HPV Life Cycle Research Articles

PRMT1 Modulates Alternative Splicing to Enhance HPV18 mRNA Stability and Promote the Establishment of Infection.

Only persistent HPV infections lead to the development of cancer. Thus, understanding the virus-host interplay that influences the establishment of viral infection has important implications for HPV biology and human cancers. The ability of papillomaviruses to establish in cells requires the strict temporal regulation of viral gene expression in sync with cellular differentiation. This control primarily happens at the level of RNA splicing and polyadenylation. However, the details of how this spatio-temporal regulation is achieved still need to be fully understood. Until recently, it has been challenging to study the early events of the HPV lifecycle following infection. We used a single-cell genomics approach to identify cellular factors involved in viral infection and establishment. We identify protein arginine N-methyltransferase 1 (PRMT1) as an important factor in viral infection of primary human cervical cells. PRMT1 is the main cellular enzyme responsible for asymmetric dimethylation of cellular proteins. PRMT1 is an enzyme responsible for catalyzing the methylation of arginine residues on various proteins, which influences processes such as RNA processing, transcriptional regulation, and signal transduction. In this study, we show that HPV18 infection leads to increased PRMT1 levels across the viral lifecycle. PRMT1 is critical for the establishment of a persistent infection in primary cells. Mechanistically, PRMT1 inhibition leads to a highly dysregulated viral splicing pattern. Specifically, reduced PRMT1 activity leads to intron retention and a change in the E6 and E7 expression ratio. In the absence of PRMT1, viral transcripts are destabilized and subject to degradation via the nonsense-mediated decay (NMD) pathway. These findings highlight PRMT1 as a critical regulator of the HPV18 lifecycle, particularly in RNA processing, and position it as a potential therapeutic target for persistent HPV18 infections.

Complete genome sequence of human papillomavirus 31 isolated from the human cervical cell line, CIN612 9E.

Human papillomavirus genomes replicate extrachromosomally in infected tissues and derived keratinocyte cells. The cell line CIN12 9E was established from a cervical CIN1 lesion, contains replicating HPV31 genomes, and is widely used as a model to study the HPV life cycle. Here, we clone and sequence the HPV31 9E genome.

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

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.

Visualization of the HPV Life Cycle in Laryngeal Papilloma by the NanoSuit-CLEM Method

Visualization of the HPV Life Cycle in Laryngeal Papilloma by the NanoSuit-CLEM Method

The First Human Vulvar Intraepithelial Neoplasia Cell Line with Naturally Infected Episomal HPV18 Genome.

Persistent infection with high-risk HPV leads to cervical cancers and other anogenital cancers and head and neck carcinomas in both men and women. There is no effective drug fortreating HPV infection and HPV-associated carcinomas, largely due to a lack of models of natural HPV infection and the complexity of the HPV life cycle. There are no available cell lines from vulvar, anal, or penile lesions and cancers in the field. In this study, we established the first human cell line from vulvar intraepithelial neoplasia (VIN) with naturally infected HPV18 by conditional reprogramming (CR) method. Our data demonstrated that VIN cells possessed different biological characteristics and diploid karyotypes from HPV18-positive cancer cells (HeLa). Then, we determined that VIN cells contained episomal HPV18 using approaches including the ratio of HPV E2copy/E7copy, rolling cycle amplification, and sequencing. The VIN cells expressed squamous epithelium-specific markers that are different from HeLa cells, a cervical adenocarcinoma cell line. When cultured under 3D air–liquid interface (ALI) system, we observed the expression of both early and late differentiation markers involucrin and filaggrin. Most importantly, we were able to detect the expression of viral late gene L1 in the cornified layer of ALI 3D culture derived from VIN cells, suggesting quite different HPV genomic status from cancer cells. We also observed progeny viral particles under transmission electron microscopy (TEM) in ALI 3D cultures, confirming the episomal HPV18 genome and active viral life cycle in the new cell line. To our knowledge, this is the first human VIN cell line with naturally infected HPV18 genome and provides a valuable model for HPV biology studies, HPV-associated cancer initiation and progression, and drug-screening platforms.

A Critical Role for p53 during the HPV16 Life Cycle.

ABSTRACTHuman 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; β-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.

The Reservoir of Persistent Human Papillomavirus Infection; Strategies for Elimination Using Anti-Viral Therapies.

Human Papillomaviruses have co-evolved with their human host, with each of the over 200 known HPV types infecting distinct epithelial niches to cause diverse disease pathologies. Despite the success of prophylactic vaccines in preventing high-risk HPV infection, the development of HPV anti-viral therapies has been hampered by the lack of enzymatic viral functions, and by difficulties in translating the results of in vitro experiments into clinically useful treatment regimes. In this review, we discuss recent advances in anti-HPV drug development, and highlight the importance of understanding persistent HPV infections for future anti-viral design. In the infected epithelial basal layer, HPV genomes are maintained at a very low copy number, with only limited viral gene expression; factors which allow them to hide from the host immune system. However, HPV gene expression confers an elevated proliferative potential, a delayed commitment to differentiation, and preferential persistence of the infected cell in the epithelial basal layer, when compared to their uninfected neighbours. To a large extent, this is driven by the viral E6 protein, which functions in the HPV life cycle as a modulator of epithelial homeostasis. By targeting HPV gene products involved in the maintenance of the viral reservoir, there appears to be new opportunities for the control or elimination of chronic HPV infections.

P–578 Human pappiloma virus 16,18 genome methylation patterns in subfertile women

Abstract Study question A comparison between L1 gene and LCR region methylation status of HPV16 and HPV18 viruses in subfertile women, investigating HPV methylation pattern in cervical cancer and asymptomatic HPV infection. Summary answer CpG methylation was more frequent in L1 gene compared to LCR in both HPV types. Methylation levels were associated with the grade of cervical dysplasia. What is known already HPV infection is a common sexually transmitted disease, related to genital warts and cancer. DNA methylation as a dynamic and strictly controlled process can be involved in numerous cellular processes, cell differentiation, gene expression regulation and genome reprogramming. Human pappiloma virus genome epigenetic alterations may play a key role in HPV life cycle as well as in the oncogenic process in general. However, whether the prevalence of high risk HPV is correlated with female infertility, has yet to be elucidated. Study design, size, duration From January 2015 to December 2019, about 2505 infertile couples were referred to the Human Reproduction Unit of Ioannina University Hospital. A total of 212 clinical and laboratory data from female partners were included in the study. Participants/materials, setting, methods Cervical smears were studied for HPV DNA methylation. CpG methylation was compared among L1 gene and LCR region in both HPV types. A bisulfite modification assay followed by DNA amplification and sequencing was performed to analyse HPV16 and HPV18 genome. Main results and the role of chance In HPV16 types, L1 gene and promoter region indicated high methylation levels in cervical cancer cases. LCR regions methylation levels ranged from 0,5% to 24,2% in asymptomatic HPV16 infection or cervical intraepithelial neoplasia and cervical cancer, respectively. As for L1 gene, the differences between asymptomatic HPV16 infection and cervical cancer cases were statistically significant (P = 0.003). In HPV18 types, L1 gene was methylated in cervical intraepithelial neoplasia and cervical cancer cases. Promoter region methylation levels were high in cervical cancer cases while LCR region methylation levels were low. Limitations, reasons for caution Main limitation is the relatively small size of the collected samples. Wider implications of the findings: HPV genome investigation, as for methylation status, may lead to better understanding and earlier diagnostics of cervical pathology in infertile population. These observations point out the importance of fertility preservation in women at high risk for cervical neoplasia. Trial registration number Not applicable

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.

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.

A Conserved Amino Acid in the C Terminus of Human Papillomavirus E7 Mediates Binding to PTPN14 and Repression of Epithelial Differentiation.

The human papillomavirus (HPV) E7 oncoprotein is a primary driver of HPV-mediated carcinogenesis. The E7 proteins from diverse HPVs bind to the host cellular nonreceptor protein tyrosine phosphatase type 14 (PTPN14) and direct it for degradation through the activity of the E7-associated host E3 ubiquitin ligase UBR4. Here, we show that a highly conserved arginine residue in the C-terminal domain of diverse HPV E7 mediates the interaction with PTPN14. We found that disruption of PTPN14 binding through mutation of the C-terminal arginine did not impact the ability of several high-risk HPV E7 proteins to bind and degrade the retinoblastoma tumor suppressor or activate E2F target gene expression. HPVs infect human keratinocytes, and we previously reported that both PTPN14 degradation by HPV16 E7 and PTPN14 CRISPR knockout repress keratinocyte differentiation-related genes. Now, we have found that blocking PTPN14 binding through mutation of the conserved C-terminal arginine rendered both HPV16 and HPV18 E7 unable to repress differentiation-related gene expression. We then confirmed that the HPV18 E7 variant that could not bind PTPN14 was also impaired in repressing differentiation when expressed from the complete HPV18 genome. Finally, we found that the ability of HPV18 E7 to extend the life span of primary human keratinocytes required PTPN14 binding. CRISPR/Cas9 knockout of PTPN14 rescued keratinocyte life span extension in the presence of the PTPN14 binding-deficient HPV18 E7 variant. These results support the model that PTPN14 degradation by high-risk HPV E7 leads to repression of differentiation and contributes to its carcinogenic activity.IMPORTANCE The E7 oncoprotein is a primary driver of HPV-mediated carcinogenesis. HPV E7 binds the putative tumor suppressor PTPN14 and targets it for degradation using the ubiquitin ligase UBR4. PTPN14 binds to a C-terminal arginine highly conserved in diverse HPV E7. Our previous efforts to understand how PTPN14 degradation contributes to the carcinogenic activity of high-risk HPV E7 used variants of E7 unable to bind to UBR4. Now, by directly manipulating E7 binding to PTPN14 and using a PTPN14 knockout rescue experiment, we demonstrate that the degradation of PTPN14 is required for high-risk HPV18 E7 to extend keratinocyte life span. Our data show that PTPN14 binding by HPV16 E7 and HPV18 E7 represses keratinocyte differentiation. HPV-positive cancers are frequently poorly differentiated, and the HPV life cycle depends upon keratinocyte differentiation. The finding that PTPN14 binding by HPV E7 impairs differentiation has significant implications for HPV-mediated carcinogenesis and the HPV life cycle.

Differential phosphorylation determines the repressor and activator potencies of GLI1 proteins and their efficiency in modulating the HPV life cycle

The Sonic Hedgehog (Shh) signalling pathway plays multiple roles during embryonic development and under pathological conditions. Although the core components of the Shh pathway are conserved, the regulation of signal transduction varies significantly among species and cell types. Protein kinases Ulk3 and Pka are involved in the Shh pathway as modulators of the activities of Gli transcription factors, which are the nuclear mediators of the signal. Here, we investigate the regulation and activities of two GLI1 isoforms, full-length GLI1 (GLI1FL) and GLI1ΔN. The latter protein lacks the first 128 amino acids including the conserved phosphorylation cluster and the binding motif for SUFU, the key regulator of GLI activity. Both GLI1 isoforms are co-expressed in all human cell lines analysed and possess similar DNA binding activity. ULK3 potentiates the transcriptional activity of both GLI1 proteins, whereas PKA inhibits the activity of GLI1ΔN, but not GLI1FL. In addition to its well-established role as a transcriptional activator, GLI1FL acts as a repressor by inhibiting transcription from the early promoters of human papillomavirus type 18 (HPV18). Additionally, compared to GLI1ΔN, GLI1FL is a more potent suppressor of replication of several HPV types. Altogether, our data show that the N-terminal part of GLI1FL is crucial for the realization of its full potential as a transcriptional regulator.

Single-cell RNA sequencing reveals cell type-specific HPV expression in hyperplastic skin lesions

Human Papillomavirus infection is highly prevalent worldwide. While most types of HPV cause benign warts, some high-risk types are known to cause cervical cancer, as well as cancer of the oral cavity and head and neck. Persistent cutaneous HPV infection can be particularly problematic in patients with chronic immunosuppression, for example following organ transplantation. Due to unknown mechanisms, these patients may develop numerous warts, as well as present with a dramatically increased skin cancer prevalence.Despite an association between HPV persistence in the epidermis and excessive wart or squamous cancer development, the molecular mechanisms linking immunosuppression, HPV expression and excessive epidermal proliferation have not been determined, largely due to low-sensitivity methodology to capture rare viral transcription events. Here, we use single-cell RNA sequencing to profile HPV-positive skin lesions from an immunosuppressed patient that were found to express the alphapapillomavirus HPV78 in basal keratinocytes, suprabasal keratinocytes and hair follicle stem cells. This method can be applied to detect and investigate HPV transcripts in cutaneous lesions, allowing mechanistic links between immunosuppression-induced HPV life cycle and epidermal hyperproliferation to be uncovered.

HPV type 16 E6 and NFX1–123 augment JNK signaling to mediate keratinocyte differentiation and L1 expression

The HPV life cycle is differentiation-dependent, with cellular differentiation driving initiation of the late, productive stage of the viral life cycle. Here, we identify a role for the protein NFX1–123 in regulating keratinocyte differentiation and events of the late HPV life cycle. NFX1–123 itself increased with differentiation of epithelial cells. Greater NFX1–123 augmented differentiation marker expression and JNK phosphorylation in differentiating 16E6-expressing human foreskin keratinocytes (16E6 HFKs). This was associated with altered expression of MKK4 and MKK7, upstream kinase regulators of JNK phosphorylation. Modulating levels of NFX1–123 in HPV16-positive W12E cells recapitulated the effects on differentiation markers, JNK phosphorylation, and MKK4/7 seen in 16E6 HFKs. Crucially, levels of NFX1–123 also correlated with expression of L1, the capsid protein of HPV. Altogether, these studies define a role for NFX1–123 in mediating epithelial differentiation through the JNK signaling pathway, potentially linking expression of cellular genes and HPV genes during differentiation.

Production and characterization of a novel HPV anti-L2 monoclonal antibody panel

The major capsid protein of HPV, L1, assembles into pentamers that form a T = 7 icosahedral particle, but the location of the co-assembled minor capsid protein, L2, remains controversial. Several researchers have developed useful monoclonal antibodies targeting L2, but most react with linear epitopes toward the N-terminus. As a means to better define the virus capsid and better assess the localization and exposure of L2 epitopes in the context of assembled HPV, we have developed a panel of 30 monoclonal antibodies (mAbs) which target the N-terminus of L2 amino acids 11–200, previously defined as a broadly protective immunogen. Select mAbs were processed with enzymes and anti-L2 Fabs were generated. These new mAb/Fab probes will be beneficial in future studies to unravel the placement of L2 and to help better define the role of L2 in the HPV lifecycle and the nature of the broadly protective epitopes.

Expression of the HPV18/E6 oncoprotein induces DNA damage.

This study investigated possible variations in DNA damage in HeLa cells with silenced expression of the HPV/E6 oncogene compared with HeLa cells with normal expression of the E6 oncogene using the DNA breakage detection-fluorescence in situ hybridization (DBD-FISH) technique and a whole human genome DNA probe. The variable levels of DNA breaks present were measured quantitatively using image analysis after whole-genome DNA hybridization. HeLa cells with silenced expression of the HPV18/E6 oncogene showed a significant decrease in DNA damage compared with parental cells with normal expression of the E6 oncogene. These results were confirmed by alkaline comet assay. In conclusion, we demonstrated a decrease in DNA damage in HeLa clones associated with low expression of the HPV/E6 oncogene. The significance of this decrease regarding the HPV life cycle and carcinogenesis requires further exploration.

An oral keratinocyte life cycle model identifies novel host genome regulation by human papillomavirus 16 relevant to HPV positive head and neck cancer.

Many aspects of the HPV life cycle have been characterized in cervical cell lines (W12, CIN612) and in HPV immortalized primary foreskin keratinocytes. There is now an epidemic of HPV positive oropharyngeal cancers (HPV16 is responsible for 80-90% of these); therefore increased understanding of the HPV16 life cycle in oral keratinocytes is a priority. To date there have been limited reports characterizing the HPV16 life cycle in oral keratinocytes. Using TERT immortalized “normal” oral keratinocytes (NOKs) we generated clonal cell lines maintaining the HPV16 genome as an episome, NOKs+HPV16. Organotypic raft cultures demonstrated appropriate expression of differentiation markers, E1^E4 and E2 expression along with amplification of the viral genome in the upper layers of the epithelium. Using this unique system RNA-seq analysis revealed extensive gene regulation of the host genome by HPV16; many of the changes have not been observed for HPV16 before. The RNA-seq data was validated on a key set of anti-viral innate immune response genes repressed by HPV16 in NOKs+HPV16. We show that the behavior of these NOKs+HPV16 lines is identical to HPV16 immortalized human tonsil keratinocytes with regards innate gene regulation. Finally, using The Cancer Genome Atlas (TCGA) data we examined gene expression patterns from HPV positive and negative head and neck cancers and demonstrate this innate immune gene signature set is also downregulated in HPV positive cancers versus negative. Our system provides a model for understanding HPV16 transcriptional regulation of oral keratinocytes that is directly relevant to HPV positive head and neck cancer.

Association of Human Papillomavirus 16 E2 with Rad50-Interacting Protein 1 Enhances Viral DNA Replication.

ABSTRACTRad50-interacting protein 1 (Rint1) associates with the DNA damage response protein Rad50 during the transition from the S phase to the G2/M phase and functions in radiation-induced G2 checkpoint control. It has also been demonstrated that Rint1 is essential in vesicle trafficking from the Golgi apparatus to the endoplasmic reticulum (ER) through an interaction with Zeste-White 10 (ZW10). We have isolated a novel interaction between Rint1 and the human papillomavirus 16 (HPV16) transcription and replication factor E2. E2 binds to Rint1 within its ZW10 interaction domain, and we show that in the absence of E2, Rint1 is localized to the ER and associates with ZW10. E2 expression results in a disruption of the Rint1-ZW10 interaction and an accumulation of nuclear Rint1, coincident with a significant reduction in vesicle movement from the ER to the Golgi apparatus. Interestingly, nuclear Rint1 and members of the Mre11/Rad50/Nbs1 (MRN) complex were found in distinct E2 nuclear foci, which peaked during mid-S phase, indicating that the recruitment of Rint1 to E2 foci within the nucleus may also result in the recruitment of this DNA damage-sensing protein complex. We show that exogenous Rint1 expression enhances E2-dependent virus replication. Conversely, the overexpression of a truncated Rint1 protein that retains the E2 binding domain but not the Rad50 binding domain acts as a dominant negative inhibitor of E2-dependent HPV replication. Put together, these experiments demonstrate that the interaction between Rint1 and E2 has an important function in HPV replication.IMPORTANCE HPV infections are an important driver of many epithelial cancers, including those within the anogenital and oropharyngeal tracts. The HPV life cycle is tightly regulated and intimately linked to the differentiation of the epithelial cells that it infects. HPV replication factories formed in the nucleus are locations where viral DNA is copied to support virus persistence and amplification of infection. The recruitment of specific cellular protein complexes to these factories aids efficient and controlled viral replication. We have identified a novel HPV-host interaction that functions in the cellular response to DNA damage and cell cycle control. We show that the HPV E2 protein targets Rad50-interacting protein 1 (Rint1) to facilitate virus genome replication. These findings add to our understanding of how HPV replicates and the host cell pathways that are targeted by HPV to support virus replication. Understanding these pathways will allow further research into novel inhibitors of HPV genome replication.

Extrachromosomal HPV-16 LCR transcriptional activation by HDACi opposed by cellular differentiation and DNA integration.

Histone deacetylase inhibitors (HDACi) have been shown to render HPV-carrying cells susceptible to intrinsic and extrinsic apoptotic signals. As such, these epigenetic drugs have entered clinical trials in the effort to treat cervical cancer. Here, we studied the effect of common HDACi, with an emphasis on Trichostatin A (TSA), on the transcriptional activity of the HPV-16 Long Control Region (LCR) in order to better understand the impact of these agents in the context of the HPV life cycle and infection. HDACi strongly induced transcription of the firefly luciferase reporter gene under the control of the HPV-16 LCR in a variety of cell lines. In the HaCaT keratinocyte cell line undergoing differentiation induced by TSA, we observed a reduction in LCR-controlled transcription. Three major AP-1 binding sites in the HPV-16 LCR are involved in the regulation by TSA. However, whatever the status of differentiation of the HaCaT cells, TSA induced integration of extra-chromosomal transfected DNA into the cellular genome. Although these data suggest caution using HDACi in the treatment of HR HPV infection, further in vivo studies are necessary to better assess the risk.

Interplay Between NF-κB/interferon Signaling and the Genome Replication of HPV

HPV infection can persist within the infected epithelium for years. The viral persistence is primarily attributed to the ability of the virus to maintain its genome as nuclear episomes in the basal cells. Recent studies have revealed that HPV induces DNA damage response to facilitate productive amplification of the viral genome. DNA damage response comprises a part of the cellular defense mechanism against viral infection and its activation can result in induction of innate immune responses. The activation of NF-κB and interferon (IFN) signals has been shown to suppress the genome replication of HPV while the viral proteins inhibit NF-κB/IFN signaling. This review intends to focus on illustrating the interplay between NFκB/IFN signaling and HPV genome replication in the HPV life cycle.