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1.  RISK FOR CERVICAL INTRAEPITHELIAL NEOPLASIA GRADE 3 OR WORSE IN RELATION TO SMOKING AMONG WOMEN WITH PERSISTENT HUMAN PAPILLOMAVIRUS INFECTION 
Background
Smoking has been associated with cervical cancer. We examined whether smoking increases the risk for high-grade cervical lesions in women with high-risk human papillomavirus (HPV) infection.
Methods
In a population-based cohort study, 8,656 women underwent a structured interview, and subsequently cervical cells were obtained for HPV DNA testing. Women with high-risk HPV infection and no prevalent cervical disease at baseline (n=1,353) were followed through the Pathology Data Bank for cervical lesions for up to 13 years. Separate analyses of women with persistent high-risk HPV infection were also conducted. Hazard ratios (HRs) for a diagnosis of cervical intraepithelial neoplasia grade 3 or worse/high-grade squamous intraepithelial lesions or worse (CIN3+) and the corresponding 95% confidence intervals (CIs) were calculated in the 2 groups.
Results
Among high-risk HPV positive women an increased risk for CIN3+ was associated with long-term smoking (≥10 years) and heavy smoking (≥20 cigarettes/day). In the subgroup of women with persistent HPV infection heavy smoking was also associated with a statistically significantly higher risk for CIN3+ than never smoking (HR, 1.85; 95% CI, 1.05–3.22, adjusted for length of schooling, parity and HPV type at baseline). The average number of cervical cytology screening tests per year during follow-up did not explain the differences in risk in relation to smoking (p=0.4).
Conclusions
Smoking is associated with an increased risk for subsequent high-grade cervical lesions in women with persistent high-risk HPV infection.
Impact
Our study adds to the understanding of the role of smoking in the natural history of HPV and cervical carcinogenesis.
doi:10.1158/1055-9965.EPI-12-0663
PMCID: PMC3970163  PMID: 23019238
cervical intraepithelial lesions; smoking; human papillomavirus
2.  Cutaneous Human Papillomaviruses Found in Sun-Exposed Skin: Beta-papillomavirus Species 2 Predominates in Squamous Cell Carcinoma 
The Journal of infectious diseases  2007;196(6):876-883.
Background
A spectrum of cutaneous human papillomaviruses (HPVs) is detectable in nonmelanoma skin cancers, as well as in healthy skin, but the significance that the presence of these types of HPV DNA has for the pathogenesis of skin cancer remains unclear.
Methods
We studied 349 nonimmunosuppressed patients with skin lesions (82 with squamous cell carcinomas, 126 with basal cell carcinomas, 49 with actinic keratoses, and 92 with benign lesions). After superficial skin had been removed by tape, paired biopsy samples—from the lesion and from healthy skin from the same patient—were tested for HPV DNA. Risk factors for HPV DNA were analyzed in multivariate models.
Results
Overall, 12% of healthy skin samples were positive for HPV DNA, compared with 26% of benign lesions, 22% of actinic keratoses, 18% of basal cell carcinomas, and 26% of squamous cell carcinomas. HPV DNA was associated with sites extensively exposed to the sun, both for the lesions (odds ratio [OR], 4.45 [95% confidence interval {CI}, 2.44–8.11]) and for the healthy skin samples (OR, 3.65 [95% CI 1.79–7.44]). HPV types of Beta-papillomavirus species 2 predominate in squamous cell carcinomas (OR, 4.40 [95% CI, 1.92–10.06]), whereas HPV types of Beta-papillomavirus species 1 are primarily found in benign lesions (OR, 3.47 [95% CI, 1.72–6.99]).
Conclusions
Cutaneous HPV types are primarily detected at sites extensively exposed to the sun. HPV types of Beta-papillomavirus species 2, but not of species 1, are associated with squamous cell carcinoma.
doi:10.1086/521031
PMCID: PMC3795387  PMID: 17703418
3.  Annual Papanicolaou screening for 5 years among human papillomavirus-negative women 
BMC Cancer  2013;13:379.
Background
Primary human papilloma virus (HPV) screening is more effective than cytology in reducing the risk of cervical cancer, but screening intervals should be extended in HPV-negative women. However, some Markov models predicted that long intervals are associated with an excess risk of cervical cancer. The aim of this analysis was to estimate the real-life risks and benefits of annual Papanicolaou (Pap) screening in HPV-negative women with normal cytology.
Methods
Women with negative Hybrid Capture 2 (HC2) results and normal cytology at the time of inclusion in the Hannover HPV screening trial underwent annual Pap smears for 5 years. A subgroup was randomly selected for retesting with cytology, HC2, and colposcopy 60–68 months after recruitment.
Results
Of 4236 women included, 3406 had at least one Pap smear, but only 1185 attended all five annual screening visits. The proportion of women with at least one abnormal smear was 14.4% in 60 months. The probability of abnormal smears increased continuously over time. No case of ≥ CIN2+ was observed during 5 years. Of 605 women selected for subgroup analysis, 292 agreed to be retested (48.3%). The rate of high-risk HPV at 60–68 months was 3.0% (9/296).
Conclusions
The long-term risk of high-grade neoplasia after an initial negative HC2 test and normal cytology result was low, while the rate of false-positive abnormal Pap smears was significant and increased constantly over time. Pap smear screening of HPV-negative women more frequently than every 5 years could be potentially harmful and seems to be of little clinical value.
doi:10.1186/1471-2407-13-379
PMCID: PMC3751119  PMID: 23937771
Annual papanicolaou smear; Cervical cancer screening; Human papillomavirus (HPV); HR-HPV DNA test; Screening intervals
4.  Prevalence of high-risk HPV types and associated genital diseases in women born in 1988/89 or 1983/84 – results of WOLVES, a population-based epidemiological study in Wolfsburg, Germany 
BMC Infectious Diseases  2013;13:135.
Background
High-risk human papilloma virus (HR-HPV) infection is associated with the development of cervical cancer. HPV vaccination reduces the risk of developing malignant lesions and is expected to change the dynamics of HPV transmission. Data from non-vaccinated women may provide an important benchmark to allow the impact of HPV vaccination programs to be assessed.
This study was designed to prospectively determine the changing dynamics of HR-HPV infection and associated genital diseases in young women, most of whom were non-vaccinated.
Methods
Data from a population-based cohort study, comprising women of two predefined birth cohorts (women born in 1983/84 or 1988/89), were analyzed between 19 October 2009 and 31 December 2010 to determine risk factors for high-risk HPV infection and the association between specific HR-HPV types and atypical Pap smear test results. HPV status was determined by Hybrid Capture 2 (HC2) assay and genotyping.
Results
The prevalence of HR-HPV was 22.8% in the 1983/84 cohort (150/659) and 23.7% in the 1988/99 cohort (142/599). Only the number of sexual partners was a significant risk factor for HPV infection (odds ratios 22.687 and 6.124 for more than five versus one partner 84 cohort,/84 and 1988/89 cohorts, respectively) in multivariate analysis. HPV16 positive-women were significantly more likely to have abnormal Pap smears of any degree than HPV16-negative women (22.0% versus 3.61%, p < 0.0001 for the 1983/84 cohort and 9.09% versus 2.52%, p = 0.0482 for the 1988/89 cohort). CIN3 was diagnosed in six women 84 cohort,/84 cohort and two in the 1988/89 cohort. All women with CIN3 tested positive for HC2-HR and all six CIN3 cases 84 cohort,/84 cohort tested positive for HPV16. In the 1988/89 cohort, the rate of HPV16 infection was significantly lower in vaccinated than non-vaccinated women (1.59% versus 8.88%; p = 0.003).
Conclusions
HR-HPV infection was highly prevalent in both cohorts and associated with an increased risk of abnormal Pap smears and biopsy proven CIN2+. HPV16 infection was associated with a high risk of clinically relevant lesions. HPV vaccination significantly decreased the risk of HPV16 infection.
doi:10.1186/1471-2334-13-135
PMCID: PMC3623770  PMID: 23497108
High-risk HPV; HPV16; CIN; HPV vaccination
5.  Prevalence of low-risk HPV types and genital warts in women born 1988/89 or 1983/84 -results of WOLVES, a population-based epidemiological study in Wolfsburg, Germany 
BMC Infectious Diseases  2012;12:367.
Background
Wolfsburg HPV Epidemiological Study (WOLVES) is a population-based cohort study on HPV infections and associated diseases in the pre-vaccination era in young women in Wolfsburg, Germany.
Methods
Women born 1983/84 or 1988/89 were invited to participate. Participants were recruited in gynecology practices, and completed a questionnaire with socioeconomic, sexual and medical data including vaccination status. Pelvic examination with Pap smear and HPV testing (HC2 = Hybrid Capture 2) was obligatory. HC2-positive and 10% of HC2-negative samples were tested for specific HPV types with SPF-10-PCR, and in inconclusive cases with DNA sequencing. Women with genital warts (GW) and those with atypical Pap smears were transferred for colposcopy. GWs were classified as typical condylomata acuminata (TCA), flat condyloma (FC) and seborrheic wart-like (SWL).
Results
In total, 1258 subjects were recruited from the target population of 2850 (44.1%). Overall the prevalence of HC2 low-risk (LR) types was 8.5%. HPV6 was the most frequent LR type (2.1%), followed by HPV42 (1.1%), HPV11 and HPV44 (each 0.4%). LiPA showed a low sensitivity for HPV types 42, 90 and 91, which were detected only by HC2 and HPV sequencing. Nine women (0.7%) were transferred with incident GW: five TCA, two FC and two SWL. All TCA were associated with HPV6 in corresponding cervical swabs and warts. Tissues of SWL contained HPV6 (n = 1) and HPV16 (n = 1). The cumulative life-risk for GW was 1.4% in the 1988/89 and 4.8% in the 1983/84 cohort. Eight of 107 HC2-LR + and five of nine cases of GW had concomitant abnormal Pap smears. All CIN lesions could be linked to high-risk HPV types but borderline and low-grade abnormal smears were explained by vaginal and cervical TCA in four cases.
Conclusions
HC2 was a specific test for the detection of established and potential LR types. In this first WOLVES analysis, HPV6 was the most frequent HPV type and the single LR type linked to disease. The observed GW incidence of 715 per 100,000 fits well with estimates of healthcare providers. Although life risks for GW were lower than in Scandinavian analyses, the societal burden within the WOLVES populations was considerable.
doi:10.1186/1471-2334-12-367
PMCID: PMC3536688  PMID: 23259726
6.  Risk factors for VIA positivity and determinants of screening attendances in Dar es Salaam, Tanzania 
BMC Public Health  2012;12:1055.
Background
Tanzania is among the countries in the world where the cervical cancer incidence is estimated to be highest. Acknowledging an increase in the burden of cervical cancer, VIA was implemented as a regional cervical cancer screening strategy in Tanzania in 2002. With the aim of describing risk factors for VIA positivity and determinants of screening attendances in Tanzania, this paper present the results from a comparative analysis performed among women who are reached and not reached by the screening program”.
Methods
14 107 women aged 25–59 enrolled in a cervical cancer screening program in Dar es Salaam in the period 2002 – 2008. The women underwent VIA examination and took part in a structured questionnaire interview. Socioeconomic characteristics, sexual behavior, HIV status and high-risk (HR) HPV infection were determined in a subpopulation of 890 who participated and 845 who did not participate in the screening.
Results
Being widowed/separated OR=1.41 (95% CI: 1.17-1.66), of high parity OR=3.19 (95% CI: 1.84-5.48) of low education OR= 4.30 (95% CI: 3.50-5.31) and married at a young age OR=2.17 (95% CI: 1.37-3.07) were associated with being VIA positive. Women who participated in the screening were more likely to be HIV positive OR= 1.59 (95% CI. 1.14-2.25) in comparison with women who had never attended screening, while no difference was found in the prevalence of HR-HPV infection among women who had attended screening and women who had not attended screening.
Conclusion
Women who are widowed/separated, of high parity, of low education and married at a young age are more likely to be VIA positive and thus at risk of developing cervical cancer. The study further documents that a referral linkage between the HIV care and treatment program and the cervical cancer screening program is in place in the setting studied, where HIV positive were more likely to participate in the cervical cancer screening program than HIV negative women.
doi:10.1186/1471-2458-12-1055
PMCID: PMC3552680  PMID: 23216752
Cervical cancer; Screening; VIA; HPV; HIV; Tanzania
7.  Predictors of human papillomavirus infection in women undergoing routine cervical cancer screening in Spain: the CLEOPATRE study 
BMC Infectious Diseases  2012;12:145.
Background
Human papillomavirus (HPV) is a sexually transmitted infection that may lead to development of precancerous and cancerous lesions of the cervix. The aim of the current study was to investigate socio-demographic, lifestyle, and medical factors for potential associations with cervical HPV infection in women undergoing cervical cancer screening in Spain.
Methods
The CLEOPATRE Spain study enrolled 3 261 women aged 18–65 years attending cervical cancer screening across the 17 Autonomous Communities. Liquid-based cervical samples underwent cytological examination and HPV testing. HPV positivity was determined using the Hybrid Capture II assay, and HPV genotyping was conducted using the INNO-LiPA HPV Genotyping Extra assay. Multivariate logistic regression was used to identify putative risk factors for HPV infection.
Results
A lifetime number of two or more sexual partners, young age (18–25 years), a history of genital warts, and unmarried status were the strongest independent risk factors for HPV infection of any type. Living in an urban community, country of birth other than Spain, low level of education, and current smoking status were also independent risk factors for HPV infection. A weak inverse association between condom use and HPV infection was observed. Unlike monogamous women, women with two or more lifetime sexual partners showed a lower risk of infection if their current partner was circumcised (P for interaction, 0.005) and a higher risk of infection if they were current smokers (P for interaction, 0.01).
Conclusion
This is the first large-scale, country-wide study exploring risk factors for cervical HPV infection in Spain. The data strongly indicate that variables related to sexual behavior are the main risk factors for HPV infection. In addition, in non-monogamous women, circumcision of the partner is associated with a reduced risk and smoking with an increased risk of HPV infection.
doi:10.1186/1471-2334-12-145
PMCID: PMC3447664  PMID: 22734435
HPV infection; Prevalence; Risk factors; Sexual behavior; Questionnaire; Spain
8.  High-Risk Human Papillomaviruses Repress Constitutive Kappa Interferon Transcription via E6 To Prevent Pathogen Recognition Receptor and Antiviral-Gene Expression ▿ †  
Journal of Virology  2011;85(21):11372-11380.
Persistent infections with human papillomavirus type 16 (HPV16), HPV18, or HPV31 are necessary for the development of cervical cancer, implying that HPVs have evolved immunoevasive mechanisms. Recent global transcriptome analyses indicated that these HPV types downregulate the constitutive expression of interferon (IFN)-stimulated genes (ISGs), but the underlying mechanism is not well understood. Comparative analyses of ISG transcription in keratinocytes with complete HPV16, -18, and -31 genomes revealed that antiviral genes (IFIT1 and MX1), genes involved in IFN signaling (STAT1), proapoptotic genes (TRAIL and XAF1), and pathogen recognition receptors (TLR3, RIG-I, and MDA5) are inhibited to similar extents by HPV16, -18, and -31. The lower expression of pathogen receptors in HPV-positive cells correlated with a greatly impaired induction of IFN-β and also of IFN-λ1, -2, and -3 upon receptor stimulation. IFN-κ is constitutively expressed in normal keratinocytes and is strongly repressed by HPV16, -18, and -31. ISGs downregulated in HPV-positive cells can be reactivated by IFN-κ expression. The viral E6 and E7 oncogenes are sufficient for IFN-κ repression, with E6 being mainly responsible. E6 inhibits IFN-κ transcription independently from binding to PDZ proteins. IFN-κ expression can be activated in only one cell line by E6AP knockdown but can be activated in all tested HPV-positive cells by addition of a DNA methyltransferase inhibitor, suggesting that HPVs modulate DNA methylation. Taken together, these results suggest that carcinogenic HPVs target IFN-κ by different pathways in keratinocytes to inhibit both antiviral ISGs and pathogen recognition receptors, which in turn reduces the expression of inducible IFNs.
doi:10.1128/JVI.05279-11
PMCID: PMC3194958  PMID: 21849431
9.  Knock down of p53 or its ubiquitin ligase E6AP does not affect the sensitivity of human papillomavirus-positive cervical cancer cells to cisplatin 
The persistent infection with high risk human papillomaviruses (hrHPV) is a necessary risk factor for the development of cervical cancer, which is the second most frequent cancer in women worldwide. Cisplatin-based radiotherapy represents the current treatment regimen. However, the results for advanced and recurrent disease are far from optimal. Since almost all cervical cancers contain wild type (wt) p53, which is degraded by the complex of hrHPV E6 and the ubiquitin ligase E6AP, we addressed if the reconstitution of p53 via silencing of E6AP sensitizes cervical cancer cells towards cisplatin treatment. For this we established and characterized two novel cervical cancer cell lines that contain integrated HPV16 genomes. Long-term established HeLa and SiHa cells and the novel cervical cancer cell lines at low passage numbers were treated with different concentrations of cisplatin. Cell viability was measured by the WST-1 assay. In addition, single cisplatin treatment was combined with the silencing of E6AP or p53. The comparison to HeLa and SiHa cells revealed a higher sensitivity of the novel cell lines to cisplatin treatment, which caused p53 accumulation and transcriptional induction of p21. Silencing of E6AP further increased p53 protein levels, but had no effect on cell viability when combined with cisplatin treatment. Interestingly, silencing of p53 had also no effect. We therefore conclude that reactivation of p53 via silencing of E6AP does not increase the sensitivity of cervical cancer cells towards cisplatin treatment.
PMCID: PMC3365809  PMID: 22679561
Cervical cancer; HPV; cisplatin; p53; E6AP; chemoresistance
10.  Physical state and viral load as predictive biomarkers for persistence and progression of HPV16-positive cervical lesions: results from a population based long-term prospective cohort study 
Persistent infection with a high risk (hr) human papillomavirus (HPV) has been established as the main cause of cervical cancer and high-grade cervical intraepithelial neoplasia (CIN3). Because most infections are transient, testing for hrHPV lacks specificity and has a low positive predictive value. It has been suggested that additional parameters like viral load and physical status of the viral genome could improve the effectiveness of HPV-based screening. We investigated the association between HPV16 viral load and physical state with viral persistence or risk of incident CIN3 or worse in a population-based prospective cohort study comprising 8656 women (20-29 years). All participants had two gynecological examinations two years apart and were followed through the nationwide Danish Pathology Data Bank (median follow-up: 12.9 yrs). Seventynine cervical swabs from women with a persistent HPV16 infection were available for analysis. For comparison we selected a random age-matched sample of transiently HPV16 infected women (N=91). Persistently infected women with incident CIN3 or cancer (CIN3+; N=31) were compared to women with normal cytology during follow up (non-progressors; N=39). Quantitative real-time PCR for HPV16E6, E2 and IFNb1 was done to determine the HPV16 viral load and the E2/E6 ratio was used as a surrogate marker for integration. Women with normal cytology who became persistently HPV16 infected had a significantly lower HPV16 load at baseline than women who cleared the infection (median 4.72 copies/cell versus median 20.0 copies/cell, respectively; p=0.0003). There was no difference in viral load at enrollment between women who progressed to CIN3+ and women who stayed cytologically normal (p=0.85). At the second examination viral load tended to be higher in women who progressed, but the difference was not statistically significant (p=0.39). The E2/E6 ratio was shown to be lower in the persistently infected group (p<0.0001) already at the first examination, but no difference between non-progressors and CIN3+ cases was observed at any of the two examinations (p=0.61 and 0.86). Lower viral load and integration of the viral genome are predictive for the persistence of HPV16 DNA, but not for the progression of a persistent HPV16 infection to CIN3+ in women with normal cytology.
PMCID: PMC3304573  PMID: 22432058
Cervical cancer; HPV; viral load; viral integration
11.  Long-term Absolute Risk of Cervical Intraepithelial Neoplasia Grade 3 or Worse Following Human Papillomavirus Infection: Role of Persistence 
Background
Infection with high-risk human papillomavirus (HPV) is the main cause of high-grade cervical intraepithelial neoplasia (CIN) and cancer. It has been suggested that information about high-risk HPV type–specific infection might make cervical cancer screening more effective. Persistent HPV infection could also be a useful screening marker. We estimated the long-term risk of high-grade CIN after one-time detection of high-risk HPV DNA and after persistent infection with individual high-risk HPV types.
Methods
A cohort of 8656 women from the general population of Denmark was examined twice, 2 years apart (first study examination: May 15, 1991, to January 31, 1993; second study examination: October 1, 1993, to January 31, 1995). The women underwent a gynecological examination and cervical cytology and had swabs taken for HPV DNA analysis by the Hybrid Capture 2 and line probe assays. The women were followed up through the nationwide Danish Pathology Data Bank for cervical neoplasia for up to 13.4 years. The absolute risk of developing cervical lesions before a given time was estimated as a function of time.
Results
For women with normal cytological findings who were concurrently HPV16 DNA positive at the second examination, the estimated probability of developing CIN grade 3 (CIN3) or worse within 12 years of follow-up was 26.7% (95% confidence interval [CI] = 21.1% to 31.8%). The corresponding risks among those infected with HPV18 was 19.1% (95% CI = 10.4% to 27.3%), with HPV31 was 14.3% (95% CI = 9.1% to 19.4%), and with HPV33 was 14.9% (95% CI = 7.9% to 21.1%). The absolute risk of CIN3 or worse after infection with high-risk HPV types other than HPV16, HPV18, HPV31, or HPV33 was 6.0% (95% CI = 3.8% to 8.3%). The estimated absolute risk for CIN3 or cancer within 12 years of the second examination among women who were HPV16 DNA positive at both examinations was 47.4% (95% CI = 34.9% to 57.5%); by contrast, the risk of CIN3 or worse following a negative Hybrid Capture 2 test was 3.0% (95% CI = 2.5% to 3.5%).
Conclusion
HPV16, HPV18, HPV31, and HPV33 infection and especially HPV16 persistence were associated with high absolute risks for progression to high-grade cervical lesions. The results indicate the potential value of genotyping in cervical cancer screening. Given that HPV DNA–negative women retained their low risk of CIN3 or worse for many years, frequent screening of these women may be unnecessary.
doi:10.1093/jnci/djq356
PMCID: PMC2950170  PMID: 20841605
12.  Growth Inhibition of HeLa Cells Is a Conserved Feature of High-Risk Human Papillomavirus E8^E2C Proteins and Can Also Be Achieved by an Artificial Repressor Protein▿ †  
Journal of Virology  2010;85(6):2918-2926.
Infections with certain human papillomaviruses (HPV), such as type 16 (HPV16), 18, or 31, are a necessary risk factor for the development of cervical cancer. Transcript analyses of several HPV revealed that the viral E2 gene encodes both the E2 regulator protein and the E8∧E2C protein, which differ in their amino termini. Up to now, functional studies have focused on HPV31 E8∧E2C and demonstrated that it is a potent repressor of viral transcription and replication. However, recent analyses of HPV16 genomes have suggested that E8∧E2C proteins may differ in their activities. Therefore, we performed a comparative analysis of E8∧E2C proteins of HPV16, -18, and -31. All E8∧E2C proteins potently inhibited HPV E6/E7 oncogene promoters, and also displayed long-distance transcriptional-repression activities. Furthermore, the expression of all E8∧E2C proteins inhibited the growth of HeLa cells. Expression of E8∧E2C proteins rapidly increased the protein levels of the E6 and E7 targets p53 and p21, consistent with the repression of the endogenous HPV18 E6/E7 promoter. All E8∧E2C proteins induced G1 arrest more efficiently than E2 proteins and activated senescence markers. Furthermore, we demonstrate that the 31E8 domain can be functionally replaced by the KRAB repression domain derived from KOX1. The KRAB-E2C fusion protein possesses long-distance transcriptional-repression activity and inhibits the growth of HeLa cells comparably to E8∧E2C. Taken together, our results suggest that the E8∧E2C proteins of HPV16, -18, and -31 are highly conserved transcriptional repressors that inhibit the growth of HeLa cells by repression of E6/E7 transcription but do not have proapoptotic activities.
doi:10.1128/JVI.01647-10
PMCID: PMC3067960  PMID: 21191025
13.  Interaction of the Papillomavirus E8∧E2C Protein with the Cellular CHD6 Protein Contributes to Transcriptional Repression▿ †  
Journal of Virology  2010;84(18):9505-9515.
Expression of the E6 and E7 oncogenes of high-risk human papillomaviruses (HPV) is controlled by cellular transcription factors and by viral E2 and E8∧E2C proteins, which are both derived from the HPV E2 gene. Both proteins bind to and repress the HPV E6/E7 promoter. Promoter inhibition has been suggested to be due to binding site competition with cellular transcription factors and to interactions of different cellular transcription modulators with the different amino termini of E2 and E8∧E2C. We have now identified the cellular chromodomain helicase DNA binding domain 6 protein (CHD6) as a novel interactor with HPV31 E8∧E2C by using yeast two-hybrid screening. Pull-down and coimmunoprecipitation assays indicate that CHD6 interacts with the HPV31 E8∧E2C protein via the E2C domain. This interaction is conserved, as it occurs also with the E8∧E2C proteins expressed by HPV16 and -18 and with the HPV31 E2 protein. Both RNA knockdown experiments and mutational analyses of the E2C domain suggest that binding of CHD6 to E8∧E2C contributes to the transcriptional repression of the HPV E6/E7 oncogene promoter. We provide evidence that CHD6 is also involved in transcriptional repression but not activation by E2. Taken together our results indicate that the E2C domain not only mediates specific DNA binding but also has an additional role in transcriptional repression by recruitment of the CHD6 protein. This suggests that repression of the E6/E7 promoter by E2 and E8∧E2C involves multiple interactions with host cell proteins through different protein domains.
doi:10.1128/JVI.00678-10
PMCID: PMC2937640  PMID: 20631145
14.  NCoR1 Mediates Papillomavirus E8^E2C Transcriptional Repression▿ †  
Journal of Virology  2010;84(9):4451-4460.
The papillomavirus E2 open reading frame encodes the full-length E2 protein as well as an alternatively spliced product called E8^E2C. E8^E2C has been best studied for the high-risk human papillomaviruses, where it has been shown to regulate viral genome levels and, like the full-length E2 protein, to repress transcription from the viral promoter that directs the expression of the viral E6 and E7 oncogenes. The repression function of E8^E2C is dependent on the 12-amino-acid N-terminal sequence from the E8 open reading frame (ORF). In order to understand the mechanism by which E8^E2C mediates transcriptional repression, we performed an unbiased proteomic analysis from which we identified six high-confidence candidate interacting proteins (HCIPs) for E8^E2C; the top two are NCoR1 and TBLR1. We established an interaction of E8^E2C with an NCoR1/HDAC3 complex and demonstrated that this interaction requires the wild-type E8 open reading frame. Small interfering RNA (siRNA) knockdown studies demonstrated the involvement of NCoR1/HDAC3 in the E8^E2C-dependent repression of the viral long control region (LCR) promoter. Additional genetic work confirmed that the papillomavirus E2 and E8^E2C proteins repress transcription through distinct mechanisms.
doi:10.1128/JVI.02390-09
PMCID: PMC2863743  PMID: 20181716
15.  Acquisition of high-risk human papillomavirus infection in a population-based cohort of Danish women 
Sexually transmitted diseases  2009;36(10):609-615.
Background
Human papillomavirus (HPV) is the cause of cervical cancer. To better understand the natural history of HPV, we assessed the incidence of type-specific HPV infection and examined risk factors for acquisition of high-risk (HR) HPV infection in Danish women.
Methods
A population-based prospective cohort study of women aged 20 – 29 years was conducted. Participants were interviewed and underwent two gynaecological examinations 2 years apart. Women for whom Hybrid Capture 2 results were available at both visits were included in the analysis (n = 7454).
Results
A HR HPV infection was acquired by 12.8% of the women during follow-up. The incidence decreased with increasing age. The commonest types were HPV16, HPV31 and HPV52. HPV66, HPV58 and HPV53 were mainly acquired with other HR types. Multiple HR types were acquired in 50% of the women who became HPV positive during follow-up. In initially HPV negative women age, number of sexual partners and oral contraceptive use were the main risk factors for acquisition, particularly of multiple HR HPV types.
Conclusions
HPV infections were commonly acquired. We confirmed the sexually transmitted nature of the infection. Our findings show that both the level of potential exposure and other behavioural factors increase the risk for HR HPV acquisition.
doi:10.1097/OLQ.0b013e3181a96d0e
PMCID: PMC2789348  PMID: 19955872
Human papillomavirus; type-specific incidence; multiple HR HPV types; risk factors
16.  Study Comparing Human Papillomavirus (HPV) Real-Time Multiplex PCR and Hybrid Capture II INNO-LiPA v2 HPV Genotyping PCR Assays ▿  
Journal of Clinical Microbiology  2009;47(7):2106-2113.
Human papillomavirus (HPV) DNA genotyping is an essential test to establish efficacy in HPV vaccine clinical trials and HPV prevalence in natural history studies. A number of HPV DNA genotyping methods have been cited in the literature, but the comparability of the outcomes from the different methods has not been well characterized. Clinically, cytology is used to establish possible HPV infection. We evaluated the sensitivity and specificity of HPV multiplex PCR assays compared to those of the testing scheme of the Hybrid Capture II (HCII) assay followed by an HPV PCR/line hybridization assay (HCII-LiPA v2). SurePath residual samples were split into two aliquots. One aliquot was subjected to HCII testing followed by DNA extraction and LiPA v2 genotyping. The second aliquot was shipped to a second laboratory, where DNA was extracted and HPV multiplex PCR testing was performed. Comparisons were evaluated for 15 HPV types common in both assays. A slightly higher proportion of samples tested positive by the HPV multiplex PCR than by the HCII-LiPA v2 assay. The sensitivities of the multiplex PCR assay relative to those of the HCII-LiPA v2 assay for HPV types 6, 11, 16, and 18, for example, were 0.806, 0.646, 0.920, and 0.860, respectively; the specificities were 0.986, 0.998, 0.960, and 0.986, respectively. The overall comparability of detection of the 15 HPV types was quite high. Analyses of DNA genotype testing compared to cytology results demonstrated a significant discordance between cytology-negative (normal) and HPV DNA-positive results. This demonstrates the challenges of cytological diagnosis and the possibility that a significant number of HPV-infected cells may appear cytologically normal.
doi:10.1128/JCM.01907-08
PMCID: PMC2708522  PMID: 19420164
17.  Long term predictive values of cytology and human papillomavirus testing in cervical cancer screening: joint European cohort study 
Objective To obtain large scale and generalisable data on the long term predictive value of cytology and human papillomavirus (HPV) testing for development of cervical intraepithelial neoplasia grade 3 or cancer (CIN3+).
Design Multinational cohort study with joint database analysis.
Setting Seven primary HPV screening studies in six European countries.
Participants 24 295 women attending cervical screening enrolled into HPV screening trials who had at least one cervical cytology or histopathology examination during follow-up.
Main outcome measure Long term cumulative incidence of CIN3+.
Results The cumulative incidence rate of CIN3+ after six years was considerably lower among women negative for HPV at baseline (0.27%, 95% confidence interval 0.12% to 0.45%) than among women with negative results on cytology (0.97%, 0.53% to 1.34%)). By comparison, the cumulative incidence rate for women with negative cytology results at the most commonly recommended screening interval in Europe (three years) was 0.51% (0.23% to 0.77%). The cumulative incidence rate among women with negative cytology results who were positive for HPV increased continuously over time, reaching 10% at six years, whereas the rate among women with positive cytology results who were negative for HPV remained below 3%.
Conclusions A consistently low six year cumulative incidence rate of CIN3+ among women negative for HPV suggests that cervical screening strategies in which women are screened for HPV every six years are safe and effective.
doi:10.1136/bmj.a1754
PMCID: PMC2658827  PMID: 18852164
18.  Inhibition of Transcription and DNA Replication by the Papillomavirus E8̂E2C Protein Is Mediated by Interaction with Corepressor Molecules▿  
Journal of Virology  2008;82(11):5127-5136.
Papillomavirus genomes replicate as nuclear plasmids at a low copy number in undifferentiated keratinocytes. Papillomaviruses encode the E1 and E2 proteins that bind to the origin of replication and are required for the activation of replication. In addition to E2, several papillomaviruses express an E8̂E2C protein, which is generated by alternative splicing and functions as a transcriptional repressor and inhibitor of the E1/E2-dependent replication of the viral origin. Previous analyses suggested that the E8 domain functions as a transferable repression domain. In this report we present evidence that the E8 domain is responsible for the interaction with cellular corepressor molecules such as histone deacetylases, the histone methyltransferase SETDB1, and the TRIM28/KAP-1/TIF1β/KRIP-1 protein. Whereas the interaction with histone deacetylases is involved only in transcriptional repression, the interaction with TRIM28/KAP-1/TIF1β/KRIP-1 contributes to the inhibition of E1/E2-dependent replication. The corepressor TRIM28/KAP-1/TIF1β/KRIP-1 has been described to be part of multicomponent complexes involved in transcriptional regulation and functions as a scaffold protein. Since neither histone deacetylases nor the histone methyltransferase SETDB1 appears to be involved in the inhibition of E1/E2-dependent replication, most likely the modification of non-histone proteins contributes to the replication repression activity of E8̂E2C.
doi:10.1128/JVI.02647-07
PMCID: PMC2395219  PMID: 18353941
19.  Gene Profiling of Cottontail Rabbit Papillomavirus-Induced Carcinomas Identifies Upregulated Genes Directly Involved in Stroma Invasion as Shown by Small Interfering RNA-Mediated Gene Silencing 
Journal of Virology  2004;78(14):7478-7489.
To investigate changes in cellular gene expression associated with malignant progression, we identified differentially expressed genes in a cottontail rabbit papillomavirus (CRPV) squamous carcinoma model employing New Zealand White rabbits. The technique of suppression subtractive cDNA hybridization was applied to pairs of mRNA isolates from CRPV-induced benign papillomas and carcinomas, with each pair derived from the same individual rabbit. The differential expression of 23 subtracted cDNAs was further confirmed by quantitative reverse transcription-PCR (RT-PCR) with additional biopsies. Eight papilloma-carcinoma pairs examined showed a constant upregulation of the transcripts for the multifunctional adaptor protein 14-3-3 ζ and the Y-box binding transcription factor YB-1, whereas transcripts for m-type calpain 2 and NB thymosin β, which are involved in cell motility and tissue invasion, as well as casein kinase 1 α, chaperonin, and annexin I, were found to be upregulated in the majority of the cases. RNA-RNA in situ hybridization and laser capture microdissection in combination with quantitative RT-PCR analysis verified the deregulated expression of the transcripts in the tumor cells. In contrast, CRPV E7 transcript levels remained rather constant indicating no requirement for a further upregulation of E7 expression following tumor induction. Small interfering RNA-mediated interference with expression of genes encoding YB-1, m-type calpain 2, or NB thymosin β in a CRPV-positive cell line established from New Zealand White rabbit keratinocytes resulted in decreased cell invasion in matrigel chamber assays.
doi:10.1128/JVI.78.14.7478-7489.2004
PMCID: PMC434115  PMID: 15220421
20.  The Papillomavirus E8∧E2C Protein Represses DNA Replication from Extrachromosomal Origins 
Molecular and Cellular Biology  2003;23(22):8352-8362.
Carcinogenic DNA viruses such as high-risk human papillomaviruses (HPV) and Epstein-Barr-Virus (EBV) replicate during persistent infections as low-copy-number plasmids. EBV DNA replication is restricted by host cell replication licensing mechanisms. In contrast, copy number control of HPV genomes is not under cellular control but involves the viral sequence-specific DNA-binding E2 activator and E8∧E2C repressor proteins. Analysis of HPV31 mutant genomes revealed that residues outside of the DNA-binding/dimerization domain of E8∧E2C limit viral DNA replication, indicating that binding site competition or heterodimerization among E2 and E8∧E2C proteins does not contribute to copy number control. Domain swap experiments demonstrated that the amino-terminal 21 amino acids of E8∧E2C represent a novel, transferable DNA replication repressor domain, whose activity requires conserved lysine and tryptophan residues. Furthermore, E8∧E2C(1-21)-GAL4 fusion proteins inhibited the replication of the plasmid origin of replication of EBV, suggesting that E8∧E2C functions as a general replication repressor of extrachromosomal origins. This finding could be important for the development of novel therapies against persistent DNA tumor virus infections.
doi:10.1128/MCB.23.22.8352-8362.2003
PMCID: PMC262328  PMID: 14585992
21.  Identification of the E9^E2C cDNA and Functional Characterization of the Gene Product Reveal a New Repressor of Transcription and Replication in Cottontail Rabbit Papillomavirus 
Journal of Virology  2003;77(16):8736-8744.
Cottontail rabbit papillomavirus (CRPV) genomes mutated in the trans-activation domain of the E2 protein, which stimulates both viral DNA replication and transcription, are severely impaired in their ability to induce tumors in New Zealand White rabbits. A number of papillomaviruses encode, in addition to full-length E2, a shortened E2 protein or an E2 protein fused to a short stretch of amino acids derived from the small E8 open reading frame that counteract the activities of E2. We identified and cloned the novel cDNA E9^E2C of CRPV from papillomas of New Zealand White and cottontail rabbits and characterized the functions of the encoded gene product. E9^E2C was shown to be a bona fide repressor of minimal viral promoters, with the E9 domain being essential for this activity, and to repress E1/E2-dependent replication of a CRPV origin construct. In addition, E9^E2C counteracted the transactivation effect of the full-length E2 on minimal promoters containing several E2 binding sites. To investigate the role of E9^E2C in tumorigenesis, we constructed two CRPV genomes mutated in E9^E2C. One, designated CRPV-E9atgmut-pLAII, contained a mutation in the unique start codon in the E9 open reading frame, and the second E9^E2C mutant was constructed by the introduction of a stop codon close to the splice donor site at nucleotide 3714 that additionally prevented the correct splicing of the transcript. When we infected New Zealand White rabbits with these constructs, we surprisingly noted no differences in tumor induction efficiency, viral genome copy number, and viral transcription in comparison to wild-type CRPV.
doi:10.1128/JVI.77.16.8736-8744.2003
PMCID: PMC167252  PMID: 12885893
22.  A Transactivator Function of Cottontail Rabbit Papillomavirus E2 Is Essential for Tumor Induction in Rabbits 
Journal of Virology  2002;76(22):11209-11215.
Infection of domestic rabbits with cottontail rabbit papillomavirus (CRPV) causes local papillomas which progress to carcinomas in more than 80% of cases. This animal model system therefore allows the identification of molecular mechanisms required for the induction and progression of epithelial tumors. The viral E2 protein stimulates both viral DNA replication and transcription, and these functions can be genetically separated. We introduced the respective mutations into CRPV E2 and found, in line with published data for other papillomavirus E2 proteins, that mutation of the highly conserved amino acid 37 or 73 resulted in replication-competent but transactivation-deficient E2 proteins, whereas E2 proteins with mutations at residue 39 were replication deficient and transactivation competent. The R37A, I73L, and I73A E2 mutants, showing a loss of transactivation function, and the R37K E2 mutant, which is still transactivation competent, were introduced into the whole genome of CRPV, which was then injected into the skin of rabbits. Strikingly, the ability to induce tumors within 6 weeks was abolished by each of the E2 mutations, in contrast to the tumor induction rate (93%) obtained with wild-type CRPV DNA. Two small papillomas induced by mutant E2 I73A CRPV DNA appeared as late as 12 or 24 weeks postinjection, were significantly smaller, and showed no further extension of growth. These data suggest that functionally conserved amino acids in the transactivation domain of E2 are also required for the induction and growth of epithelial tumors in rabbits infected with CRPV.
doi:10.1128/JVI.76.22.11209-11215.2002
PMCID: PMC136747  PMID: 12388680
23.  The E8 Domain Confers a Novel Long-Distance Transcriptional Repression Activity on the E8̂E2C Protein of High-Risk Human Papillomavirus Type 31 
Journal of Virology  2001;75(9):4139-4149.
Infections with high-risk human papillomaviruses (HPVs) are the major risk factor for the development of anogenital cancers. Viral E2 proteins are involved in viral DNA replication and regulation of transcription. Repression of the viral P97 promoter by E2 proteins has been implicated in the modulation of the immortalization capacity and DNA replication properties of high-risk HPVs. Analysis of the cis and trans requirements for repression of the HPV type 31 (HPV31) P97 promoter, however, revealed striking differences between the full-length E2 and the E8̂E2C fusion protein which were due to conserved residues W6 and K7 of the E8 domain. In contrast to E2, E8̂E2C completely inhibited the P97 promoter from a single promoter-distal E2 binding site. This novel long-distance repression activity of the E8 domain also enabled E8̂E2C to inhibit the HPV6a P2 promoter and minimal-promoter constructs containing E2 binding sites. Thus, E8̂E2C may represent the master repressor of viral gene expression during a high-risk HPV infection, and changes in the activity of E8̂E2C might contribute to the progression of high-risk HPV-induced lesions.
doi:10.1128/JVI.75.9.4139-4149.2001
PMCID: PMC114159  PMID: 11287563
24.  Primary screening for human papillomavirus compared with cytology screening for cervical cancer in European settings: cost effectiveness analysis based on a Dutch microsimulation model 
Objectives To investigate, using a Dutch model, whether and under what variables framed for other European countries screening for human papillomavirus (HPV) is preferred over cytology screening for cervical cancer, and to calculate the preferred number of examinations over a woman’s lifetime.
Design Cost effectiveness analysis based on a Dutch simulation model. Base case analyses investigated the cost effectiveness of more than 1500 different screening policies using the microsimulation model. Subsequently, the policies were compared for five different scenarios that represent different possible scenarios (risk of cervical cancer, previous screening, quality associated test characteristics, costs of testing, and prevalence of HPV).
Setting Various European countries.
Population Unvaccinated women born between 1939 and 1992.
Main outcome measures Optimal screening strategy in terms of incremental cost effectiveness ratios (costs per quality adjusted life years gained) compared with different cost effectiveness thresholds, for two levels of sensitivity and costs of the HPV test.
Results Primary HPV screening was the preferred primary test over the age of 30 in many considered scenarios. Primary cytology screening was preferred only in scenarios with low costs of cytology and in scenarios with a high prevalence of HPV in combination with high costs of HPV testing.
Conclusions Most European countries should consider switching from primary cytology to HPV screening for cervical cancer. HPV screening must, however, only be implemented in situations where screening is well controlled.
doi:10.1136/bmj.e670
PMCID: PMC3293782  PMID: 22391612

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