Mus musculus papillomavirus 1 E8

High-risk human papillomaviruses (PV) account for approximately 600,000 new cancers per year. The early protein E8^E2 is a conserved repressor of PV replication, whereas E4 is a late protein that arrests cells in G2 and collapses keratin filaments to facilitate virion release. While inactivation of the Mus musculus PV1 (MmuPV1) E8 start codon (E8-) increases viral gene expression, surprisingly, it prevents wart formation in FoxN1nu/nu mice. To understand this surprising phenotype, the impact of additional E8^E2 mutations was characterized in tissue culture and mice. MmuPV1 and HPV E8^E2 similarly interact with cellular NCoR/SMRT-HDAC3 co-repressor complexes. Disruption of the splice donor sequence used to generate the E8^E2 transcript or E8^E2 mutants (mt) with impaired binding to NCoR/SMRT-HDAC3 activates MmuPV1 transcription in murine keratinocytes. These MmuPV1 E8^E2 mt genomes also fail to induce warts in mice. The phenotype of E8^E2 mt genomes in undifferentiated cells resembles productive PV replication in differentiated keratinocytes. Consistent with this, E8^E2 mt genomes induced aberrant E4 expression in undifferentiated keratinocytes. In line with observations for HPV, MmuPV1 E4-positive cells displayed a shift to the G2 phase of the cell cycle. In summary, we propose that in order to enable both expansion of infected cells and wart formation in vivo, MmuPV1 E8^E2 inhibits E4 protein expression in the basal keratinocytes that would otherwise undergo E4-mediated cell cycle arrest. IMPORTANCE Human papillomaviruses (PVs) initiate productive replication, which is characterized by genome amplification and expression of E4 protein strictly within suprabasal, differentiated keratinocytes. Mus musculus PV1 mutants that disrupt splicing of the E8^E2 transcript or abolish the interaction of E8^E2 with cellular NCoR/SMRT-HDAC3 co-repressor complexes display increased gene expression in tissue culture but are unable to form warts in vivo. This confirms that the repressor activity of E8^E2 is required for tumor formation and genetically defines a conserved E8 interaction domain. E8^E2 prevents expression of E4 protein in basal-like, undifferentiated keratinocytes and thereby their arrest in G2 phase. Since binding of E8^E2 to NCoR/SMRT-HDAC3 co-repressor is required to enable expansion of infected cells in the basal layer and wart formation in vivo, this interaction represents a novel, conserved, and potentially druggable target.

Deregulation of host gene expression by HPV16 E8

Productive replication of human papillomaviruses (HPV) only takes place in differentiating keratinocytes. The HPV16 E8^E2 protein acts as a repressor of viral gene expression and genome replication and HPV16 E8^E2 knock-out (E8-) genomes display enhanced viral late protein expression in differentiated cells. Global transcriptome analysis of differentiated HPV16 wild-type and E8-cell lines revealed a small number of differentially expressed genes which are not related to cell cycle, DNA metabolism or keratinocyte differentiation. The analysis of selected genes suggested that deregulation requires cell differentiation and positively correlated with the expression of viral late, not early transcripts. Consistent with this, the additional knock-out of the viral E4 and E5 genes, which are known to enhance productive replication, attenuated the deregulation of these host cell genes. In summary, these data reveal that productive HPV16 replication modulates host cell transcription.

Detection of circulating cell-free HPV DNA of 13 HPV types for patients with cervical cancer as potential biomarker to monitor therapy response and to detect relapse.

BACKGROUND: HPV-related cervical cancer (CC) is the fourth most frequent cancer in women worldwide. Cell-free tumour DNA is a potent biomarker to detect treatment response, residual disease, and relapse. We investigated the potential use of cell-free circulating HPV-DNA (cfHPV-DNA) in plasma of patients with CC. METHODS: cfHPV-DNA levels were measured using a highly sensitive next-generation sequencing-based approach targeting a panel of 13 high-risk HPV types. RESULTS: Sequencing was performed in 69 blood samples collected from 35 patients, of which 26 were treatment-naive when the first liquid biopsy sample was retrieved. cfHPV-DNA was successfully detected in 22/26 (85%) cases. A significant correlation between tumour burden and cfHPV-DNA levels was observed: cfHPV-DNA was detectable in all treatment-naive patients with advanced-stage disease (17/17, FIGO IB3-IVB) and in 5/9 patients with early-stage disease (FIGO IA-IB2). Sequential samples revealed a decrease of cfHPV-DNA levels in 7 patients corresponding treatment response and an increase in a patient with relapse. CONCLUSIONS: In this proof-of-concept study we demonstrated the potential of cfHPV-DNA as a biomarker for therapy monitoring in patients with primary and recurrent CC. Our findings facilitate the development of a sensitive and precise, non-invasive, inexpensive, and easily accessible tool in CC diagnosis, therapy monitoring and follow-up.

Human Papillomavirus 42 Drives Digital Papillary Adenocarcinoma and Elicits a Germ Cell-like Program Conserved in HPV-Positive Cancers.

The skin is exposed to viral pathogens, but whether they contribute to the oncogenesis of skin cancers has not been systematically explored. Here we investigated 19 skin tumor types by analyzing off-target reads from commonly available next-generation sequencing data for viral pathogens. We identified human papillomavirus 42 (HPV42) in 96% (n = 45/47) of digital papillary adenocarcinoma (DPA), an aggressive cancer occurring on the fingers and toes. We show that HPV42, so far considered a nononcogenic, "low-risk" HPV, recapitulates the molecular hallmarks of oncogenic, "high-risk" HPVs. Using machine learning, we find that HPV-driven transformation elicits a germ cell-like transcriptional program conserved throughout all HPV-driven cancers (DPA, cervical carcinoma, and head and neck cancer). We further show that this germ cell-like transcriptional program, even when reduced to the top two genes (CDKN2A and SYCP2), serves as a fingerprint of oncogenic HPVs with implications for early detection, diagnosis, and therapy of all HPV-driven cancers. SIGNIFICANCE: We identify HPV42 as a uniform driver of DPA and add a new member to the short list of tumorigenic viruses in humans. We discover that all oncogenic HPVs evoke a germ cell-like transcriptional program with important implications for detecting, diagnosing, and treating all HPV-driven cancers. See related commentary by Starrett et al., p. 17. This article is highlighted in the In This Issue feature, p. 1.

Transcription Properties of Beta-HPV8 and HPV38 Genomes in Human Keratinocytes.

Persistent infections with high-risk human papillomaviruses (HR-HPV) from the genus alpha are established risk factors for the development of anogenital and oropharyngeal cancers. In contrast, HPV from the genus beta have been implicated in the development of cutaneous squamous cell cancer (cSCC) in epidermodysplasia verruciformis (EV) patients and organ transplant recipients. Keratinocytes are the in vivo target cells for HPV, but keratinocyte models to investigate the replication and oncogenic activities of beta-HPV genomes have not been established. A recent study revealed, that beta-HPV49 immortalizes normal human keratinocytes (NHK) only, when the viral E8^E2 repressor (E8-) is inactivated (T. M. Rehm, E. Straub, T. Iftner, and F. Stubenrauch, Proc Natl Acad Sci U S A 119:e2118930119, 2022, https://doi.org/10.1073/pnas.2118930119). We now demonstrate that beta-HPV8 and HPV38 wild-type or E8- genomes are unable to immortalize NHK. Nevertheless, HPV8 and HPV38 express E6 and E7 oncogenes and other transcripts in transfected NHK. Inactivation of the conserved E1 and E2 replication genes reduces viral transcription, whereas E8- genomes display enhanced viral transcription, suggesting that beta-HPV genomes replicate in NHK. Furthermore, growth of HPV8- or HPV38-transfected NHK in organotypic cultures, which are routinely used to analyze the productive replication cycle of HR-HPV, induces transcripts encoding the L1 capsid gene, suggesting that the productive cycle is initiated. In addition, transcription patterns in HPV8 organotypic cultures and in an HPV8-positive lesion from an EV patient show similarities. Taken together, these data indicate that NHK are a suitable system to analyze beta-HPV8 and HPV38 replication. IMPORTANCE High-risk HPV, from the genus alpha, can cause anogenital or oropharyngeal malignancies. The oncogenic properties of high-risk HPV are important for their differentiation-dependent replication in human keratinocytes, the natural target cell for HPV. HPV from the genus beta have been implicated in the development of cutaneous squamous cell cancer in epidermodysplasia verruciformis (EV) patients and organ transplant recipients. Currently, the replication cycle of beta-HPV has not been studied in human keratinocytes. We now provide evidence that beta-HPV8 and 38 are transcriptionally active in human keratinocytes. Inactivation of the viral E8^E2 repressor protein greatly increases genome replication and transcription of the E6 and E7 oncogenes, but surprisingly, this does not result in immortalization of keratinocytes. Differentiation of HPV8- or HPV38-transfected keratinocytes in organotypic cultures induces transcripts encoding the L1 capsid gene, suggesting that productive replication is initiated. This indicates that human keratinocytes are suited as a model to investigate beta-HPV replication.

Cottontail rabbit papillomavirus E6 proteins: Interaction with MAML1 and modulation of the Notch signaling pathway.

Animal models are necessary to study how cutaneous human papillomaviruses (HPVs) are associated with carcinogenesis. The cottontail rabbit papillomavirus (CRPV) induces papilloma in the -cutaneous skin of rabbits and serves as an established animal model for HPVlinked carcinogenesis where viral E6 proteins play crucial roles. Several studies have reported the dysregulation of the Notch signaling pathway by cutaneous beta HPV, bovine PV and mouse PV E6 via their association with Mastermind-like 1 protein (MAML1), thus interfering with cell proliferation and differentiation. However, the CRPV E6 gene encodes an elongated E6 protein (long E6, LE6) and an N-terminally truncated product (short E6, SE6) making it unique from other E6 proteins. Here, we describe the interaction between both CRPV E6 proteins and MAML1 and their ability to downregulate the Notch signaling pathway which could be a way CRPV infection induces carcinogenesis similar to beta HPV.

Functions of Papillomavirus E8

Papillomaviruses (PV) replicate in undifferentiated keratinocytes at low levels and to high levels in differentiated cells. The restricted replication in undifferentiated cells is mainly due to the expression of the conserved viral E8^E2 repressor protein, a fusion protein consisting of E8 and the hinge, DNA-binding, and dimerization domain of E2. E8^E2 binds to viral genomes and represses viral transcription and genome replication by recruiting cellular NCoR/SMRT-HDAC3 corepressor complexes. Tissue culture experiments have revealed that E8^E2 modulates long-term maintenance of extrachromosomal genomes, productive replication, and immortalization properties in a virus type-dependent manner. Furthermore, in vivo experiments have indicated that Mus musculus PV1 E8^E2 is required for tumor formation in immune-deficient mice. In summary, E8^E2 is a crucial inhibitor whose levels might determine the outcome of PV infections.

Restriction of viral gene expression and replication prevents immortalization of human keratinocytes by a beta-human papillomavirus.

SignificanceHigh-risk (HR) human papillomaviruses (HPV) from the genus alpha cause anogenital and oropharyngeal cancers, whereas the contribution of HPV from the genus beta to the development of cutaneous squamous cell cancer is still under debate. HR-HPV genomes display potent immortalizing activity in human keratinocytes, the natural target cell for HPV. This paper shows that immortalization of keratinocytes by the beta-HPV49 genome requires the inactivation of the viral E8^E2 repressor protein and the presence of the E6 and E7 oncoproteins but also of the E1 and E2 replication proteins. This reveals important differences in the carcinogenic properties of HR-HPV and beta-HPV but also warrants further investigations on the distribution and mutation frequencies of beta-HPV in human cancers.

An enhanced triple fluorescence flow-cytometry-based assay shows differential activation of the Notch signaling pathway by human papillomavirus E6 proteins.

Human papillomaviruses are DNA tumor viruses. A persistent infection with high-risk HPV types is the necessary risk factor for the development of anogenital carcinoma. The E6 protein is a viral oncoprotein that directly interacts with different cellular regulatory proteins mainly affecting the cell cycle, cellular differentiation and polarization of epithelial cells. In dependency of the phylogenetic classification of HPV different interaction partners of E6 have been described. The Notch pathway seems to be one common target of HPV, which can be up or down regulated by different E6 proteins. Our novel triple fluorescence flow-cytometry-based assay allows a semi-quantitative comparison of the E6 proteins´ effect on the Notch pathway using a Notch-responsive reporter plasmid. As a result, all E6 proteins of beta-HPV repressed the Notch reporter expression, of which HPV38 E6 showed the greatest repression potential. In contrast, alpha-HPV E6 of HPV16, activates the reporter expression most significantly, whereas E6 of HPV31 and low-risk HPV6b showed significant activation only in a p53-null cell line. Interestingly, HPV18 E6, with the second highest carcinogenic risk, shows no effect. This high divergence within different genus of HPV is important for targeting the Notch pathway regarding a potential HPV therapy.

EXPRESSION OF E8

Human papillomavirus (HPV) E1 and E2 proteins activate genome replication. E2 also modulates viral gene expression and is involved in the segregation of viral genomes. In addition to full length E2, almost all PV share the ability to encode an E8^E2 protein, that is a fusion of E8 with the C-terminal half of E2 which mediates specific DNA-binding and dimerization. HPV E8^E2 acts as a repressor of viral gene expression and genome replication. To analyze the function of E8^E2 in vivo, we used the Mus musculus PV1 (MmuPV1)-mouse model system. Characterization of the MmuPV1 E8^E2 protein revealed that it inhibits transcription from viral promoters in the absence and presence of E1 and E2 proteins and that this is partially dependent upon the E8 domain. MmuPV1 genomes, in which the E8 ATG start codon was disrupted (E8-), displayed a 10- to 25-fold increase in viral gene expression compared to wt genomes in cultured normal mouse tail keratinocytes in short-term experiments. This suggests that the function and mechanism of E8^E2 is conserved between MmuPV1 and HPVs. Surprisingly, challenge of athymic nude Foxn1nu/nu mice with MmuPV1 E8- genomes did not induce warts on the tail in contrast to wt MmuPV1. Furthermore, viral gene expression was completely absent at E8- MmuPV1 sites 20 - 22 weeks after DNA challenge on the tail or quasivirus challenge in the vaginal vault. This reveals that expression of E8^E2 is necessary to form tumors in vivo and that this is independent from the presence of T-cells.IMPORTANCE HPV encode an E8^E2 protein which acts as repressors of viral gene expression and genome replication. In cultured normal keratinocytes, E8^E2 is essential for long-term episomal maintenance of HPV31 genomes, but not for HPV16. To understand E8^E2's role in vivo, the Mus musculus PV1 (MmuPV1)-mouse model system was used. This revealed that E8^E2's function as a repressor of viral gene expression is conserved. Surprisingly, MmuPV1 E8^E2 knock out genomes did not induce warts in T-cell deficient mice. This shows for the first time that expression of E8^E2 is necessary for tumor formation in vivo independently of T cell immunity. This indicates that E8^E2 could be an interesting target for anti-viral therapy in vivo.

Structure of High-Risk Papillomavirus 31 E6 Oncogenic Protein and Characterization of E6/E6AP/p53 Complex Formation.

The degradation of p53 is a hallmark of high-risk human papillomaviruses (HPVs) of the alpha genus and HPV-related carcinogenicity. The oncoprotein E6 forms a ternary complex with the E3 ubiquitin ligase E6-associated protein (E6AP) and tumor suppressor protein p53 targeting p53 for ubiquitination. The extent of p53 degradation by different E6 proteins varies greatly, even for the closely related HPV16 and HPV31. HPV16 E6 and HPV31 E6 display high sequence identity (∼67%). We report here, for the first time, the structure of HPV31 E6 bound to the LxxLL motif of E6AP. HPV16 E6 and HPV31 E6 are structurally very similar, in agreement with the high sequence conservation. Both E6 proteins bind E6AP and degrade p53. However, the binding affinities of 31 E6 to the LxxLL motif of E6AP and p53, respectively, are reduced 2-fold and 5.4-fold compared to 16 E6. The affinity of E6-E6AP-p53 ternary complex formation parallels the efficacy of the subsequent reaction, namely, degradation of p53. Therefore, closely related E6 proteins addressing the same cellular targets may still diverge in their binding efficiencies, possibly explaining their different phenotypic or pathological impacts.IMPORTANCE Variations of carcinogenicity of human papillomaviruses are related to variations of the E6 and E7 interactome. While different HPV species and genera are known to target distinct host proteins, the fine differences between E6 and E7 of closely related HPVs, supposed to target the same cellular protein pools, remain to be addressed. We compare the oncogenic E6 proteins of the closely related high-risk HPV31 and HPV16 with regard to their structure and their efficiency of ternary complex formation with their cellular targets p53 and E6AP, which results in p53 degradation. We solved the crystal structure of 31 E6 bound to the E6AP LxxLL motif. HPV16 E6 and 31 E6 structures are highly similar, but a few sequence variations lead to different protein contacts within the ternary complex and, as quantified here, an overall lower binding affinity of 31 E6 than 16 E6. These results align with the observed lower p53 degradation potential of 31 E6.

Contribution of HDAC3 to transcriptional repression by the human papillomavirus 31 E8

Human papillomaviruses (HPV) such as HPV16 and HPV31 encode an E8^E2 protein that acts as a repressor of viral replication and transcription. E8^E2's repression activities are mediated via the interaction with host-cell NCoR (nuclear receptor corepressor)/SMRT (silencing mediator of retinoid and thyroid receptors) corepressor complexes, which consist of NCoR, its homologue SMRT, GPS2 (G-protein pathway suppressor 2), HDAC3 (histone deacetylase 3), TBL1 (transducin b-like protein 1) and its homologue TBLR1 (TBL1-related protein 1). We now provide evidence that transcriptional repression by HPV31 E8^E2 is NCoR/SMRT-dependent but surprisingly always HDAC3-independent when analysing different HPV promoters. This is in contrast to the majority of several cellular transcription factors using NCoR/SMRT complexes whose transcriptional repression activities are both NCoR/SMRT- and HDAC3-dependent. However, NCoR/SMRT-dependent but HDAC3-independent repression has been described for specific cellular genes, suggesting that this may not be specific for HPV promoters but could be a feature of a subset of NCoR/SMRT-HDAC3 regulated genes.

A TGF-ß- and p63-Responsive Enhancer Regulates IFN-κ Expression in Human Keratinocytes.

Type I IFNs have antiviral and immune-modulating activities. IFN-α/-ß have very low basal expression levels but are strongly induced upon activation of pattern recognition receptors. In contrast, IFN-κ is constitutively expressed in uninfected keratinocytes and responds only weakly to pattern recognition receptor activation. IFN-κ expression has been implicated in the pathogenesis of inflammatory skin diseases and in limiting human papillomavirus replication in human keratinocytes. We have identified an enhancer ∼5 kb upstream of the IFNK gene driving its expression in keratinocytes. The enhancer consists of binding sites for the transcription factors jun-B, SMAD3/4, AP-2α/γ, and p63, of which the latter two are key regulators of keratinocyte biology. The jun-B and SMAD3/4 elements confer activation by the TGF-ß pathway. Furthermore, inhibition of ERK1/2 kinases activates IFN-κ expression. Our study provides a framework for the cell type-specific, constitutive expression of IFN-κ and its modulation by signal transduction pathways in human keratinocytes.

Methylation of CpG 5962 in L1 of the human papillomavirus 16 genome as a potential predictive marker for viral persistence: A prospective large cohort study using cervical swab samples.

Several studies have demonstrated that the viral genome can be methylated by the host cell during progression from persistent infection to cervical cancer. The aim of this study was to investigate whether methylation at a specific site could predict the development of viral persistence and whether viral load shows a correlation with specific methylation patterns. HPV16-positive samples from women aged 20-29 years (n = 99) with a follow-up time of 13 years, were included from a Danish cohort comprising 11 088 women. Viral load was measured by real-time PCR and methylation status was determined for 39 CpG sites in the upstream regulatory region (URR), E6/E7, and L1 region of HPV16 by next-generation sequencing. Participants were divided into two groups according to whether they were persistently (≥ 24 months) or transiently HPV16 infected. The general methylation status was significantly different between women with a persistent and women with a transient infection outcome (P = .025). One site located in L1 (nt. 5962) was statistically significantly (P = .00048) different in the methylation status after correction using the Holm-Sidak method (alpha = 0.05). Correlation analyses of samples from HPV16 persistently infected women suggest that methylation is higher although viral load is lower. This study indicates that methylation at position 5962 of the HPV16 genome within the L1 gene might be a predictive marker for the development of a persistent HPV16 infection.

Brd4 inhibition suppresses HPV16 E6 expression and enhances chemoresponse: A potential new target in cervical cancer therapy.

Although a vast amount of research underlines the roles of the HR HPV E6 and E7 oncogenes in HPV-induced carcinogenesis of cervical cancer, it remains unclear whether these oncogenes are also involved in the resistance of the cancer against chemotherapy. We examined the role of the HPV16 E6 oncogene in cisplatin resistance by analyzing its expression in newly established cisplatin-sensitive versus -resistant cervical cancer cell lines (CC7, CC10). Resistant variants were obtained by interval exposure treatment with 1-2 µM cisplatin for 8-9 months. Our results demonstrate that the expression level of HPV16 E6 directly correlates with the extent of cisplatin resistance in novel as well as established (SiHa) drug resistant cervical cancer cell lines. Overexpression of HPV16 E6 in cisplatin-naïve cells rendered these cells more resistant to cisplatin. Reducing E6 expression by JQ1 treatment reversed the drug resistant phenotype and strongly enhanced chemoresponse only in HPV-positive cisplatin-resistant variants and not in HPV-negative C33A cervical cancer cells. The level of E6 directly correlated with the extent of cisplatin sensitivity and was shown to be increased in newly established drug-resistant cell line variants, while reducing E6 expression using Brd4-inhibitors enhanced chemoresponse when co-delivered with cisplatin. Inhibition of Brd4 could represent a new therapeutic option by increasing treatment response in cervical cancer cells and might allow lower cisplatin dosages, thus reducing negative side effects.

Cottontail Rabbit Papillomavirus E1 and E2 Proteins Mutually Influence Their Subcellular Localizations.

The papillomavirus (PV) E2 protein is a nuclear, sequence-specific DNA-binding protein that regulates transcription and nuclear retention of viral genomes. E2 also interacts with the viral E1 protein to replicate the viral genome. E2 residue K111 is highly conserved among PV and has been implicated in contributing to nuclear transport, transcription, and replication. Cottontail rabbit (Sylvilagus floridanus) PV (CRPV or SfPV1) E2 K111R, A, or Q mutations are transcription deficient and localized to the cytoplasm, comparable to other PV types. The addition of a nuclear localization signal (NLS) resulted in nuclear E2 K111 mutant proteins but did not restore transcriptional activation, and this is most likely due to an impaired binding to the cellular Brd4 protein. Surprisingly, coexpression of E1 with E2 K111 mutations resulted in their nuclear localization and, for K111A and R mutations, the activation of an E1/E2-dependent reporter construct. Interestingly, the nuclear localization of E2 K111Q mutant protein was independent from the presence of the conserved bipartite NLS in E1 and the direct interaction between E1 and E2. On the other hand, the cytoplasmic E1 NLS mutation could be targeted to the nucleus by wild-type E2, and this was dependent upon an interaction between E1 and E2. In summary, our studies have uncovered that E1 and E2 control each other's subcellular localization: direct binding of E2 to E1 can direct E1 to the nucleus independently from the E1 NLS, and E1 can direct E2 to the nucleus without an intact NLS or direct binding to E2.IMPORTANCE Papillomaviruses encode the DNA-binding E1 and E2 proteins, which form a complex and are essential for genome replication. Both proteins are targeted to the nucleus via nuclear localization signals. Our studies have uncovered that cytoplasmic mutant E1 or E2 proteins can be localized to the nucleus when E1 or E2 is also present. An interaction between E1 and E2 is necessary to target cytoplasmic E1 mutant proteins to the nucleus, but cytoplasmic E2 mutant proteins can be targeted to the nucleus without a direct interaction, which points to a novel function of E1.

The Myb-related protein MYPOP is a novel intrinsic host restriction factor of oncogenic human papillomaviruses.

The skin represents a physical and chemical barrier against invading pathogens, which is additionally supported by restriction factors that provide intrinsic cellular immunity. These factors detect viruses to block their replication cycle. Here, we uncover the Myb-related transcription factor, partner of profilin (MYPOP) as a novel antiviral protein. It is highly expressed in the epithelium and binds to the minor capsid protein L2 and the DNA of human papillomaviruses (HPV), which are the primary causative agents of cervical cancer and other tumors. The early promoter activity and early gene expression of the oncogenic HPV types 16 and 18 is potently silenced by MYPOP. Cellular MYPOP-depletion relieves the restriction of HPV16 infection, demonstrating that MYPOP acts as a restriction factor. Interestingly, we found that MYPOP protein levels are significantly reduced in diverse HPV-transformed cell lines and in HPV-induced cervical cancer. Decades ago it became clear that the early oncoproteins E6 and E7 cooperate to immortalize keratinocytes by promoting degradation of tumor suppressor proteins. Our findings suggest that E7 stimulates MYPOP degradation. Moreover, overexpression of MYPOP blocks colony formation of HPV and non-virally transformed keratinocytes, suggesting that MYPOP exhibits tumor suppressor properties.

HPV16 viral load and physical state measurement as a potential immediate triage strategy for HR-HPV-infected women: a study in 644 women with single HPV16 infections.

High genome copy number (viral load) of human papillomavirus (HPV) is being discussed as a risk factor for high-grade cervical lesions. However, conflicting data about the integration status or viral load of the virus as risk factors for prevalent high-grade squamous intraepithelial lesions (HSIL) are found in the literature. To investigate whether viral load and/or integration status are indicative for prevalent ASCUS/LSIL or HSIL, we determined the HPV16 viral load and the physical state of the genome in 644 women with single HPV16 infections stratified by their cytology results from a large Danish population-based cohort consisting of 40,399 women. Cervical smear samples were tested using a multiplex quantitative real-time PCR (qPCR) with primers specific for HPV16 E2, E6 and beta actin, allowing simultaneous determination of the genome's physical state and the viral copy number per cell. The associations of viral load and physical state with cervical abnormalities were assessed using multinomial logistic regression. We found that a 10-fold increase in viral load was significantly associated with the presence of ASCUS/LSIL (OR=3.91; 95% CI, 2.49-6.13) and HSIL (OR=4.1; 95% CI, 2.45-6.68). A significant association with HSIL was observed for primarily integrated genomes (OR=6.68; 95% CI, 1.45-30.8). Among women with integrated viral genomes, we observed a trend towards increased risk of ASCUS/LSIL (OR=1.32; 95% CI -2.90-3.44) and HSIL (OR=5.10; 95% CI -0.67-38.9) per 10-fold increase in viral load, although not statistically significant. In conclusion, increasing viral load and integrated viral genomes were significantly associated with prevalent HSIL, thus indicating that viral load and physical state may potentially be useful triage markers for HPV16-positive women during cervical screening.

Identification and Functional Characterization of Phosphorylation Sites of the Human Papillomavirus 31 E8

The papillomavirus E2 protein regulates transcription, replication, and nuclear retention of viral genomes. Phosphorylation of E2 in the hinge region has been suggested to modulate protein stability, DNA-binding activity, and chromosomal attachment. The papillomavirus E8^E2 protein shares the hinge domain with E2 and acts as a repressor of viral replication. Mass spectrometry analyses of human papillomavirus 31 (HPV31) E8^E2 and E2 proteins identify phosphorylated S78, S81, and S100 in E8^E2 and S266 and S269 in E2 in their hinge regions. Phos-tag analyses of wild-type and mutant proteins indicate that S78 is a major phosphorylation site in E8^E2, but the corresponding S266 in E2 is not. Phosphorylation at S78 regulates E8^E2's repression activity of reporter constructs, whereas the corresponding E2 mutants do not display a phenotype. Phosphorylation at S78 does not alter E8^E2's protein stability, nuclear localization, or binding to DNA or to cellular NCoR/SMRT complexes. Surprisingly, in the context of HPV31 genomes, mutation of E8^E2 S78 does not modulate viral replication or transcription in undifferentiated or differentiated cells. However, comparative transcriptome analyses of differentiated HPV31 E8^E2 S78A and S78E cell lines reveal that the expression of a small number of cellular genes is changed. Validation experiments suggest that the transcription of the cellular LYPD2 gene is altered in a phospho-S78 E8^E2-dependent manner. In summary, our data suggest that phosphorylation of S78 in E8^E2 regulates its repression activity by a novel mechanism, and this seems to be important for the modulation of host cell gene expression but not viral replication.IMPORTANCE Posttranslational modification of viral proteins is a common feature to modulate their activities. Phosphorylation of serine residues S298 and S301 in the hinge region of the bovine papillomavirus type 1 E2 protein has been shown to restrict viral replication. The papillomavirus E8^E2 protein shares the hinge domain with E2 and acts as a repressor of viral replication. A large fraction of HPV31 E8^E2 is phosphorylated at S78 in the hinge region, and this is important for E8^E2's repression activity. Surprisingly, phosphorylation at S78 in E8^E2 has no impact on viral replication in tissue culture but rather seems to modulate the expression of a small number of cellular genes. This may indicate that phosphorylation of viral transcription factors serves to broaden their target gene specificity.

Control of viral replication and transcription by the papillomavirus E8

Human papillomaviruses have adjusted their replication levels to the differentiation state of the infected keratinocyte. PV genomes replicate in undifferentiated cells at low levels and to high levels in differentiated cells. Genome replication requires the viral E1 helicase and the viral E2 transcription/replication activator. The limited replication in undifferentiated cells is predominantly due to the expression of the highly conserved E8^E2 viral repressor protein, which is a fusion between E8 and the C-terminal half of the E2 protein. E8^E2 is a sequence-specific DNA binding protein that inhibits viral gene expression and viral genome replication. The E8 domain is required for repression activities, which are mainly due to the interaction with cellular NCoR/SMRT corepressor complexes. In the case of HPV16, the most carcinogenic HPV type, E8^E2 not only limits genome replication in undifferentiated cells but also productive replication in differentiated epithelium. E8^E2 is expressed from a separate promoter that is controlled by unknown cellular factors and the viral transcription and replication regulators E1, E2 and E8^E2. In summary, E8^E2 is an important negative regulator whose levels may be critical for the outcome of HPV infections.