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.

Effects of ß-HPV on DNA damage response pathways to drive carcinogenesis: a review.

The DDR is a complex signaling network responsible for the preservation of genomic integrity. Beta human papillomaviruses (ß-HPVs) are able to destabilize the host genome by attenuating the DDR machinery at the molecular scale following expression of the oncogenes E6 and E7. In the event of ß-HPV infection, the E6- and E7-mediated inhibition of the DDR enhances the oncogenicity of UV-induced mutations to enable carcinogenesis in an otherwise immunocompetent host, marking an important mechanistic divergence from the alpha genus of HPVs. In this review, we summarize recent updates to build upon the 'hit-and-run' hypothesis of ß-HPV pathomechanism and highlight strain-dependent variations. Simultaneously, we illuminate points within the ß-HPV-DDR interface that may unravel new insights for HPV viral genetics, genus-specific mechanistic models, and developments in targeted molecular therapy of ß-HPV-related cancers.

Beta human papillomaviruses infection and skin carcinogenesis.

During the last decade, the worldwide incidence of keratinocyte carcinomas (KC) has increased significantly. They are now the most common malignancy, representing approximately 30% of all cancers. The role of ultraviolet (UV) radiation as a major environmental risk factor for skin cancers is well recognized. The aim of this review is to analyse the current understanding of the nature of beta-human papillomavirus (HPV) and its association with KC and explore the implications for the management and prevention of these cancers. A comprehensive review of the literature on beta-HPV and its association with KC was undertaken, the results reported in the form of a narrative review. A subgroup of HPV that infects the mucosal epithelia of the genital tract has been firmly associated with carcinogenesis. In addition, some HPV types with cutaneous tropism have been proposed to cooperate with UV in the development of KC. The first evidence for this association was reported in 1922 in patients with epidermodysplasia verruciformis (EV). Since then, epidemiological studies have highlighted the higher risk of skin cancer in patients with EV and certain cutaneous HPV types, and in vitro studies have elucidated molecular mechanisms and transforming properties of beta-HPV. Furthermore, in vivo research conducted on transgenic mice models has shown the possible role of beta-HPV in cutaneous carcinogenesis as a co-factor with UV radiation and immunosuppression. There is good evidence supporting the role of beta-HPV in the oncogenesis of KC. The high prevalence of beta-HPV in human skin and the worldwide burden of KC makes the search for an effective vaccine relevant and worthwhile.

The Role of Beta HPV Types and HPV-Associated Inflammatory Processes in Cutaneous Squamous Cell Carcinoma.

Cutaneous squamous cell carcinoma (cSCC) is a common form of skin cancer with a complex but not fully understood pathogenesis. Recent research suggests the role of beta human papillomavirus (HPV) types and HPV-associated inflammatory processes in cSCC development. Beta HPV types are components of the normal flora; however, under the influence of certain cofactors, the virus may trigger a malignant process. Dysregulation of the immune system (chronic inflammation and immunosuppression), environmental factors (ultraviolet radiation), and genetic factors are the most important cofactors involved in beta HPV-related carcinogenesis. In addition, the oncoproteins E6 and E7 of beta HPV types differ biochemically from their counterparts in the structure of alpha HPV types, resulting in different mechanisms of action in carcinogenesis. The aim of our manuscript is to present an updated point of view on the involvement of beta HPV types in cSCC pathogenesis.

Cross-talk of cutaneous beta human papillomaviruses and the immune system: determinants of disease penetrance.

Human papillomaviruses (HPVs) infect the epithelia of skin or mucosa, where they can induce hyperproliferative lesions. More than 220 different HPV types have been characterized and classified into five different genera. Mucosal high-risk HPVs are causative for cancers of the anogenital region and oropharynx. Clinical data from patients with the rare genetic disorder epidermodysplasia verruciformis (EV) indicate that genus beta-HPVs cooperate with ultraviolet (UV) radiation in the development of cutaneous squamous cell carcinoma. In addition, epidemiological and biological findings indicate that beta-HPV types play a role in UV-mediated skin carcinogenesis also in non-EV individuals. However, the mechanisms used by these cutaneous viruses to promote epithelial carcinogenesis differ significantly from those of mucosal HPVs. Recent studies point to a delicate cross-talk of beta-HPVs with the cell-autonomous immunity of the host keratinocytes and the local immune microenvironment that eventually determines the fate of cutaneous HPV infection and the penetrance of disease. This review gives an overview of the critical interactions of genus beta-HPVs with the local immune system that allow the virus to complete its life cycle, to escape from extrinsic immunity, and eventually to cause chronic inflammation contributing to skin carcinogenesis. This article is part of the theme issue 'Silent cancer agents: multi-disciplinary modelling of human DNA oncoviruses'.

Beta and gamma human papillomaviruses in anal and genital sites among men: prevalence and determinants.

Data regarding the anogenital distribution of and type-specific concordance for cutaneous ß- and γ-HPV types in men who have sex with women is limited and geographically narrow. Knowledge of determinants of anogenital detection of cutaneous HPV types in different regions is needed for better understanding of the natural history and transmission dynamics of HPV, and its potential role in the development of anogenital diseases. Genital and anal canal samples obtained from 554 Russian men were screened for 43 ß-HPVs and 29 γ-HPVs, using a multiplex PCR combined with Luminex technology. Both ß- and γ-HPVs were more prevalent in the anal (22.8% and 14.1%) samples than in the genital (16.8% and 12.3%) samples. Low overall and type-specific concordance for ß-HPVs (3.5% and 1.1%) and γ-HPVs (1.3% and 0.6%) were observed between genital and anal samples. HIV-positive men had higher anal ß- (crude OR = 12.2, 95% CI: 5.3-28.1) and γ-HPV (crude OR = 7.2, 95% CI: 3.3-15.4) prevalence than HIV-negative men. Due to the lack of genital samples from the HIV-positive men, no comparison was possible for HIV status in genital samples. The lack of type-specific positive concordance between genital and anal sites for cutaneous ß- and γ-HPV types in heterosexual men posits the needs for further studies on transmission routes to discriminate between contamination and true HPV infection. HIV-positive status may favor the anal acquisition or modify the natural history of cutaneous HPV types.

Molecular Mechanisms of Human Papillomavirus Induced Skin Carcinogenesis.

Infection of the cutaneous skin with human papillomaviruses (HPV) of genus betapapillomavirus (ßHPV) is associated with the development of premalignant actinic keratoses and squamous cell carcinoma. Due to the higher viral loads of ßHPVs in actinic keratoses than in cancerous lesions, it is currently discussed that these viruses play a carcinogenic role in cancer initiation. In vitro assays performed to characterize the cell transforming activities of high-risk HPV types of genus alphapapillomavirus have markedly contributed to the present knowledge on their oncogenic functions. However, these assays failed to detect oncogenic functions of ßHPV early proteins. They were not suitable for investigations aiming to study the interactive role of ßHPV positive epidermis with mesenchymal cells and the extracellular matrix. This review focuses on ßHPV gene functions with special focus on oncogenic mechanisms that may be relevant for skin cancer development.

Cutaneous beta human papillomaviruses and the development of male external genital lesions: A case-control study nested within the HIM Study.

BACKGROUND: Cutaneous human papillomaviruses (HPVs) increase the risk of non-melanoma skin cancer in sun-exposed skin. We examined the role of beta-HPV in the development of male external genital lesions (EGLs), a sun-unexposed site. METHODS: In this nested case-control study (67 men with pathologically-confirmed EGLs and 134 controls), exfoliated cells collected from the surface of lesions and normal genital skin 0, 6, and 12 months preceding EGL development were tested for beta-HPV DNA using a type-specific multiplex genotyping assay. Beta-HPV prevalence was estimated and conditional logistic regression was used to evaluate the association with condyloma, the most common EGL. RESULTS: While beta-HPV prevalence among controls remained stable, the prevalence among cases was lowest on the surface of lesion. Detecting beta-HPV on the normal genital skin was not associated with the presence or development of condyloma. CONCLUSIONS: Cutaneous beta-HPV does not appear to be contributing to pathogenesis in male genital skin.

The levels of epithelial anchor proteins ß-catenin and zona occludens-1 are altered by E7 of human papillomaviruses 5 and 8.

Infection with viruses of the genus Betapapillomavirus, ß-human papillomaviruses (ß-HPV), is implicated in the development of non-melanoma skin cancer. This was first evidenced for HPV5 and HPV8 in patients with the skin disease epidermodysplasia verruciformis (EV). The relocalization of the junctional bridging proteins ß-catenin and zona occludens-1 (ZO-1) from the adherens and tight junctions are common processes of the epithelial-mesenchymal transition (EMT) associated with tumour invasion. Here, we report that ß-catenin and ZO-1 are strongly upregulated by the E7 oncoproteins of HPV5 and HPV8 in keratinocytes grown in organotypic skin cultures. Although the membrane-tethered form of ß-catenin was elevated, no signs of ß-catenin activity within the canonical Wnt signalling pathway could be detected. The upregulation of ß-catenin and ZO-1 could also be confirmed in the skin of HPV8 transgenic mice as well as in cutaneous squamous cell carcinomas of EV patients. These data provide the first evidence that ß-catenin and ZO-1 are direct targets of E7 of the oncogenic ß-HPV types 5 and 8. The ability to deregulate these epithelial junction proteins may contribute to the oncogenic potential of these viruses in human skin.

Beta genus papillomaviruses and skin cancer.

A role for the beta genus HPVs in keratinocyte carcinoma (KC) remains to be established. In this article we examine the potential role of the beta HPVs in cancer revealed by the epidemiology associating these viruses with KC and supported by oncogenic properties of the beta HPV proteins. Unlike the cancer associated alpha genus HPVs, in which transcriptionally active viral genomes are invariably found associated with the cancers, that is not the case for the beta genus HPVs and keratinocyte carcinomas. Thus a role for the beta HPVs in KC would necessarily be in the carcinogenesis initiation and not in the maintenance of the tumor.

Distinct functions of epidermal and myeloid-derived VEGF-A in skin tumorigenesis mediated by HPV8.

Beta human papillomaviruses (HPV) have been suspected to be carcinogenic in nonmelanoma skin cancers (NMSC), but the basis for potential viral contributions to these cancers is poorly understood. In particular, it is unresolved how HPV-infected keratinocytes escape cell-cycle control and whether their cross-talk with immune cells is critical for tumorigenesis. In nonviral preclinical models, the angiogenic cytokine VEGF-A has been identified as a critical regulator of NMSC. In this study, we dissected the contribution of epidermal versus myeloid cell-derived VEGF-A in HPV-mediated skin cancer by interbreeding an HPV8 transgenic mouse model with a conditional disruption of VEGF-A restricted to either epidermal or myeloid cells. Although only epidermal-derived VEGF-A was essential for initiation of skin tumor development, both spontaneously and UV-light triggered, both epidermal and myeloid cell-derived VEGF-A contributed to regeneration-induced tumorigenesis upon HPV8 overexpression, partly not only through a paracrine effect on endothelial cells, but also most probably through an additional autocrine effect on epidermal cells. Our findings offer new mechanistic insights into distinct functions of epidermal versus myeloid cell-derived VEGF-A during HPV-mediated tumorigenesis, with possible implications for preventing this disease.

Genus beta human papillomavirus E6 proteins vary in their effects on the transactivation of p53 target genes.

UNLABELLED: The genus beta human papillomaviruses (beta HPVs) cause cutaneous lesions and are thought to be involved in the initiation of some nonmelanoma skin cancers (NMSCs), particularly in patients with the genetic disorder epidermodysplasia verruciformis (EV). We have previously reported that at least two of the genus beta HPV E6 proteins bind to and/or increase the steady-state levels of p53 in squamous epithelial cells. This is in contrast to a well-characterized ability of the E6 proteins of cancer-associated HPVs of genus alpha HPV, which inactivate p53 by targeting its ubiquitin-mediated proteolysis. In this study, we have investigated the ability of genus beta E6 proteins from eight different HPV types to block the transactivation of p53 target genes following DNA damage. We find that the E6 proteins from diverse beta HPV species and types vary in their capacity to block the induction of MDM2, p21, and proapoptotic genes after genotoxic stress. We conclude that some genus beta HPV E6 proteins inhibit at least some p53 target genes, although perhaps not by the same mechanism or to the same degree as the high-risk genus alpha HPV E6 proteins. IMPORTANCE: This study addresses the ability of various human papillomavirus E6 proteins to block the activation of p53-responsive cellular genes following DNA damage in human keratinocytes, the normal host cell for HPVs. The E6 proteins encoded by the high-risk, cancer-associated HPV types of genus alpha HPV have a well-established activity to target p53 degradation and thereby inhibit the response to DNA damage. In this study, we have investigated the ability of genus beta HPV E6 proteins from eight different HPV types to block the ability of p53 to transactivate downstream genes following DNA damage. We find that some, but not all, genus beta HPV E6 proteins can block the transactivation of some p53 target genes. This differential response to DNA damage furthers the understanding of cutaneous HPV biology and may help to explain the potential connection between some beta HPVs and cancer.

[Natural history of the infection for human papillomavirus: an actualization]. / Historia natural de la infección por el virus del papiloma humano: una actualización.

In recent years, there have been major advances in our understanding of the biology and natural history of Human Papilloma Virus (HPV). Most papillomavirus infections are transmitted by close contact of either skin to skin or mucosa to mucosa. Sexual intercourse is not a requirement for genital HPV infection. Digital-oral infections occur and there is evidence that digital-genital and oral-genital contacts can result in the transmission of HPV, although in a relatively low percentage. Vertical transmission from mother to fetus is a common route of infection; in fact, it is recognized that more than 80% of infants born from mothers infected with genital HPV will be positive for HPV DNA determination in the nasal-pharyngeal region and oral mucosa. Women with transient infections often develop cytological abnormalities that take place while there is active HPV replication. This occurs because productive HPV infections result in cytological abnormalities in infected epithelial cells. The strong association between the risk of HPV infection and increased immune suppression, supports a direct biological effect of Human Immunodeficiency Virus (HIV) infection on the natural history of HPV.

Historia natural de la infección por el virus del papiloma humano: una actualización / Natural history of the infection for human papillomavirus: an actualization

Durante los últimos años, se han sucedido grandes avances en nuestro entendimiento acerca de la biología e historia natural del Virus del Papiloma Humano (VPH). La mayoría de las infecciones por papiloma virus son transmitidas por un contacto cercano bien sea de piel a piel o mucosa a mucosa. La relación sexual con penetración no es un requerimiento para la transmisión del VPH. Las infecciones orales y digitales por VPH ocurren, y existe evidencia de que el contacto digital-genital y genital-oral puede resultar en la transmisión del VPH, aunque en un porcentaje relativamente bajo. La transmisión vertical de la madre al feto es una vía frecuente de infección, de hecho, se reconoce que más del 80% de los neonatos nacido de madres infectadas con VPH genital serán positivos a la determinación del ADN del VPH en la región naso-faríngea y mucosa oral. Mujeres con infecciones transitorias frecuentemente desarrollan anormalidades citológicas mientras ocurra una replicación activa del VPH. Esto ocurre debido a que las infecciones productivas de VPH resultan en anormalidades citológicas en las células epiteliales infectadas. La fuerte asociación entre el riesgo de infección por VPH y el incremento en la supresión inmune apoya un efecto biológico directo de la infección por VIH en la historia natural del VPH.

In recent years, there have been major advances in our understanding of the biology and natural history of Human Papilloma Virus (HPV). Most papillomavirus infections are transmitted by close contact of either skin to skin or mucosa to mucosa. Sexual intercourse is not a requirement for genital HPV infection. Digital-oral infections occur and there is evidence that digital-genital and oral-genital contacts can result in the transmission of HPV, although in a relatively low percentage. Vertical transmission from mother to fetus is a common route of infection; in fact, it is recognized that more than 80% of infants born from mothers infected with genital HPV will be positive for HPV DNA determination in the nasal-pharyngeal region and oral mucosa. Women with transient infections often develop cytological abnormalities that take place while there is active HPV replication. This occurs because productive HPV infections result in cytological abnormalities in infected epithelial cells. The strong association between the risk of HPV infection and increased immune suppression, supports a direct biological effect of Human Immunodeficiency Virus (HIV) infection on the natural history of HPV.

Mapping of betapapillomavirus human papillomavirus 5 transcription and characterization of viral-genome replication function.

Betapapillomavirus replication and transcription have not been studied in detail because of a lack of suitable cellular systems supporting human papillomavirus (HPV) genome replication. We have recently shown that the human osteosarcoma cell line U2OS provides a useful environment for the genome replication of many different HPVs, including the betapapillomaviruses HPV5 and HPV8. Using mutational analysis and complementation assay, we demonstrated herein that the viral early proteins E1 and E2 are viral transfactors that are necessary and sufficient for HPV5 genome replication. We also identified four HPV5 early promoter regions with transcription start sites (TSSs) at nucleotides (nt) 184/191, 460, 840, and 1254, respectively, and the HPV late promoter with a TSS at nt 7640. In addition, we mapped the HPV5 early polyadenylation cleavage sites via 3' rapid amplification of cDNA ends (3'RACE) to nt 4457 and 4475. In total, 14 different viral mRNA species, originating from the HPV5 genome, were mapped in U2OS cells during transient and stable replication. The main splicing donor and acceptor sites identified herein are consistent with the data previously obtained in HPV5-positive skin lesions. In addition, we identified novel E8 open reading frame (ORF)-containing transcripts (E8^E1C and E8^E2C) expressed from the HPV5 genome. Similar to several other papillomaviruses, the product of the E8^E2C mRNA acts as a repressor of viral genome replication.

HPV 5 and 8 E6 expression reduces ATM protein levels and attenuates LINE-1 retrotransposition.

The expression of the E6 protein from certain members of the HPV genus ß (ß HPV 5 and 8 E6) can disrupt p53 signaling by diminishing the steady state levels of two p53 modifying enzymes, ATR and p300. Here, we show that ß-HPV 5 and 8 E6 are also capable of reducing the steady state levels of another p53 modifying enzyme, ATM, and as a result restrict LINE-1 retrotransposition. Furthermore, we show that the reduction of both ATM and LINE-1 retrotransposition is dependent upon the ability of ß-HPV 8 E6 to bind and degrade p300. We use inhibitors and dominant negative mutants to confirm that ATM is needed for efficient LINE-1 retrotransposition. Furthermore, neither sensitivity to LINE-1 expression nor LINE-1 induced DSB formation is altered in an ATM deficient background. Together, these data illustrate the broad impact some ß-HPVs have on DNA damage signaling by promoting p300 degradation.

Comparative analysis of transforming properties of E6 and E7 from different beta human papillomavirus types.

The cutaneous beta human papillomavirus (beta HPV) types appear to be involved in skin carcinogenesis. However, only a few beta HPVs have been investigated so far. Here, we compared the properties of E6 and E7 oncoproteins from six uncharacterized beta HPVs (14, 22, 23, 24, 36, 49). Only HPV49 E6 and E7 immortalized primary human keratinocytes and efficiently deregulated the p53 and pRb pathways. Furthermore, HPV49 E6, similarly to E6 from the oncogenic HPV16, promoted p53 degradation.

E6 and E7 proteins from different beta-papillomaviruses types do not interfere in UVB-induced apoptosis of HaCaT keratinocytes.

Beta-papillomaviruses (beta-HPV) have been linked to the development of skin cancer in humans. Because both E6 and E7 proteins from beta-HPV have been involved in the potential carcinogenicity of these viruses, we investigated their role on UVB-induced apoptosis in HaCaT cell line. HaCaT cells have been transduced with both E6/E7 using a retroviral system and treated with PRIMA-1. Apoptosis was assessed by flow cytometry to measure mitochondrial membrane potential and DNA fragmentation. HaCat keratinocytes transduced with both E6 and E7 genes of seven beta-HPV types (HPV5, HPV8, HPV14, HPV24, HPV36, HPV38 and HPV49) did not demonstrate any inhibition of UVB-induced apoptosis, even after p53 reactivation through PRIMA-1. Our data suggest that the expression of E6 and E7 exert different modulatory effects on UVB-induced apoptosis according to beta-HPV types and to the cellular genetic context.

[Biology of epidermodysplasia verruciformis-associated HPV]. / Biologie der Epidermodysplasia-verruciformis-assoziierten HPV.

The genus betapapillomavirus (betaPV) presently comprises more than 40 virus types including the so-called epidermodysplasia verruciformis (EV)-associated HPV, which were originally detected in EV-patients by Southern blot hybridization. BetaPV are ubiquitous in the general population and frequently establish themselves already during the first weeks of life. Hair follicles are regarded as natural reservoir. About 25% of betaPV detected in adults persist for at least 9 months. Due to very low virus production, seroconversion against betaPV starts sluggishly. Hyperproliferation of keratinocytes in psoriasis patients or after severe burns stimulates virus replication. Massive virus replication only occurs in EV-patients, associated with the induction of disseminated skin lesions with a high risk of malignant conversion. In 75% of EV-patients this can be put down to homozygous, inactivating mutations in the genes EVER1 or EVER2. A transgenic mouse model substantiated the crucial role of increased HPV8 oncogene expression, induced by UV-irradiation or wounding, for tumor induction.

Adenovirus type 5 E1A and E6 proteins of low-risk cutaneous beta-human papillomaviruses suppress cell transformation through interaction with FOXK1/K2 transcription factors.

The adenovirus (Adv) oncoprotein E1A stimulates cell proliferation and inhibits differentiation. These activities are primarily linked to the N-terminal region (exon 1) of E1A, which interacts with multiple cellular protein complexes. The C terminus (exon 2) of E1A antagonizes these processes, mediated in part through interaction with C-terminal binding proteins 1 and 2 (CtBP1/2). To identify additional cellular E1A targets that are involved in the modulation of E1A C-terminus-mediated activities, we undertook tandem affinity purification of E1A-associated proteins. Through mass spectrometric analysis, we identified several known E1A-interacting proteins as well as novel E1A targets, such as the forkhead transcription factors, FOXK1/K2. We identified a Ser/Thr-containing sequence motif in E1A that mediated interaction with FOXK1/K2. We demonstrated that the E6 proteins of two beta-human papillomaviruses (HPV14 and HPV21) associated with epidermodysplasia verruciformis also interacted with FOXK1/K2 through a motif similar to that of E1A. The E1A mutants deficient in interaction with FOXK1/K2 induced enhanced cell proliferation and oncogenic transformation. The hypertransforming activity of the mutant E1A was suppressed by HPV21 E6. An E1A-E6 chimeric protein containing the Ser/Thr domain of the E6 protein in E1A interacted efficiently with FOXK1/K2 and inhibited cell transformation. Our results suggest that targeting FOXK1/K2 may be a common mechanism for certain beta-HPVs and Adv5. E1A exon 2 mutants deficient in interaction with the dual-specificity kinases DYRK1A/1B and their cofactor HAN11 also induced increased cell proliferation and transformation. Our results suggest that the E1A C-terminal region may suppress cell proliferation and oncogenic transformation through interaction with three different cellular protein complexes: FOXK1/K2, DYRK(1A/1B)/HAN11, and CtBP1/2.