. Despite the availability of highly effective vaccines, there are still no specific anti-viral therapies targeting HPV or treatments for HPV-associated cancers. As such, a better understanding of viral-host interactions may allow the identification of novel therapeutic targets. Here, we demonstrate that the actin-binding protein LASP1 is upregulated in cervical cancer and significantly correlates with a poorer overall survival. In HPV positive cervical cancer, LASP1 depletion significantly inhibited the oncogenic phenotype in vitro, whilst having minimal effects in HPV negative cervical cancer cells. Furthermore, we demonstrate that the LASP1 SH3 domain is essential for LASP1-mediated oncogenicity in these cells. Mechanistically, we show that HPV E7 regulates LASP1 at the post-transcriptional level by repressing the expression of miR-203, which negatively regulates LASP1 mRNA levels by binding to its 3'UTR. Finally, we demonstrate that LASP1 expression is required for the growth of HPV positive cervical cancer cells in an in vivo tumourigenicity model. Together, these data demonstrate that HPV induces LASP1 expression to promote proliferation and survival in cervical cancer, thus identifying a potential therapeutic target in these cancers.

E7-mediated repression of miR-203 promotes LASP1-dependent proliferation in HPV-positive cervical cancer.

Human papillomaviruses (HPV) are a major cause of malignancy, contributing to ∼5% of all human cancers worldwide, including most cervical cancer cases and a growing number of ano-genital and oral cancers. The major HPV viral oncogenes, E6 and E7, manipulate many host cellular pathways that promote cell proliferation and survival, predisposing infected cells to malignant transformation. Despite the availability of highly effective vaccines, there are still no specific anti-viral therapies targeting HPV or treatments for HPV-associated cancers. As such, a better understanding of viral-host interactions may allow the identification of novel therapeutic targets. Here, we demonstrate that the actin-binding protein LASP1 is upregulated in cervical cancer and significantly correlates with a poorer overall survival. In HPV positive cervical cancer, LASP1 depletion significantly inhibited proliferation in vitro , whilst having minimal effects in HPV negative cervical cancer cells. Furthermore, we show that the LASP1 SH3 domain is essential for LASP1-mediated proliferation in these cells. Mechanistically, we show that HPV E7 regulates LASP1 at the post-transcriptional level by repressing the expression of miR-203, which negatively regulated LASP1 mRNA levels by binding to its 3'UTR. Finally, we demonstrated that LASP1 expression is required for the growth of HPV positive cervical cancer cells in an in vivo tumourigenicity model. Together, these data demonstrate that HPV induces LASP1 expression to promote proliferation and survival role in cervical cancer, thus identifying a potential therapeutic target in these cancers.