Deficiency in Ever2 does not increase susceptibility of mice to pathogenesis by the mouse papillomavirus, MmuPV1.

Epidermodysplasia verruciformis (EV) is a rare genetic skin disorder that is characterized by the development of papillomavirus-induced skin lesions that can progress to squamous cell carcinoma (SCC). Certain high-risk, cutaneous ß-genus human papillomaviruses (ß-HPVs), in particular HPV5 and HPV8, are associated with inducing EV in individuals who have a homozygous mutation in one of three genes tied to this disease: EVER1, EVER2, or CIB1. EVER1 and EVER2 are also known as TMC6 and TMC8, respectively. Little is known about the biochemical activities of EVER gene products or their roles in facilitating EV in conjunction with ß-HPV infection. To investigate the potential effect of EVER genes on papillomavirus infection, we pursued in vivo infection studies by infecting Ever2-null mice with mouse papillomavirus (MmuPV1). MmuPV1 shares characteristics with ß-HPVs including similar genome organization, shared molecular activities of their early, E6 and E7, oncoproteins, the lack of a viral E5 gene, and the capacity to cause skin lesions that can progress to SCC. MmuPV1 infections were conducted both in the presence and absence of UVB irradiation, which is known to increase the risk of MmuPV1-induced pathogenesis. Infection with MmuPV1 induced skin lesions in both wild-type and Ever2-null mice with and without UVB. Many lesions in both genotypes progressed to malignancy, and the disease severity did not differ between Ever2-null and wild-type mice. However, somewhat surprisingly, lesion growth and viral transcription was decreased, and lesion regression was increased in Ever2-null mice compared with wild-type mice. These studies demonstrate that Ever2-null mice infected with MmuPV1 do not exhibit the same phenotype as human EV patients infected with ß-HPVs.IMPORTANCEHumans with homozygous mutations in the EVER2 gene develop epidermodysplasia verruciformis (EV), a disease characterized by predisposition to persistent ß-genus human papillomavirus (ß-HPV) skin infections, which can progress to skin cancer. To investigate how EVER2 confers protection from papillomaviruses, we infected the skin of homozygous Ever2-null mice with mouse papillomavirus MmuPV1. Like in humans with EV, infected Ever2-null mice developed skin lesions that could progress to cancer. Unlike in humans with EV, lesions in these Ever2-null mice grew more slowly and regressed more frequently than in wild-type mice. MmuPV1 transcription was higher in wild-type mice than in Ever2-null mice, indicating that mouse EVER2 does not confer protection from papillomaviruses. These findings suggest that there are functional differences between MmuPV1 and ß-HPVs and/or between mouse and human EVER2.

Synchronous Epidermodysplasia Verruciformis and Intraepithelial Lesion of the Vulva Is Caused by Coinfection With Alpha-Human Papillomavirus and Beta-Human Papillomavirus Genotypes and Facilitated by Mutations in Cell-Mediated Immunity Genes.

CONTEXT.­: There have been exceedingly few reports of epidermodysplasia verruciformis (EV) or EV-like lesions in the vulva. We describe the first observation of vulvar lesions displaying synchronous EV-like histology and conventional high-grade squamous intraepithelial lesion (HSIL), a finding hitherto unreported in medical literature. OBJECTIVES.­: To describe this novel vulvar lesion with hybrid features of HSIL and EV, attempt to confirm the hypothesis of coinfection with α and ß human papillomavirus (α-HPV and ß-HPV) genotypes, and describe relevant underlying genetic mutations. DESIGN.­: Cases were retrospectively selected from our institutional archive. Detailed review of clinical information, histologic examination, and whole genome sequencing (WGS) were performed. RESULTS.­: Five samples from 4 different patients were included. Three of 4 patients had a history of either iatrogenic immune suppression or prior immune deficiency, and all 3 featured classic HSIL and EV changes within the same lesion. One patient had no history of immune disorders, presented with EV-like changes and multinucleated atypia of the vulva, and was the sole patient without conventional HSIL. By WGS, several uniquely mappable reads pointed toward infection with multiple HPV genotypes, including both α-HPVs and ß-HPVs. Mutations in genes implicated in cell-mediated immunity, such as DOCK8, CARMIL2, MST1, and others, were also found. CONCLUSIONS.­: We provide the first description of vulvar lesions harboring simultaneous HSIL and EV features in the English-language literature, a phenomenon explained by coinfection with α-HPV and ß-HPV genotypes. The finding of EV-like changes in a vulvar specimen should prompt assessment of the patient's immune status.

Betapapillomavirus natural history and co-detection with alphapapillomavirus in cervical samples of adult women.

Human papillomaviruses (HPV) of the genus Betapapillomavirus can infect both cutaneous and mucosal sites, but research on their natural history at mucosal sites remains scarce. We examined the risk factors and co-detection patterns of HPVs of the Betapapillomavirus and Alphapapillomavirus genera in cervical samples of the Ludwig-McGill cohort study. We assessed a subset of 505 women from the Ludwig-McGill cohort study from São Paulo, Brazil. Cervical samples over the first year of follow-up were tested for DNA of over 40 alphapapillomavirus types and 43 betapapillomavirus types using a type-specific multiplex genotyping polymerase chain reaction assay. We assessed the risk factors for prevalent and incident betapapillomavirus type detection, and whether types were detected more frequently together than expected assuming independence using permutation tests, logistic regression, and Cox regression. We observed significant within-genus clustering but not cross-genus clustering. Multiple betapapillomavirus types were co-detected in the same sample 2.24 (95% confidence interval [CI]: 1.65-3.29) times more frequently than expected. Conversely, co-detections of alphapapillomavirus and betapapillomavirus types in the same sample occurred only 0.64 (95% CI: 0.51-0.83) times as often as expected under independence. In prospective analyses, positivity to one HPV genus was associated with a nonsignificant lower incidence of detection of types in the other genus. Lifetime number of sex partners and new sex partner acquisition were associated with lower risks of prevalent and incident betapapillomavirus detection. Betapapillomaviruses are commonly found in the cervicovaginal tract. Results suggest potentially different mechanisms of transmission for betapapillomavirus genital infections other than vaginal sex.

Vaccination with human alphapapillomavirus-derived L2 multimer protects against human betapapillomavirus challenge, including in epidermodysplasia verruciformis model mice.

Human alphapapillomaviruses (αHPV) infect genital mucosa, and a high-risk subset is a necessary cause of cervical cancer. Licensed L1 virus-like particle (VLP) vaccines offer immunity against the nine most common αHPV associated with cervical cancer and genital warts. However, vaccination with an αHPV L2-based multimer vaccine, α11-88x5, protected mice and rabbits from vaginal and skin challenge with diverse αHPV types. While generally clinically inapparent, human betapapillomaviruses (ßHPV) are possibly associated with cutaneous squamous cell carcinoma (CSCC) in epidermodysplasia verruciformis (EV) and immunocompromised patients. Here we show that α11-88x5 vaccination protected wild type and EV model mice against HPV5 challenge. Passive transfer of antiserum conferred protection independently of Fc receptors (FcR) or Gr-1+ phagocytes. Antisera demonstrated robust antibody titers against ten ßHPV by L1/L2 VLP ELISA and neutralized and protected against challenge by 3 additional ßHPV (HPV49/76/96). Thus, unlike the licensed vaccines, α11-88x5 vaccination elicits broad immunity against αHPV and ßHPV.

Human Beta Papillomavirus Type 8 E1 and E2 Proteins Suppress the Activation of the RIG-I-Like Receptor MDA5.

Persistent infections of the skin with the human papillomavirus of genus beta (ß-HPV) in immunocompetent individuals are asymptomatic, but in immunosuppressed patients, ß-HPV infections exhibit much higher viral loads on the skin and are associated with an increased risk of skin cancer. Unlike with HPV16, a high-risk α-HPV, the impact of ß-HPV early genes on the innate immune sensing of viral nucleic acids has not been studied. Here, we used primary skin keratinocytes and U2OS cells expressing HPV8 or distinct HPV8 early genes and well-defined ligands of the nucleic-acid-sensing receptors RIG-I, MDA5, TLR3, and STING to analyze a potential functional interaction. We found that primary skin keratinocytes and U2OS cells expressed RIG-I, MDA5, TLR3, and STING, but not TLR7, TLR8, or TLR9. While HPV16-E6 downregulated the expression of RIG-I, MDA5, TLR3, and STING and, in conjunction with HPV16-E7, effectively suppressed type I IFN in response to MDA5 activation, the presence of HPV8 early genes showed little effect on the expression of these immune receptors, except for HPV8-E2, which was associated with an elevated expression of TLR3. Nevertheless, whole HPV8 genome expression, as well as the selective expression of HPV8-E1 or HPV8-E2, was found to suppress MDA5-induced type I IFN and the proinflammatory cytokine IL-6. Furthermore, RNA isolated from HPV8-E2 expressing primary human keratinocytes, but not control cells, stimulated a type I IFN response in peripheral blood mononuclear cells, indicating that the expression of HPV8-E2 in keratinocytes leads to the formation of stimulatory RNA ligands that require the active suppression of immune recognition. These results identify HPV8-E1 and HPV8-E2 as viral proteins that are responsible for the immune escape of ß-HPV from the innate recognition of viral nucleic acids, a mechanism that may be necessary for establishing persistent ß-HPV infections.

Beta-Genus Human Papillomavirus 8 E6 Destabilizes the Host Genome by Promoting p300 Degradation.

The beta genus of human papillomaviruses infects cutaneous keratinocytes. Their replication depends on actively proliferating cells and, thus, they conflict with the cellular response to the DNA damage frequently encountered by these cells. This review focus on one of these viruses (HPV8) that counters the cellular response to damaged DNA and mitotic errors by expressing a protein (HPV8 E6) that destabilizes a histone acetyltransferase, p300. The loss of p300 results in broad dysregulation of cell signaling that decreases genome stability. In addition to discussing phenotypes caused by p300 destabilization, the review contains a discussion of the extent to which E6 from other ß-HPVs destabilizes p300, and provides a discussion on dissecting HPV8 E6 biology using mutants.

ß-HPV 8E6 combined with TERT expression promotes long-term proliferation and genome instability after cytokinesis failure.

Human papillomavirus (HPV) is a family of viruses divided into five genera: alpha, beta, gamma, mu, and nu. There is an ongoing discussion about whether beta genus HPVs (ß-HPVs) contribute to cutaneous squamous cell carcinoma (cSCC). The data presented here add to this conversation by determining how a ß-HPV E6 protein (ß-HPV 8E6) alters the cellular response to cytokinesis failure. Specifically, cells were observed after cytokinesis failure was induced by dihydrocytochalasin B (H2CB). ß-HPV 8E6 attenuated the immediate toxicity associated with H2CB but did not promote long-term proliferation after H2CB. Immortalization by telomerase reverse transcriptase (TERT) activation also rarely allowed cells to sustain proliferation after H2CB exposure. In contrast, TERT expression combined with ß-HPV 8E6 expression allowed cells to proliferate for months following cytokinesis failure. However, this continued proliferation comes with genome destabilizing consequences. Cells that survived H2CB-induced cytokinesis failure suffered from changes in ploidy.

Comparison and evaluation of three different molecular methods for detection of human Betapapillomaviruses in skin biopsies from patients with nonmelanoma skin cancer and precancerous lesions.

Betapapillomaviruses have been linked to the development of nonmelanoma skin cancers. A great diversity of these viruses in skin specimens requires the use of sensitive and reliable detection methods. There are currently no standardized assays for diagnostic purposes. A combination of several molecular methods has great practical significance and gives the opportunity to broaden the spectrum of detected Beta-HPV types. In the present study, different molecular methods for Beta-HPVs detection and genotyping were used: PCRs with different sets of primers, PCR followed by reverse hybridization and direct sequencing of PCR amplimers; all performed in skin biopsies from lesions and perilesional healthy area of 118 patients with NMSC or precancerous lesions. Beta-HPVs were detected in 41% of 261 biopsies examined. The RHA for 25 types of Beta-HPVs showed a significantly higher sensitivity than PCR-based methods and allowed to detect 172 genotypes in 86 samples, including 39 with multiple infections. The most frequently identified types were HPV23, HPV24 and HPV93. HPV5 and HPV8, considered high-risk carcinogen types, were detected only in a small percentage of samples. Direct sequencing confirmed the presence of Beta-HPV genotypes from outside of RHA panel in the analysed biopsies. This allowed detecting thirty-two additional genotypes in 5 samples, that were positive only in RHA with the universal probe, which failed to identify the virus genotypes. Our findings confirmed the need to apply different methods to detect Beta-HPV infections.

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.

Presence of human papillomavirus DNA in voriconazole-associated cutaneous squamous cell carcinoma.

BACKGROUND: Voriconazole and genus beta human papillomavirus (HPV) are independently associated with the development of photo-exposed cutaneous squamous cell carcinoma (SCC) but have not been evaluated concurrently. The objective of this study is to determine the prevalence and type of detectable HPV DNA in voriconazole-associated SCC. METHODS: SCCs from immunosuppressed patients, in those with and without voriconazole exposure, were evaluated by PCR analysis for HPV DNA and compared to SCC from non-immunosuppressed patients. An additional expanded PCR analysis of all SCC that developed in the voriconazole group was also performed. RESULTS: HPV DNA was detected by PCR in all groups regardless of the immunosuppression status (80.5%) with beta HPV most prevalent (64.3-78.6%). However, immunosuppressed patients were significantly more likely to be infected by beta HPV types 5, 8, 14, 20, and 21 (P-value 0.014), and represented the majority of beta HPV types found in the voriconazole group. CONCLUSIONS: In this retrospective study, beta HPV 5, 8, 14, 20, and 21 were commonly detected in voriconazole-associated SCC. The results indicate a possible role of beta HPV in the pathogenesis of cutaneous SCC in photo-exposed areas. Further studies are needed to establish the role of HPV and voriconazole in the pathogenesis of the lesion.