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Anogenital and oropharyngeal infections with human papilloma viruses (HPV) are common. Clinically manifest disease may significantly impact quality of life; the treatment of HPV-associated lesions is associated with a high rate of recurrence and invasive neoplasms, such as cervical, anal, vulvar, penile, and oropharyngeal cancers, which are characterized by significant morbidity and mortality. Vaccination against HPV is an effective and safe measure for the primary prevention of HPV-associated lesions, but immunization rates are still low in Germany. The present publication is an abridged version of the German evidence and consensus-based guideline "Vaccination recommendations for the prevention of HPV-associated lesions", which is available on the website of the German Association of the Scientific Medical Societies (AWMF). On the basis of a systematic review with meta-analyses, a representative panel developed and agreed upon recommendations for the vaccination of different populations against HPV. In addition, consensus-based recommendations were developed for specific issues relevant to everyday practice. Based on current evidence and a representative expert consensus, these recommendations are intended to provide guidance in a field in which there is often uncertainty and in which both patients and health care providers are sometimes confronted with controversial and emotionally charged points of view.

Data about the prevalence of human papillomaviruses (HPV) in African women with normal and abnormal cervical cytology are still scarce. Current HPV vaccines contain HPV types, which mainly represent the HPV epidemiology of industrial countries. As further developments of HPV vaccines are going on, it is necessary to regard regional differences in HPV type prevalence to ensure optimal protection by the vaccine. Vaginal swabs of Ghanaian pregnant women, routinely collected before delivery to rule out bacterial infections causing early onset sepsis, were screened for 12 high-risk (HR), 13 probably/possibly (pHR), and 18 low-risk (LR) HPV types. Most pregnant women come for delivery to the hospital. This was considered as appropriate possibility to have an unselected group of women. HPV DNA were detected in 55/165 women (33.3, 95 % CI 26.3-41.1 %). Thirty-four out of fifty-five (61.8, 95 % CI 47.7-74.3 %) of HPV-positive women were infected with HR and/or pHR HPV types. The five most prevalent HR or pHR HPV types were HPV-52 and HPV-67 (7 women each, 4.2, 95 % CI 1.9-8.9 %), HPV-53 (six women, 3.6, 95 % CI 1.5-8.1 %), HPV-45 (five women, 3.0, 95 % CI 1.1-7.3 %), and HPV-18 (four women, 2.4, 95 % CI 0.8-6.5 %), respectively. HPV-16 was found in two women only (1.2, 95 % CI 0.2-4.8 %). Future HPV vaccine research may devote special interest to HPV-67 and HPV-53 provided further studies confirm their high prevalence in the general population of Sub-Saharan African countries. The true carcinogenic potential of HPV-67, which is a member of species alpha9 including HPV-16, and so far categorized as pHR, should be clarified.

// Jayesh Deshmukh 1 , Ruth Pofahl 1 , Herbert Pfister 2 , Ingo Haase 1 1 Department of Dermatology, University of Cologne, Cologne, 50937, Germany 2 Institute of Virology, University of Cologne, Cologne, 50935, Germany Correspondence to: Jayesh Deshmukh, email: jayesh.deshmukh@uk-koeln.de Keywords: HPV-8, Rac1, skin cancer, SCC, UV (Ultraviolet) light Received: April 01, 2016 Accepted: July 19, 2016 Published: August 05, 2016 ABSTRACT Overexpression and increased activity of the small Rho GTPase Rac1 has been linked to squamous cell carcinoma of the epidermis and mucosa in humans. Targeted deletion of Rac1 or inhibition of Rac1 activity in epidermal keratinocytes reduced papilloma formation in a chemical skin carcinogenesis mouse model. However, a potential role of Rac1 in HPV- and UV-light induced skin carcinogenesis has not been investigated so far, solar UV radiation being an important carcinogen to the skin. To investigate this, we deleted Rac1 or modulated its activity in mice with transgenic expression of Human papilloma virus type-8 (HPV-8) in epidermal keratinocytes. Our data show that inhibition or deletion of Rac1 results in reduced papilloma formation upon UV-irradiation with a single dose, whereas constitutive activation of Rac1 strongly increases papilloma frequency in these mice. Surprisingly, we observed that, upon chronic UV-irradiation, the majority of mice with transgenic expression of HPV-8 and epidermis specific Rac1 deletion developed squamous cell carcinomas. Taken together, our data show that Rac1 exerts a dual role in skin carcinogenesis: its activation is, on one hand, required for HPV-8- and UV-light induced papilloma formation but, on the other, suppresses the development of squamous cell carcinomas.

Organ transplant recipients (OTRs) have a 100‐fold increased risk of cutaneous squamous cell carcinoma (cSCC). We prospectively evaluated the association between β genus human papillomaviruses (βPV) and keratinocyte carcinoma in OTRs. Two OTR cohorts without cSCC were assembled: cohort 1 was transplanted in 2003‐2006 (n = 274) and cohort 2 was transplanted in 1986‐2002 (n = 352). Participants were followed until death or cessation of follow‐up in 2016. βPV infection was assessed in eyebrow hair by using polymerase chain reaction–based methods. βPV IgG seroresponses were determined with multiplex serology. A competing risk model with delayed entry was used to estimate cumulative incidence of histologically proven cSCC and the effect of βPV by using a multivariable Cox regression model. Results are reported as adjusted hazard ratios (HRs). OTRs with 5 or more different βPV types in eyebrow hair had 1.7 times the risk of cSCC vs OTRs with 0 to 4 different types (HR 1.7, 95% confidence interval 1.1‐2.6). A similar risk was seen with high βPV loads (HR 1.8, 95% confidence interval 1.2‐2.8). No significant associations were seen between serum antibodies and cSCC or between βPV and basal cell carcinoma. The diversity and load of βPV types in eyebrow hair are associated with cSCC risk in OTRs, providing evidence that βPV is associated with cSCC carcinogenesis and may present a target for future preventive strategies., In two cohorts of organ transplant recipients, those with five and more different beta‐genus human papillomavirus types and high virus loads in eyebrow hairs subsequently develop significantly more cutaneous squamous cell carcinomas than others, suggesting that these virus types may increase the risk of cutaneous squamous cell carcinoma in these patients.

HIV-positive men who have sex with men (MSM) have an increased risk for anal human papillomavirus (HPV) infection, anal high-grade intraepithelial lesions (HSIL), and anal cancer. Smoking is associated with abnormal anal cytology and with an increased risk for anal cancer. We collected 3736 intraanal swabs from 803 HIV-positive MSM who participated in an anal cancer screening program between October 2003 and August 2014. HPV prevalence, anal cytology and HPV DNA load of high-risk (HR) HPV-types 16, 18, 31 and 33 of non-smokers and smokers were compared. HPV-typing was performed by alpha-HPV genus-specific PCR and hybridization with 38 type-specific probes using a multiplex genotyping assay. In samples positive for HPV16, 18, 31, or 33, HPV DNA loads were determined by type-specific real-time PCRs and expressed as HPV DNA copies per betaglobin gene copy. At baseline, HR-HPV DNA (80.5 vs. 89.0%, p=0.001), HPV16 DNA (41.6 vs. 52.3%, p=0.003), HPV18 DNA (15.5 vs. 26.0%, p

Activation-induced cytidine deaminase (AID) not only promotes immune diversity by initiating somatic hypermutation and class switch recombination in immunoglobulin genes but also provokes genomic instability by introducing translocations and mutations into non-immunoglobulin genes. To test whether AID is essential for virus-induced tumor development, we used two transgenic tumor models: mice expressing hepatitis C virus (HCV) core proteins (HCV-Tg), driven by the hepatitis B virus promoter, and mice expressing human papillomavirus type 8 proteins (HPV8-Tg), driven by the Keratin 14 promoter. Both strains were analyzed in the absence and presence of AID by crossing each with AID−/− mice. There was no difference in the liver tumor frequency between the HCV-Tg/AID+/+ and HCV-Tg/AID−/− mice at 20 months of age although the AID+/+ mice showed more severe histological findings and increased cytokine expression. Furthermore, a low level of AID transcript was detected in the HCV-Tg/AID+/+ liver tissue that was not derived from hepatocytes themselves but from intra-hepatic immune cells. Although AID may not be the direct cause of HCV-induced oncogenesis, AID expressed in B cells, not in hepatocytes, may prolong steatosis and cause increased lymphocyte infiltration into HCV core protein-induced liver lesions. Similarly, there was no difference in the time course of skin tumor development between the HPV8-Tg/AID−/− and HPV8-Tg/AID+/+ groups. In conclusion, AID does not appear to be required for tumor development in the two virus-induced tumor mouse models tested although AID expressed in infiltrating B cells may promote inflammatory reactions in HCV core protein-induced liver pathogenesis.

Background Anal human papillomavirus (HPV) infection and anal dysplasia are frequent in HIV-positive men who have sex with men (HIV + MSM), and progression of low-grade (LSIL) to high-grade squamous intraepithelial lesions (HSIL) or anal cancer (AC) occurs faster than in HIV-negative individuals. High-risk (HR)-HPV-E6/E7 oncogene mRNA testing has a higher specificity and a higher positive predictive value (PPV) than HR-HPV-DNA testing for detecting high-grade cervical lesions. Objective To evaluate the diagnostic accuracy of the NucliSENS-EasyQ HPV1.1 E6/E7-mRNA-assay for the detection of anal dysplasia in HIV + MSM. Study design 289 intraanal swabs from HIV + MSM participating in a screening program that included anal cytology, high-resolution anoscopy and histology were analyzed. HR-HPV-DNA detection was performed by PCR and hybridization using a bead-based multiplex genotyping assay. E6/E7-mRNA detection of HR-HPV-types 16, 18, 31, 33 and 45 was performed using the NucliSENS-EasyQ assay. Results 269 swabs had valid results in both test formats (111 normal, 10 ASCUS, 105 LSIL, 42 HSIL, 1 AC). For the detection of LSIL + (LSIL + HSIL + cancer) sensitivity, specificity, negative predictive value (NPV) and PPV were 80.4%, 26.4%, 52.5%, and 57.2% for HR-HPV-DNA testing, respectively, compared to 75.7%, 57.9%, 66.0% and 68.7% for E6/E7-mRNA testing. The respective values for the detection of HSIL/cancer were 95.3%, 26.1%, 96.7%, 19.7% for HR-HPV-DNA and 95.3%, 46.0%, 98.1%, 25.2% for E6/E7-mRNA detection. Conclusion Compared to HR-HPV-DNA detection, E6/E7-mRNA testing has an increased specificity (approximately two-fold), similar sensitivity and higher NPV and PPV for the detection of low- and high-grade anal dysplasia in HIV + MSM.

Epidermodysplasia verruciformis (EV) is a rare recessive genodermatosis characterized by high susceptibility to infections with human papillomaviruses (HPVs) of genus beta. Knowledge about seroreactivity against HPV in these patients and their first-degree relatives is scarce. Using multiplex serology, we analyzed antibodies to 38 HPV types from five genera in 32 EV patients, 22 first-degree relatives, and 64 and 44 age- and sex-matched, non-related, healthy controls, respectively. EV patients showed higher seroprevalences than non-related controls with statistically significant odds ratios (ORs) for 5 of 10 investigated alpha (OR range 6.9-21.3), all 16 beta (OR range 12.3-61.3), 3 of 9 gamma (OR range 6.4-11.7), and 1 of 2 micro HPVs (OR 5.8). In comparison to their relatives, antibodies in EV patients were significantly more prevalent for 4 of 16 beta HPVs (OR range 12.5-25.6), but for none of the other genera. A significantly increased seroprevalence in relatives compared with their controls was only seen for HPV 5 (OR 22.1). The considerably elevated HPV seroprevalence in EV patients, especially for beta papillomaviruses (PVs), reflects the high viral load described for these individuals. Whether the observed differences between relatives and healthy controls depend on heterozygosity for EV-associated alleles requires further investigation.

Summary Background Antimicrobial peptides and proteins (AMPs) are widely distributed effector molecules of the innate immune system with well-known antibacterial activity. However, there is a paucity of information regarding antiviral effects of AMPs. Objectives The present study was performed to analyse expression of AMPs in human papillomavirus (HPV)-associated anal skin lesions of human immunodeficiency virus (HIV)-positive men who have sex with men (MSM), a special high-risk group for persistent HPV infections and anal dysplasia. Methods Skin lesions were analysed for the presence of LL-37, RNase 7, and human β-defensin (hBD)-1, hBD-2 and hBD-3. Moreover, HPV typing and HPV DNA load determination for HPV types 6, 11, 16, 18, 31 and 33 were performed to evaluate possible correlations between expression of AMPs and lesional HPV types. Results Skin biopsies of 45 HIV-positive MSM with anal intraepithelial neoplasia (AIN), anal condylomata acuminata or unaffected anal mucosa, as well as condylomata acuminata of eight HIV-negative MSM, were analysed for AMP mRNA expression. Additionally, immunohistochemical analysis for hBD-2 and hBD-3 was performed in a total of 45 samples. hBD-2 and hBD-3 gene and protein expression was significantly increased in both AIN and condyloma, whereas LL-37, RNase 7 and hBD-1 gene expression did not differ significantly from unaffected anal mucosa. AMP expression correlated neither with the number of HPV types nor with the high-risk and low-risk HPV DNA loads of the quantified types. No significant differences in AMP expression were observed in condylomata of HIV-positive and HIV-negative MSM. Conclusions hBD-2 and hBD-3 expression was shown to be significantly upregulated in HPV-associated anal skin lesions of both HIV-positive and HIV-negative MSM. Their biological significance in the innate immunity against these lesions needs further research.

Anal intraepithelial neoplasia (AIN), a human papillomavirus (HPV)-associated precursor lesion of anal carcinoma, is highly prevalent in HIV-infected men having sex with men (MSM). This prospective follow-up study evaluated the long-term results of imiquimod treatment of AIN in 19 HIV-infected MSM. Standardized follow-up examinations included high-resolution anoscopy, anal cytology/histology, HPV typing, and DNA load determination for HPV types 16, 18, 31, and 33. Mean follow-up time was 30.3 months. A total of 74% (14/19) of the patients remained free of AIN at the previously treated site. Five patients (26%) had recurrent high-grade AIN after a mean time of 24.6 months. At the end of follow-up, the numbers of HPV types as well as high-risk HPV-DNA loads were significantly lower than before therapy. During follow-up, 58% of all patients (11/19) developed new anal cytological abnormalities in previously normal, untreated anal regions. 55% of these new AIN lesions were high-grade lesions and most of them were located intra-anally and associated with high-risk HPV types not detectable before therapy. These results demonstrate that imiquimod leads to a high rate of long-term clearance of AIN in HIV-positive men together with a prolonged decrease of high-risk HPV-DNA load. However, new AIN lesions associated with previously undetected HPV types frequently occur in untreated areas.

Molecular prognostic indicators for oropharyngeal squamous cell carcinoma (OSCC), including HPV-DNA detection, epidermal growth factor receptor (EGFR) and p16 expression, have been suggested in the literature, but none of these are currently used in clinical practice. To compare these predictors, 106 newly diagnosed OSCC for the presence of HPV-DNA and expression of p16 and EGFR were analyzed. The 5-year disease-free survival (DFS) and overall survival (OS) were calculated in relation to these markers and a multivariate Cox analysis was performed. Twenty-eight percent of the cases contained oncogenic HPV-DNA and 30% were positive for p16. The p16 expression was highly correlated with the presence of HPV-DNA (p < 0.001). Univariate analysis of the 5-year DFS revealed a significantly better outcome for patients with p16-positive tumors (84% vs. 49%, p = 0.009). EGFR-negative tumors showed a tendency toward a better prognosis in DFS (74% vs. 47%, p = 0.084) and OS (70% vs. 45%, p = 0.100). Remarkable and highly significant was the combination of p16 and EGFR expression status, leading to 5-year DFS of 93% for p16+/EGFR- tumors vs. 39% for p16-/EGFR+ tumors (p = 0.003) and to a 5-year OS of 79% vs. 38%, respectively (p = 0.010). In multivariate analysis p16 remained a highly significant prognostic marker for DFS (p = 0.030) showing a 7.5-fold increased risk for relapse in patients with p16-negative tumors. Our data indicate that p16 expression is the most reliable prognostic marker for OSCC and further might be a surrogate marker for HPV-positive OSCC. HPV+/p16+ tumors tended to have decreased EGFR expression, but using both immunohistological markers has significant prognostic implications.

Background The failure to mount an effective DNA damage response to repair UV induced cyclobutane pyrimidine dimers (CPDs) results in an increased propensity to develop cutaneous squamous cell carcinoma (cSCC). High-risk patient groups, such as organ transplant recipients (OTRs) frequently exhibit field cancerization at UV exposed body sites from which multiple human papillomavirus (HPV)-associated cSCCs develop rapidly, leading to profound morbidity and increased mortality. In vitro molecular evidence indicates that HPV of genus beta-papillomavirus (β-PV) play an important role in accelerating the early stages of skin tumorigenesis. Methods We investigated the effects of UV induced DNA damage in murine models of β-PV E6 oncoprotein driven skin tumorigenesis by crossing K14-HPV8-E6wt mice (developing skin tumors after UV treatment) with K14-CPD-photolyase animals and by generating the K14-HPV8-E6-K136N mutant mouse strain. Thymine dimers (marker for CPDs) and γH2AX (a marker for DNA double strand breaks) levels were determined in the murine skin and organotypic skin cultures of E6 expressing primary human keratinocytes after UV-irradiation by immunohistochemistry and in cell lines by In Cell Western blotting. Phosphorylation of ATR/Chk1 and ATM were assessed in cell lines and organotypic skin cultures by Western blots and immunohistochemistry. Results Skin tumor development after UV-irradiation in K14-HPV8-E6wt mice could completely be blocked through expression of CPD-photolyase. Through quantification of thymine dimers after UV irradiation in cells expressing E6 proteins with point mutations at conserved residues we identified a critical lysine in the C-terminal part of the protein for prevention of DNA damage repair and p300 binding. Whereas all K14-HPV8-E6wt animals develop skin tumors after UV expression of the HPV8-E6-K136N mutant significantly blocked skin tumor development after UV treatment. The persistence of CPDs in hyperproliferative epidermis K14-HPV8-E6wt skin resulted in the accumulation of γH2AX foci. DNA damage sensing was impaired in E6 positive cells grown as monolayer culture and in organotypic cultures, due to lack of phosphorylation of ATM, ATR and Chk1. Conclusion We showed that cells expressing E6 fail to sense and mount an effective response to repair UV-induced DNA lesions and demonstrated a physiological relevance of E6-mediated inhibition of DNA damage repair for tumor initiation. These are the first mechanistical in vivo data on the tumorigenicity of HPV8 and demonstrate that the impairment of DNA damage repair pathways by the viral E6 protein is a critical factor in HPV-driven skin carcinogenesis. Electronic supplementary material The online version of this article (doi:10.1186/s12943-015-0453-7) contains supplementary material, which is available to authorized users.

Human papillomaviruses (HPV) infect keratinocytes of skin and mucosa. Persistent infection can lead to the formation of benign tumors. In cases of high-risk HPV, such as HPV16 or 18, these may further progress to cancer. In order to support viral replication in suprabasal keratinocytes, the HPV E7 protein employs various strategies to keep keratinocytes in cycle and counteracts anti-proliferative signals from outside. HPV16 E7 can directly interfere with transforming growth factor-beta (TGF-beta) signalling by binding to Smad proteins mediating growth arrest. It has been speculated that this property of HPV16 E7 contributes to HPV-associated carcinogenesis. Here, we show that E7 proteins from different low- and high-risk HPV types bind to Smad 1 to 4. The E7 protein from HPV1, a low-risk HPV causing plantar warts, efficiently inhibited Smad 3-induced transcription. Our data strongly indicate that the Smad-binding capacity of E7 proteins from different HPVs may preserve keratinocyte proliferation required for the productive viral life cycle rather than promoting carcinogenesis.

Objectives: The aim of this study was analysis of the results of use of interferon-α (IFN-α) in patients with recurrent respiratory papillomatosis (RRP) and correlation of the results with human papillomavirus (HPV) type. Methods: A multicenter prospective series (42 patients from 22 hospitals) yielded 20 years of follow-up of patients with RRP and HPV typing who were treated with IFN-α in doses of 3 MU/m2 3 times per week. Results: During long-term follow-up (mean ± SD, 172 ± 36.8 months), the rate of event-free survival evaluated by Kaplan-Meier analysis was 42.8%, and the overall survival rate was 82.6%. The HPV typing revealed an association of HPV 11 with a more aggressive disease course (64% of HPV 11 patients versus 24% of HPV 6 patients), a lower incidence of long-term response to IFN-α therapy (14% of HPV 11 patients versus 64% of HPV 6 patients), and a higher incidence of malignant transformation and mortality during follow-up (36% and 24%, respectively, of HPV 11 patients versus 0% of HPV 6 patients). Conclusions: The obtained results revealed maximal effectiveness of IFN-α therapy in RRP patients with HPV 6 as compared with HPV 11. The association of HPV 11 with a worse long-term response to IFN-α therapy and a higher incidence of malignant transformation and mortality is clinically important and indicates the necessity of HPV typing in RRP patients after the first biopsy.

Background Anal intraepithelial neoplasia (AIN) represents a precursor lesion of invasive squamous cell carcinoma with a clear association to high-risk human papillomavirus (HPV) types. HIV infection is strongly associated with a higher prevalence of genital HPV infection, a higher incidence of AIN, and, consecutively, an increased risk for anal cancer. Objective The aim of this study was to determine the clinical spectrum of AIN and lesional HPV colonization in a cohort of homosexual men who were HIV positive and had a history of receptive anal intercourse. Methods In all, 103 men who were HIV-1 positive were screened by using clinical, proctologic, cytologic, histologic, and HPV DNA testing. Results Of all patients, 86% had anal HPV infection at their first visit. HPV-16 (53%), HPV-18 (27%), HPV-58 (22%), and HPV-83 (22%) were the most frequently found HPV types. AIN was diagnosed in 20 of the 103 patients (19.4%). High-risk HPV types were present in all AIN cases with up to 7 different high-risk and up to 5 different low-risk types per lesion. Histologically, 7 (35%), 7 (35%), and 6 (30%) of the patients had AIN grade I, II, or III, respectively. Four different types of clinical presentation could be distinguished in the 20 patients with AIN: bowenoid (1 case, 5%); erythroplakic (2 cases, 10%); verrucous (6 cases, 30%); and leukoplakic (11 cases, 55%). All verrucous lesions were graded as high-grade intraepithelial lesions in cytology, whereas 6 of the 11 leukoplakic lesions (55%) were low grade. All verrucous AIN carried at least 4 different HPV types, always including HPV-16, and the mean number of HPV types was higher in verrucous lesions than in leukoplakic lesions (5.5 vs 3.8, respectively). Conclusion These data confirm the high incidence and prevalence of AIN in patients who are HPV positive with HIV infection. Four different clinical types of AIN can be distinguised that might have prognostic implications. Standardized screening programs for anal cancer prevention and treatment protocols for AIN in patients infected with HIV must be implemented.

Using PCR, the overall prevalence of human papillomavirus (HPV) DNA in penile carcinoma is about 40-45%, which is similar to the detection rate of HPV-DNA in vulvar carcinoma (50%). In analogy to vulvar cancer two different pathways of penile carcinogenesis seem to exist. In contrast to basaloid and warty penile cancers which are regularly HPV-associated (about 80-100%), only a part of keratinizing and verrucous penile carcinomas appear to be related with HPV (33-35%). Penile intraepithelial neoplasias comprising Bowen's disease, erythroplasia of Queyrat and bowenoid papulosis are precursor lesions of basaloid and warty carcinomas of the penis. Precursors of keratinizing carcinomas and verrucous carcinomas are not established. Whether lichen sclerosus and squamous-cell hyperplasia precede penile keratinizing carcinoma is a matter of discussion. Giant condylomata acuminata may precede the development of verrucous carcinomas in some cases. Since high risk HPVs are more frequently found in verrucous carcinomas than in giant condylomas, HPV typing may be a helpful diagnostic step to differentiate giant condyloma from verrucous carcinoma.

Esophageal squamous cell carcinomas ( n=23) and adenocarcinomas ( n=14) from German patients were tested for the presence of human papillomavirus (HPV) and Epstein-Barr-Virus (EBV) DNA. No DNA for either genital HPV or epidermodysplasia verruciformis-associated HPV was detected in any tumor. In contrast, 35% of squamous cell carcinomas and 36% of adenocarcinomas were shown to contain EBV DNA by nested polymerase chain reaction.

Human papillomavirus (HPV) DNA loads of six oncogenic HPV types were measured by real-time PCR in cervical scrapes of human immunodeficiency virus (HIV)-infected and uninfected women. In both groups, HPV loads increased with the grade of cervical disease. HIV infection did not affect HPV loads in low-grade lesions but was associated with significantly higher HPV loads in severe dysplasia; highest loads were found in advanced HIV disease. Our data reflect the aggressive course of HPV infection in HIV-positive women.

The papillomavirus life cycle is closely linked to the differentiation program of the host keratinocyte. Thus, late gene expression and viral maturation are restricted to terminally differentiated keratinocytes. A variety of cellular transcription factors including those of the C/EBP family are involved in the regulation of keratinocyte differentiation. In this study we show that the papillomavirus transcription factor E2 cooperates with C/EBPα and -β in transcriptional activation. This synergism was independent of an E2 binding site. E2 and C/EBP factors synergistically transactivated a synthetic promoter construct containing classical C/EBPβ sites and the C/EBPα-responsive proximal promoter of the involucrin gene, which is naturally expressed in differentiating keratinocytes. C/EBPα or -β coprecipitated with E2 proteins derived from human papillomavirus type 8 (HPV8), HPV16, HPV18, and bovine papillomavirus type 1 in vitro and in vivo, indicating complex formation by the cellular and viral factors. The interaction domains could be mapped to the C terminus of E2 and amino acids 261 to 302 located within the bZIP motif of C/EBPβ. Our data suggest that E2, via its interaction with C/EBP factors, may contribute to enhancing keratinocyte differentiation, which is suppressed by the viral oncoproteins E6 and E7 in HPV-induced lesions.

Recent analyses of head and neck squamous cell carcinomas revealed frequent infections by oncogenic human papillomavirus (HPV) type 16 in tonsillar carcinomas. Concerning involvement of risk factors, clinical course of the disease, and prognosis there are strong indications arguing that the HPV-positive tonsillar carcinomas may represent a separate tumor entity. Looking for a surrogate marker, which in further epidemiological studies could replace the laborious and expensive HPV detection and typing we analyzed p16 protein expression in 34 tonsillar carcinoma for correlation to HPV status and load of viral DNA. p16 has been shown to be of diagnostic value for clinical evaluation of cervical dysplasia. We found 53% of the tested tonsillar carcinomas to be HPV-positive. Fifty-six percent of all tumors tested were immunohistochemically positive for the p16 protein. In 16 of 18 of the HPV-positive carcinomas diffuse p16 expression was observed. In contrast, only one of the HPV-negative carcinomas showed focal p16 staining (P < 0.001). As determined by laser-assisted microdissection and quantitative real-time polymerase chain reaction, p16 expression correlated with the presence of HPV-DNA in the individual tumor specimens. Clinical outcome analysis revealed significant correlation of p16 expression with increased disease-free survival (P = 0.02). These data indicate that p16 is a technically simple immunohistological marker, applicable for routine pathological histology, and its prognostic value for survival is fully equivalent to HPV-DNA detection.

Repression of transcription by the full-length E2 protein of papillomaviruses (PV) seems to occur when the E2 binding sites and those of positively acting cellular factors overlap. Previously, we showed that RUNX1 (formerly called CBF) binds to the repression-mediating E2 binding site P2 of human PV type 8 (HPV8). By a yeast one-hybrid system we could identify an unknown protein binding also to P2, tentatively called PBF (papillomavirus binding factor). PBF recognizes the sequence CCGG, which represents the 3′ half of the E2 binding site just adjacent to the RUNX1 motif. PBF also binds to the repression-mediating E2 BS-1 in BPV1, which is conserved to P2 of HPV8. Point mutations destroying PBF binding to HPV8 P2 and BPV-1 E2 BS-1 in vitro reduce promoter activity in corresponding reporter constructs. Our results suggest that PBF might play a role in transcription of PV genes and in E2-mediated repression.

High-risk human papillomavirus types, especially type 16, are risk factors for cervical cancer. Preliminary studies suggest that HPV16 polymorphisms in the long control region or in the E6 gene may alter the oncogenic potential of the virus. This could partially explain why some lesions progress to cancer while others do not. A systematic study combining the long control region and E6 has not been undertaken. This prompted us to investigate the long control region and the E6 in northern European women infected with human papillomavirus 16. We identified the sequence variations of both regions and investigated the long control region promoter activity among various isolates. In addition, we correlated the distribution of long control region and E6 polymorphisms with disease status. We analyzed 45 samples from Swedish and Finnish women. The long control region and the E6 gene were sequenced after polymerase chain reaction long control region fragments of six European isolates covering the majority of polymorphisms in this region were ligated into the pALuc vector and used for luciferase assays. In European HPV16 isolates, polymorphisms in the long control region are more frequent than in the E6 gene. Nevertheless, the promoter function was slightly increased in only one of the tested European long control region variants. In addition, we found a specific European E6 variant, L83V, to be enriched in high-grade lesions and cancer rather than a specific European long control region variant. The difference in oncogenicity between European HPV16 genotypes is more probably due to an altered property of the corresponding E6 proteins rather than to an altered activity of the P97 promoter. British Journal of Cancer (2002) 86, 269–273. DOI: 10.1038/sj/bjc/6600024 www.bjcancer.com © 2002 The Cancer Research Campaign

Chronic immunosuppression for various reasons is an important risk factor for persistent infections with human papillomaviruses (HPV) and HPV-associated disease. Anogenital cancers and their precursor lesions play an important role in modern HIV medicine. Anal intraepithelial neoplasia and anal cancer are especially prevalent in HIV-positive men who have sex with men, and experts have advocated screening similarly to cervical cancer screening for this high-risk group. HPV-induced skin warts are a frequent problem of organ transplant recipients (OTRs) and a severe manifestation (generalized verrucosis) of certain primary immunodeficiencies. HIV-positive individuals are very often affected by genital warts (condylomata acuminata). Iatrogenic immunosuppression in OTRs allows more active replication of the commensal β-papillomavirus spectrum in the entire skin. A high viral DNA load in plucked eyebrow hairs was shown to imply a significant risk for the development of cutaneous squamous cell carcinoma. This may explain the up to 250-fold increased risk of OTRs to develop precancerous actinic keratoses and skin cancer.

Chronic immunosuppression for various reasons is an important risk factor for persistent infections with human papillomaviruses (HPV) and HPV-associated disease. Anogenital cancers and their precursor lesions play an important role in modern HIV medicine. Anal intraepithelial neoplasia and anal cancer are especially prevalent in HIV-positive men who have sex with men, and experts have advocated screening similarly to cervical cancer screening for this high-risk group. HPV-induced skin warts are a frequent problem of organ transplant recipients (OTRs) and a severe manifestation (generalized verrucosis) of certain primary immunodeficiencies. HIV-positive individuals are very often affected by genital warts (condylomata acuminata). Iatrogenic immunosuppression in OTRs allows more active replication of the commensal β-papillomavirus spectrum in the entire skin. A high viral DNA load in plucked eyebrow hairs was shown to imply a significant risk for the development of cutaneous squamous cell carcinoma. This may explain the up to 250-fold increased risk of OTRs to develop precancerous actinic keratoses and skin cancer.

Background Netherton’s syndrome (NS) is a hereditary disorder with dermatological signs (e.g. ichthyosis) and a complex immunological dysfunction. In immunodeficient individuals human papillomavirus (HPV) types are associated with carcinomas on non-mucosal sites. Objectives To study the presence of HPV infection in different skin lesions of three male NS patients and to investigate a possible association between HPV and malignancies in NS. Methods Patient 1 had extraordinary widespread multiple skin carcinomas on sunlight-exposed areas, as well as common viral warts. Patient 2 showed disseminated viral plane warts that resolved spontaneously, and patient 3 was free of skin lesions suspicious for HPV infection; only pseudoepitheliomatous wart-like lesions as a symptom of ichthyosis were apparent. We performed nested polymerase chain reaction analysis of DNA from benign and malignant skin lesions and HPV-8 serology in these three patients. Results Antibodies to HPV-8 were not detectable in our patients; however, seven of 22 (31%) biopsies of the three NS patients were positive for HPV DNA. Epidermodysplasia verruciformis (EV) -associated HPV types and normal cutaneous types (HPV-2, HPV-28) were detected. Interestingly, only the patient with cutaneous carcinomas harboured, preferentially in malignant lesions, EV-HPV types (HPV-19, 23, 38 and HPV-RTRX9, closely related to EV-HPVs), whereas plane warts of patient 2 were positive for HPV-28. The pseudoepitheliomatous skin lesions were HPV-DNA negative in all investigated probes. Conclusions These data in NS patients further confirm an association of EV-HPVs with non-melanoma skin cancer (NMSC) and suggest a possible carcinogenic role similar to that assumed for NMSC in transplant recipients. A complex immunological disorder facilitating EV-HPV infection, negative HPV serology and photochemotherapy may all have contributed to the unusual occurrence of multiple cancers in one of our NS patients.

Human papillomavirus (HPV) type 7 is frequently found in butchers' warts and has been demonstrated in oral and facial warts of HIV-infected patients. The reservoirs of HPV7 and the route of transmission are still unclear. Here we describe an HIV-negative, otherwise healthy patient with extensive, recurrent orofacial papillomatosis whose immune status proved to be normal and who had no history of meat handling. HPV7 L1 gene DNA that differed in 3 point mutations from the HPV7 prototype could be detected in 2 morphologically distinct, perioral lesions by different PCR protocols. In situ hybridization confirmed the presence of HPV7 DNA in the nuclei of vacuolated cells of the granular layer. Our data show that HPV7 can lead to perioral, spiky warts and brownish plaques in immunocompetent patients who had never been working as a meat or fish handler.

The 18th international papillomavirus conference took place in Barcelona, Spain in July 2000. The HPV clinical workshop was jointly organised with the annual meeting of the Spanish Association of Cervical Pathology and Colposcopy. The conference included 615 abstracts describing ongoing research in epidemiology, diagnosis/screening, treatment/prognosis, immunology/human immunodeficiency virus, vaccine development/trials, transformation/progression, replication, transcription/translation, viral protein functions, and viral and host interactions. This leader summarises the highlights presented at the conference (the full text of the abstracts and lectures can be found at www.hpv2000.com). Relevant material in Spanish can be found at www.aepcc.org.

BACKGROUND: The development of a reproducible, sensitive, and standardised human papillomavirus (HPV) polymerase chain reaction (PCR) test is required to implement HPV testing in cervical cancer screening programmes and for triaging women with mild to moderate dysplasia. AIMS: To determine the intermethod agreement between different GP5+/6+ and MY09/11 PCR based protocols for the detection and typing of high risk (HR) HPV DNA in cervical smears and to assess the intramethod reproducibility of the GP5+/6+ PCR enzyme immunoassay (EIA) for HR-HPV detection. METHODS: For the intermethod comparison, crude aliquots of 20 well characterised cervical smears comprising five HPV negative samples, and six and nine samples containing single and multiple HPV infections, respectively, were coded and sent from reference laboratory (A) to three other laboratories. One of these (laboratory B) used the GP5+/6+ PCR-EIA and was provided with standard protocols. Another laboratory (C) used GP5+/6+ PCR combined with sequence analysis and type specific PCR, whereas two laboratories (D and E) used MY09/11 PCR followed by restriction fragment length polymorphism (RFLP) analysis for the detection and typing of HR-HPV. The intramethod agreement of GP5+/6+ PCR-EIA was analysed in a subsequent study with four other laboratories (F to I) on crude aliquots of 50 well characterised cervical smears, consisting of 32 HR-HPV positive and 18 HPV negative samples. Standardised protocols, primers, and probes were also provided by the reference laboratory for HR-HPV detection. RESULTS: In the intermethod comparison, pairwise agreement of the different laboratories with reference laboratory A for the detection of HR-HPV varied between 75% and 100% (kappa values: 0.5 to 1). Typing data revealed a broader range in pairwise agreement rates between 32% and 100%. The highest agreement was found between laboratories A and B using standardised protocols and validated reagents. In the intramethod evaluation, pairwise comparison of the laboratories F to I with reference laboratory A revealed excellent agreement rates from 92% to 100% (kappa values: 0.88 to 1.0) with an overall sensitivity of 97.5% (195/200) and specificity of 99.5% (199/200). CONCLUSIONS: The detection of HR-HPV as a group is highly reproducible with GP5+/6+ PCR-EIA provided that standardised protocols and validated reagents are used.

The long control region (LCR) and the E2 protein of human papillomaviruses (HPV) are the most important viral factors regulating transcription of the viral oncogenes E6 and E7. Sequence variation within these genomic regions may have an impact on the oncogenic potential of the virus. Sequence variation in the LCR and in the E2 gene of human papillomavirus type 16 (HPV-16) isolates originating from cervical cancer patients from East Hungary was studied. In 30 samples, sequencing and/or single-strand conformation polymorphism analysis revealed variants belonging to the European variant lineage of HPV-16. These variants differed from the reference European clone only slightly in their E2 and LCR sequences. Three samples represented variants belonging to the Asian-American group. These differed from the published reference sequence at several positions in the LCR and E2 regions. Compared to the reference clone, the LCR clones of the European isolates showed very similar transcriptional activities, while that of an Asian-American isolate had approximately 1.7-fold increased activity. Most of the increased activity of the Asian-American isolate could be ascribed to nucleotide changes found at the 3' end of the LCR (nt 7660-7890). The transcriptional transactivation potentials of the HPV-16 E2 isolates differed only slightly from each other, and the differences seemed to be independent of the taxonomic position of the isolates.

Evidence of the functional significance of two naturally occurring mutations at nt 40 or 41 in the Sp1 motif in the promoter proximal segment of the upstream regulatory region (URR) of human papillomavirus (HPV) type 18 is presented. In electrophoretic mobility shift assays, Sp1 protein bound more efficiently to the Sp1 mutant motifs than to the prototype; while in both HeLa and HT3 cells, luciferase activity controlled by the mutant URRs was upregulated 2- and 3-fold, or 4- and 6-fold, in comparison with the prototype URR or HeLa cell-derived URR respectively. The HeLa URR represents a more appropriate baseline for promoter activity, containing a series of point mutations representative of most HPV-18 cancer isolates, including one in the Yin Yang 1 (YY1) site at the P105 promoter. The effect of the Sp1 mutations was found to be largely maintained in the context of the HeLa URR containing the prototype YY1 site.

Human papillomavirus (HPV) DNA, originally isolated from patients suffering from the skin disease epidermodysplasia verruciformis (EV), and a growing number of related sequences have recently been detected in a high percentage of benign and malignant skin lesions of both immunosuppressed and immunocompetent people. HPV L1 DNA fragments (374-389 bp long) from a solar keratosis and a squamous cell carcinoma (SCC) of a renal transplant recipient were amplified, cloned and sequenced. In 54 clones, six different HPV sequences were identified. One of these six corresponded to the known type HPV-8 and two (RTRX3 and RTRX7) have been described previously in cutaneous lesions of immunosuppressed patients. The remaining three sequences were different from all known HPV types: an HPV-9-related sequence (77.4% identity), an RTRX2-related sequence (82.6% identity), and an HPV-22-related sequence (83.7% identity). These three sequences, representing putatively new HPV types, were named RTRX8, RTRX9 and RTRX10, respectively. RTRX7 was found in the majority of clones from both lesions. The complete genome of RTRX7 (7731 bp) was cloned as six overlapping subgenomic fragments, generated by nested PCR with DNA extracts from the SCC. RTRX7 showed a genome organization typical of HPVs associated with EV. The L1 DNA sequence differed by 15% from the corresponding region of its closest known relative, HPV-12; thus, RTRX7 can be regarded as a new HPV type. RTRX7 DNA could not be detected by Southern blot hybridization with the homologous probe, indicating that the DNA concentration was below one copy per 10 cells in the investigated SCC.

The promoter P7535 of human papillomavirus type 8 and the promoter P7185 of bovine papillomavirus type 1 are negatively regulated by viral E2 proteins via the promoter proximal binding sites P2 and BS1, respectively. Mutations of these E2 binding sites can reduce basal promoter activity. This suggests binding of a transcription-stimulating factor and may indicate that repression by E2 is due to competitive binding of viral and cellular proteins. A computer search revealed putative binding sites for core-binding factor (CBF; also referred to as PEA2, PEBP2, or AML), overlapping with P2 and BS1. Binding of recombinant CBF proteins to these sites was confirmed by band shift analysis. Competition of CBF and E2 protein for DNA binding was shown for both human papillomavirus type 8 and bovine papillomavirus type 1. The importance of CBF-E2 competition in E2-mediated repression could be demonstrated by comparing the E2 effect on P7185 activity in two cell lines containing different amounts of endogenous CBF. In cells with large amounts of CBF, E2 repressed P7185 wild-type constructs to the basal promoter activity of a mutant (50%) that could not bind this protein any more. In contrast, in a cell line containing small amounts of CBF, the promoter activities of constructs with wild-type and mutated CBF binding sites hardly differed and specific repression by E2 was not detectable.

This study describes sequence variation in both the enhancer and promoter segments of the upstream regulatory region (URR) of 28 human papillomavirus (HPV) type 18 isolates from cervical cancers, 25 from women treated at an Australian center and three from overseas included for comparison. No large-scale changes were found in either region. Fourteen substitutions were identified in the enhancer region with the number in individual isolates ranging from one to eight. Four substitutions impacted cellular transcription factor binding sites but there were no obvious associations with clinicopathological variables. The promoter segment was found to be more highly conserved than the enhancer, but four of the five point mutations identified involved cellular transcription factor binding motifs including a substitution of C for T at nt 104 which affected 21 samples. This change was found to impact upon a previously unrecognized Yin Yang (YY1) binding site. Electrophoretic mobility shift assays (EMSA) showed that this substitution significantly reduced protein-DNA binding and evidence was sought for its possible clinical implications. Of the 24 women with less than Stage III disease and known clinical outcome, tumor recurrence was observed in all of the 6 women whose isolates had the "prototype" T at nt 104, whereas only 8 of the 18 cancers with the mutation at this YY1 site recurred. This is the first report on URR variation in HPV 18 isolates from the South Pacific region. The study also provides initial data on diversity in the promoter region and preliminary evidence suggesting that a specific point mutation in this region may be clinically significant.

The detection of human papillomavirus (HPV) types originally isolated from patients with epidermodysplasia verruciformis (EV) in skin tumors of transplant recipients may point to a role of this HPV subgroup in non-melanoma skin cancer in immunosuppressed people. We analyzed 17 formalin-fixed, paraffin-embedded biopsies of benign or malignant skin tumors of a renal transplant patient with unusually widespread cutaneous carcinomas. Using a nested polymerase chain reaction (PCR), HPV-specific DNA was demonstrated in 11 specimens (65%). Analysis of nine PCR amplification products revealed four different sequences related to EV-associated HPVs. Three sequences occurred only in one lesion. In six samples identical sequences were found that differed from all HPV sequences published to date and may therefore represent a novel EV-HPV type, preliminarily labeled RTRX7. RTRX7 was found in benign, premalignant, and malignant skin lesions. Alignments identified HPV12 as the closest relative of RTRX7, both in the DNA (81% homology) and in the amino acid sequence (84% homology).

The late gene promoter P7535 of the epidermodysplasia verruciformis-associated human papillomavirus type 8 (HPV8) is regulated by the viral E2 protein. Transfection experiments performed with the human skin keratinocyte cell line RTS3b and P7535 reporter plasmids revealed transactivation at low amounts and a repression of basal promoter activity at high amounts of E2 expression vector. This repression was promoter specific and correlated with the amount of transiently expressed E2 protein. Mutational analyses revealed that the negative regulation of P7535 activity is mediated by the low-affinity E2 binding site P2, which is separated by one nucleotide from the P7535 TATA box. Biochemical and genetic analyses suggested that repression is due to a displacement of the TATA-box binding protein by E2 and an interference of E2 with promoter-activating cellular factors that specifically recognize the P2 sequence. The high conservation of the P2 sequence among several papillomaviruses (epidermodysplasia verruciformis-associated HPVs, HPV1, cottontail rabbit papillomavirus, and bovine papillomavirus type 1) in the vicinity of the late gene promoter cap site suggests that an interplay of E2 and cellular factors at this sequence element is important for the expression of structural proteins.

Persistent infections with human papilloma viruses (HPV) can lead to a wide range of benign and malignant epithelial tumors as cutaneous warts or anogenital cancers. Methods principally available for the laboratory diagnosis of HPV infections are cytology and histology, immunocytochemistry, electron microscopy, serology and methods for the detection of HPV-specific DNA. Among the latter are polymerase chain reaction (PCR), Southern blot, dot blot, filter in situ hybridization, in situ hybridization and direct hybridization assays as Hybrid Capture. The advantages and disadvantages of the different assays are reviewed in this article. HPV DNA testing (and typing) by PCR and Hybrid Capture is clinically useful when combined with cytology in screening programs and for managing patients with equivocal cytology or cervical disease.

Human papillomavirus type 5 (HPV 5) induces cutaneous lesions and persists in skin carcinomas of patients with epidermodysplasia verruciformis (EV). We investigated the expression pattern of HPV 5 in biopsies from benign skin lesions of EV patients by cDNA analysis andin situhybridization. Nine different cDNAs could be generated from total RNA of one of these lesions by reverse transcription and PCR amplification with HPV 5-specific primers. We could identify two major splice donors: one was found in the E6-proximal part of the noncoding region (NCR), and the other just downstream of the first ATG codon of ORF E1. Each of the characterized transcripts was processed at one or the other donor site and the two corresponding leader exons were found in combination with both 3′-early and late exons. Two transcripts appear to be specific for EV-associated papillomaviruses: one species might encode an E1 up angle E2C fusion protein, and the other mRNA (NCR/E2) is probably encoding for the full-length E2 protein. According to the results of the cDNA analysis, riboprobes were designed forin situhybridization experiments to study the cell differentiation-dependent expression of the different exons. Only the E7/E1 and E4 probes led to strong signals almost throughout the epithelium. The signals generated by the 5′-E2 and E1 probe increased with cell differentiation and were mainly confined to the nucleus. The NCR, E6, E7, L2, and L1 probes yielded more or less strong signals in the terminally differentiated epidermal layers. The difference in the cell differentiation-dependent expression of the 5′-early region exon (probe E7/1) and L2/L1 exons may point to a differentiation-dependent processing of transcripts.

Importance Human papillomavirus (HPV)-induced anogenital lesions are very frequent in men who have sex with men (MSM) who are HIV-positive (HIV+). Anogenital warts (AGWs) are considered benign lesions caused by low-risk HPV-types, whereas anogenital dysplasias are potential cancer precursors associated with high-risk HPV-types. Both types of lesions can usually be distinguished clinically. Objective To describe a case series of HIV+ MSM with typical AGW that harbored different grades of dysplasia. Design, Setting, and Participants For this retrospective virological analysis, we recruited 25 HIV+ MSM with AGWs (n = 38) harboring areas of dysplasia and 22 patients who were HIV-negative (HIV−) with AGWs seen between February 2013 and March 2015 at a tertiary dermatological referral center for anal cancer screening. Dysplasia-containing AGW tissue of HIV+ MSM were compared with randomly selected AGWs of patients who were HIV−. Main Outcomes and Measures Histopathological analysis, immunohistochemical staining for p16 INK4a and Ki67, HPV-typing, and viral load determination in AGWs of HIV+ compared with patients who were HIV−. Results Overall, 25 HIV+ MSM with AGWs (mean [SD] age, 47.3 [11.1] years) harboring areas of dysplasia and 22 patients who were HIV− (5 women, 17 men; mean [SD] age, 35.5 [12.8] years) with AGWs were included in this study. The 38 dysplasia-containing AGWs of HIV+ MSM harbored low-grade dysplasia in 6 cases (16%), high-grade dysplasia in 31 cases (81%), and areas of invasive anal carcinoma in 1 (3%) case. With the exception of 1 biopsy, all low-grade lesions were p16 INK4a -negative, whereas 25 of 31 (81%) AGWs with high-grade lesions or an anal carcinoma were p16 INK4a -positive. Only low-risk HPV-types were present in 11 samples (29%; 2 low-grade lesions and 9 high-grade lesions), low-risk and high-risk types were found in 19 samples (50%; 1 low-grade lesion and 18 high-grade lesions), and only high-risk HPV-types were present in 8 samples (21%; 3 low-grade lesions, 4 high-grade lesion, and 1 cancer-containing lesion). High low-risk HPV DNA loads were found in low-grade and high-grade lesions, while high high-risk HPV DNA loads were only found in AGWs harboring high-grade lesions. The 22 AGWs of patients who were HIV− showed no signs of dysplasia, and p16 INK4a -staining was always negative. All of these samples carried low-risk HPV types, and in 2 cases high-risk HPV-types were detected additionally. Conclusions and Relevance In contrast to immunocompetent patients, AGWs of HIV+ MSM may harbor high-grade dysplasia or even invasive squamous cell carcinoma. A substantial proportion of theses lesions may only contain low-risk HPV-types. Anogenital warts in patients who are HIV+ should be evaluated histopathologically to exclude cancer precursors.

The constitutively active promoter P7535 of the epidermodysplasia verruciformis-associated human papillomavirus type 8 (HPV8) is transactivated by the viral E2 protein. The distribution of potential E2-binding sites (ACCN6GGT) in the viral transcription control region is highly conserved among epidermodysplasia verruciformis-associated human papillomaviruses and differs completely from that of other papillomaviruses. To investigate the role of E2-binding sites P0 to P4 in P7535 regulation, we analyzed their binding affinities in gel retardation experiments using a full-length HPV8 E2 protein expressed from a recombinant baculovirus. Binding site P1 within a transcriptional silencer showed the highest affinity, followed by P0 within the L1 gene and P3 downstream of P7535. P2, 33 nucleotides upstream of the mRNA cap site, and P4 were very weak binders. There is some indication that the number of A/T pairs in the nonconserved core of the recognition sequence is critical for the binding of HPV8 E2. Transient transfection experiments were carried out with an HPV8 E2 expression vector and reporter plasmids containing mutated E2-binding sites in the context of the HPV8 regulatory region. The knockout of the strongest binding site P1 sufficed to clearly diminish transactivation. P0, P3, and P4 mutations had little effect on their own, whereas double mutations P01 and P34 strongly reduced E2 inducibility. Both mutations in P2 severely affected constitutive promoter activity but had opposite effects on transactivation. They revealed an inverse correlation between E2-binding strength and the extent of E2 transactivation. This finding suggests that P2 mediates a negative control of P7535 by E2, counteracting E2 transactivation exerted via the four distal E2 target sequences.

Human papillomavirus type 16 (HPV16) induces squamous intraepithelial lesions of the cervical mucosa which may develop into invasive cancer. The expression of viral oncogenes in advanced neoplasias appears increased relative to the proliferating cell layers of low grade lesions raising questions about molecular mechanisms of deregulation of transcription. In a lymph node metastasis of a cervical cancer, we observed full-length HPV16 plasmids and molecules with a small deletion, which was mapped to the long control region (LCR). Both wild type and shortened LCR were amplified by PCR, cloned into the promoter test plasmid pBLCAT6 and sequenced to identify a 107 bp deletion from position 7794 to 7901 in the short LCR. CAT expression in cervical cancer-derived HT3, SiHa and CaSki cells appeared 5- to 6-fold increased under the control of the short LCR. This could be traced back to elevated levels of mRNA initiated at the viral oncogene promoter. A slight further increase in CAT expression was noted in the presence of the HPV16 E2 protein which is probably due to the deletion of one E2 binding site and consequent relief from E2 repression. Computer-assisted sequence analysis and band-shift experiments with purified YY1 protein and wild type or mutated oligonucleotides identified four binding sites for this cellular transcriptional repressor within the promoter-proximal segment of the HPV16 LCR, three of which were removed by the deletion. A LCR fragment comprising these YY1 binding sites was cloned in front of the heterologous thymidine kinase gene promoter and suppressed CAT expression 3- to 4-fold.(ABSTRACT TRUNCATED AT 250 WORDS)

Human papillomavirus (HPV) type 16 is strongly implicated in the development of progressive neoplasias of the uterine cervix. Its oncogenic potential is decisively determined by the activity of the early gene products E6 and E7. To look for changes in the expression of these genes during tumour progression we cloned subgenomic fragments of HPV16 into RNA expression vectors, which allowed the generation of 35S-labelled riboprobes specific for distinct mRNA classes. Four constructs were made to differentiate between transcripts starting upstream of the E6 ORF or the E1 ORF, and one probe was specific for unspliced E6/E7 region transcripts. Five other constructs were used to identify transcripts covering the E1, E2, E4, L1 and L2 regions. With the help of these constructs, we analyzed by in situ hybridization 2 low-grade intraepithelial neoplasias of the vulva, 1 high-grade neoplasia of the cervix as well as 4 vulvar and 3 cervical carcinomas. Transcripts from the E1, E2, E4, L1 and L2 region that were consistently detected in the differentiated layers of benign lesions were variably expressed in precancers and carcinomas. None of the investigated cases revealed detectable amounts of unspliced E6/E7 transcripts with a coding potential for a full-length E6 protein. In benign lesions, the E7 transcripts were confined to isolated nuclei of differentiated cells, whereas high-grade lesions and invasive cancers showed elevated levels of equally distributed E7-specific signals in the cytoplasm of all tumour cells. The most abundant transcripts observed in intraepithelial neoplasias and in invasive cancers appear to initiate within ORF E7 and therefore have no coding potential for full-length E6 and E7 proteins. Our data show that the actual level of E7-specific transcripts in cancers is lower than anticipated from earlier studies using an ORF E6/E7-specific probe that hybridizes with the 5'-ends of the abundant mRNA class.

Human papillomavirus (HPV) 8 belongs to the HPV types frequently associated with skin cancers of Epidermodysplasis verruciformis (EV)-patients. There are 33 nucleotides (M33 motif) in the 5′-part of the non-coding regulatory region of HPV8, which appear highly conserved among EV-specific HPVs and are consistently followed by an AP1 binding site. These sequences were shown to constitute an essential activator of transcription driven by the HPV8 late promoter P 7535 . The M33/AP1 element displayed properties of a constitutive enhancer, being also able to stimulate the activity of the heterologous thymidine kinase promoter in a position-independent manner. No protein binding could be detected within the 5′-part of the M33/AP1 region, which contributed significantly to the overall activity of the HPV8 enhancer. As shown by DNasel-footprinting, the central and the 3′-part of the enhancer region were involved in interactions with nuclear proteins. Three specific complexes could be observed in gel retardation tests with nuclear extracts from epithelial cells. One of these interactions involved the AP1 protein. Analysis of deletion and point mutations revealed binding of the AP1 protein to be essential for transcriptional activation, but DNA-protein interactions within M33 were important for maximal stimulation. The response to the phorbol ester TPA also required a cooperation of M33 and AP1.

Background Dysregulation of microRNA (miRNA) expression is regularly found in various types of cancer and contributes to tumorigenic processes. However, little is known about miRNA expression in non-melanoma skin cancer in which a pathogenic role of beta human papillomaviruses (HPV) is discussed. A carcinogenic potential of beta HPV8 could be demonstrated in a transgenic mouse model, expressing all early genes of HPV8 (HPV8-CER). A single UVA/B-dose induced oncogene expression and led to papilloma growth within three weeks. Objective Expression of miRNAs and their targets during HPV8-mediated tumor formation in mice. Methods Skin of untreated or UV-irradiated wild-type and HPV8-CER mice was analyzed for miRNA expression and localization by qPCR and in situ hybridization. MiRNA target protein expression was analyzed by immunohistochemical staining. Results Early steps in skin tumor formation in HPV8-CER mice were associated with an upregulation of the oncogenic miRNA-17-5p, -21 and -106a and a downregulation of the tumor-suppressive miRNA-155 and -206, which could be demonstrated by qPCR and in situ hybridization. The respective targets of miRNA-21 and -106a, the tumor suppressors PTEN, PDCD4 and Rb with their pivotal role in cell cycle regulation, apoptosis and proliferation were found to be downregulated. Conclusion This is the first report demonstrating that a cutaneous HPV type deregulates the expression of miRNAs. These deregulations are closely related to the UV-induced upregulation of HPV8 oncogene levels, which suggest a direct or indirect HPV8-specific effect on miRNA expression. These data presume that HPV8 interferes with the miRNA mediated gene regulation to induce tumorigenesis.

Human papillomavirus type 13 (H PV-13) is associated with oral focal epithelial hyperplasia (FEH) in humans. A recent epidemic of a FEH-like disease in a pygmy chimpanzee (Pan paniscus) colony allowed us to clone a novel papillomavirus genome. To assess the homology between HPV-13 and the pygmy chimpanzee papillomavirus type 1 (PCPV-1), the complete nucleotide sequences of both FEH-related viruses were determined. In both viruses, all eight major open reading frames were located on one strand and the genomic organization was similar to that of other mucosal papillomaviruses. The genomes of PCPV-1 and HPV-13 showed extensive overall sequence homology (85%). They could be classified, using phylogenetic analysis, together with HPV types 6, 11, 43, and 44 in a group associated with benign orogenital lesions. These data indicate that two phylogenetically related papillomavinuses can elicit similar pathology in different primate host species, reflecting viral genomic similarities.

The E6 and E7 genes of human genital papillomaviruses (HPVs) appear to transform cells by different mechanisms. They seem to act synergistically but are not equally important when tested under diverse experimental conditions. We were therefore tempted to investigate the E6- and E7-specific transcription pattern in HPV6-infected condylomas separately, by in situ hybridization. Recent studies have identified three promoters within the E6-E7 region of HPV6 and HPV11 by applying S1, exonuclease VII, and cDNA analyses. On the basis of these data, we cloned subgenomic fragments of HPV6 into plasmid pBS to obtain riboprobes that differentiated between transcripts starting upstream of the E6 and E7 open reading frames, respectively. These different species of mRNAs were analyzed in serial thin sections of eight HPV6-positive anogenital condylomas. The E6 probe (nucleotides 7862 to 241) led to weak signals within the basal layer. In three cases, rather strong signals were confined to a few basal cells. The E7 probe (nucleotides 242 to 534) gave rise to a more pronounced labeling of all cells within the two to three lowest epidermal layers. In situ hybridization with a riboprobe for human c-fos revealed an expression pattern similar to that observed with the E7 probe. In contrast to the preferential expression of the transforming E6 and E7 genes in the lower epithelium, the major transcriptional activity of the virus was detected in the middle and upper third by probes colinear with the 3' moiety of the early region.

Human papillomavirus type 8 (HPV8) belongs to the HPV types associated with skin carcinomas of patients with epidermodysplasia verruciformis (EV). Its noncoding regulatory sequences (NCR) were shown to drive the expression of the reporter gene chloramphenicol acetyltransferase (cat) in transient assays with human epithelial cells (HT3 cells). This constitutive activity could be enhanced by coexpression of the HPV8 transactivator protein E2. The analysis of 5' deletions of the NCR showed that the EV-specific sequence motif M33 and the neighboring AP1 site are essential for the promoter activity, whereas 44 nucleotides located immediately upstream of M33 are strongly inhibitory. The same effects were observed in simian virus 40-immortalized fetal keratinocytes (SV61 cells) and spontaneously immortalized skin keratinocytes (HaCaT cells). By using primer extension and RNase protection analyses two promoters could be identified within the HPV8 NCR. A nested set of weak signals, corresponding to start sites between positions 175 to 179, represented the previously described E6 promoter. The vast majority of transcripts was initiated at position 7535 and shown to undergo processing at an NCR-internal splice donor (positions 1 to 8). The promoter P7535 is similar to late promoters of other skin-associated papillomaviruses as far as localization, transcript structure, and sequence characteristics are concerned. To confirm that P7535-initiated transcripts proceed indeed to the L1 gene for the major capsid protein, viral mRNAs from an HPV8-induced lesion of a patient with EV were characterized by RNase protection and sequence analysis of polymerase chain reaction-amplified cDNAs. The NCR leader (positions 7535 to 4) appeared in two messages with three exons each. The third exon started with the second ATG codon of L1 in both cases; the short central exons from the 3' part of the early coding region were defined by a common splice acceptor site (position 3303) and different splice donor sites (positions 3443 and 3704).

DNA tumorviruses like adenoviruses (AdV) or human papillomaviruses (HPV) have adopted various strategies to interfere with antiproliferative transforming growth factor-beta (TGF-beta) signalling. Here we report that the AdV E1A oncoprotein is sufficient to induce Smad7 expression, an inhibitor of TGF-beta signalling. E1A but not HPV oncoproteins activated the Smad7 promoter. A promoter proximal E-box was crucial for E1A-mediated transcriptional activity. E1A but not HPV oncoproteins induced specific binding activity at this E-box, which was identified as upstream stimulatory factor. In conclusion, these results unravel a novel mechanism of how the AdV E1A oncoprotein induces a cellular inhibitor of TGF-beta signalling.

The non-coding region (NCR) of the epidermodysplasia verruciformis (EV)-associated human papillomavirus 8 (HPV-8) has been investigated for sequence-specific DNA-protein interactions with nuclear proteins from epithelial HeLa cells. Using DNase I protection analysis 18 footprints could be found within the HPV-8 NCR, covering altogether over 60% of both DNA strands. Several footprints coincided with the known binding sites of transcription factors (NF1, AP1, octamer and PEA3 consensus sequences); the other displayed no obvious similarities in this regard. The overall distribution of sequences involved in DNA-protein interactions differed clearly from the binding patterns reported for other HPVs. Parts of the two binding sites for the viral trans-activator protein E2 were shown to interact with non-E2 factors. The EV-specific NCR motifs M33, M29 and A/T box were all involved in protein binding. By comparing the footprints within the respective motifs of the closely related types HPV-8 and -19, quantitative and qualitative differences were detected for M33 and the A/T box.

Background Epidermodysplasia verruciformis (EV) is a rare genodermatosis associated with infections with specific human papillomaviruses (HPVs) belonging to the β genus of HPV. Patients with EV usually have a selective defect in cell-mediated immunity. Although skin cancer frequently develops in the sun-exposed cutaneous lesions of patients with EV, the anogenital area is usually not affected by squamous cell carcinomas related to mucosal HPV types. Observations We report the case of a patient with clinical similarities to EV who also presented with primary lymphedema, anogenital dysplasias, and depressed cell-mediated immunity. Swab samples and biopsy specimens from various body sites collected over a 28-month period were screened by different protocols for DNA of the HPV groups alpha, beta, and mu/nu. Seventeen α-HPV types could be demonstrated. Interestingly, β-HPVs (HPV-22 and HPV-23) were detectable only in plucked eyebrows and in 1 skin swab sample. None of the specimens from lesional biopsies carried β-HPV. Consistently found α-HPV types included HPV types 6, 51, 52, 61, and 84 in the genitoanal region and HPV-57 in skin lesions. Histological and cytological evaluation revealed multifocal anogenital dysplasia and benign genital and cutaneous warts. Conclusions To our knowledge, only 1 other similar case of an EV-like syndrome with impaired, cell-mediated immunity and primary lymphedema has been described in the literature. Based on the characteristic clinical and virological findings in the present case and the previously published case, we speculate that both patients could have a previously unknown syndrome that has clinical similarities to EV but notably differs in the associated HPV types. We suggest the acronym WILD (warts, immunodeficiency, lymphedema, dysplasia) to characterize this syndrome.