Evaluation of an automated real-time transcription-mediated amplification (TMA) assay for detection and quantification of cytomegalovirus DNA in different clinical specimens.

BACKGROUND: Reliable and fast detection and quantification of human cytomegalovirus (CMV) DNA in various diagnostic specimens is essential for care of immunocompromised or congenitally infected individuals. OBJECTIVES: To evaluate the analytical and clinical performance of the Panther Aptima® CMV (Hologic) quantitative real-time transcription mediated amplification (TMA) assay. STUDY DESIGN: Performance of the TMA assay run on the Hologic Panther Fusion was analysed for 32 proficiency testing samples and 21 quantitative reproducibility panel samples; additionally, we compared results of TMA assay and routine quantitative real-time PCR assays ("PCR-A"= Biomérieux CMV R-gene® or "PCR-B"= Laboratory-developed CMV-PCR) in 518 diagnostic specimens (254 plasma, 120 EDTA whole blood, 43 urine, 45 amniotic fluid and 56 breast milk) at two university hospital laboratories. RESULTS: All proficiency panel samples were correctly identified and quantified by the TMA assay; replicate testing of the reproducibility panel samples showed good reproducibility within and between the two laboratories. Sensitivity in plasma and WB was higher for the TMA assay detecting low-level CMV-DNAemia in samples tested negative by routine PCR. Quantitative CMV-DNAemia values correlated well between TMA and real-time PCR. Similarly, urine, AF and BM specimens showed a high rate of concordant results (91%, 98% and 98%, respectively) among TMA and PCR with good correlation of quantitative values. CONCLUSION: The performance of the Aptima® CMV TMA assay for viral blood load testing compared well to established real-time PCRs. In addition, it can be useful for diagnostics in urine, amniotic fluid and breast milk specimens.

BRD4S interacts with viral E2 protein to limit human papillomavirus late transcription.

The E2 protein encoded by human papillomaviruses (HPV) is a sequence-specific DNA-binding protein that recruits viral and cellular proteins. Bromodomain-containing protein 4 (BRD4) is a highly conserved interactor for E2 proteins that has been linked to E2's functions as transcription modulator, activator of viral replication and segregation factor for viral genomes. In addition to BRD4, a short form of BRD4 (BRD4S) is expressed from the BRD4 gene which lacks the C-terminal domain of BRD4. E2 proteins interact with the C-terminal motif (CTM) of BRD4, but a recent study suggested that the phospho-dependent interaction domain (PDID) and the basic interaction domain (BID) in BRD4 also bind to E2. These domains are also present in BRD4S. We now find that HPV31 E2 interacts with the isolated PDID domain in living cells and also with BRD4S which is present in detectable amounts in HPV-positive cell lines and is recruited into HPV31 E1 and E2 induced replication foci. Overexpression and knockdown experiments surprisingly indicate that BRD4S inhibits activities of E2. In line with that, the specific knockdown of BRD4S in the HPV31-positive CIN612-9E cell line induces mainly late viral transcripts. This occurs only in undifferentiated but not differentiated cells in which the productive viral replication cycle is induced. These data suggest that the BRD4S-E2 interaction is important to prevent HPV late gene expression in undifferentiated keratinocytes which may contribute to immune evasion and HPV persistence.ImportanceHuman papillomaviruses (HPV) have coevolved with their host by using cellular factors like bromodomain-containing protein 4 (BRD4) to control viral processes such as genome maintenance, gene expression and replication. We here show that, in addition to the C-terminal motif in BRD4, the phospho-dependent interaction domain in BRD4 interacts with E2 proteins which enable the recruitment of BRD4S, the short isoform of BRD4, to E2. Knock-down and overexpression of BRD4S reveals that BRD4S is a negative regulator of E2 activities. Importantly, the knockdown of BRD4S induces mainly L1 transcripts in undifferentiated CIN612-9E cells, which maintain replicating HPV31 genomes. Our study reveals an inhibitory role of BRD4S on HPV transcription, which may serve as an immune escape mechanism by the suppression of L1 transcripts and thus contribute to the establishment of persistent HPV infections.

Orf Virus-Based Therapeutic Vaccine for Treatment of Papillomavirus-Induced Tumors.

Orf virus (ORFV) represents a suitable vector for the generation of efficient, prophylactic antiviral vaccines against different pathogens. The present study investigated for the first time the therapeutic application of ORFV vector-based vaccines against tumors induced by cottontail rabbit papillomavirus (CRPV). ORFV-CRPV recombinants were constructed expressing the early CRPV gene E1, E2, E7, or LE6. In two independent experiments we used in total 23 rabbits which were immunized with a mixture of the four ORFV-CRPV recombinants or empty ORFV vector as a control 5 weeks after the appearance of skin tumors. For the determination of the therapeutic efficacy, the subsequent growth of the tumors was recorded. In the first experiment, we could demonstrate that three immunizations of rabbits with high tumor burden with the combined four ORFV-CRPV recombinants resulted in significant growth retardation of the tumors compared to the control. A second experiment was performed to test the therapeutic effect of 5 doses of the combined vaccine in rabbits with a lower tumor burden than in nonimmunized rabbits. Tumor growth was significantly reduced after immunization, and one vaccinated rabbit even displayed complete tumor regression until the end of the observation period at 26 weeks. Results of delayed-type hypersensitivity (DTH) skin tests suggest the induction of a cellular immune response mediated by the ORFV-CRPV vaccine. The data presented show for the first time a therapeutic potential of the ORFV vector platform and encourage further studies for the development of a therapeutic vaccine against virus-induced tumors.IMPORTANCE Viral vectors are widely used for the development of therapeutic vaccines for the treatment of tumors. In our study we have used Orf virus (ORFV) strain D1701-V for the generation of recombinant vaccines expressing cottontail rabbit papillomavirus (CRPV) early proteins E1, E2, LE6, and E7. The therapeutic efficacy of the ORFV-CRPV vaccines was evaluated in two independent experiments using the outbred CRPV rabbit model. In both experiments the immunization achieved significant suppression of tumor growth. In total, 84.6% of all outbred animals benefited from the ORFV-CRPV vaccination, showing reduction in tumor size and significant tumor growth inhibition, including one animal with complete tumor regression without recurrence.

Vaginal prevalence of human papillomavirus infections in women with uterovaginal aplasia before and after laparoscopically assisted creation of a neovagina: a prospective epidemiological observational study.

OBJECTIVE: To study vaginal as opposed to cervical human papillomavirus (HPV) acquisition with regard to true prevalence, HPV types, and the role of co-factors in virgins and after their sexual debut. DESIGN: Prospective epidemiological observational study. SETTING: University hospital specialised in genital malformations. POPULATION: Women diagnosed with Mayer-Rokitansky-Küster-Hauser syndrome (MRKHS) and undergoing neovaginoplasty between November 2011 and July 2017. METHODS: This is a prospective study including 186 women with MRKHS before and after sexual debut. MAIN OUTCOME MEASURES: Conventional vaginal cytology and different HPV tests were performed at surgery and during routine gynaecological follow-up 1, 3, 6 and ≥ 11 months after surgery and risk factors were documented. RESULTS: The mean age of all women at surgery was 20.1 years (SD 5.4), mean body mass index (BMI) was 22.1 kg/m2 (SD 4.6). In 83 vaginal samples from 41 different women at least one of the HPV tests was positive. Thirty-three different HPV types were detected. The prevalence of 41/186 = 22.0% as well as type distribution are comparable with those found in a young German female population. The overall rate of acquisition was clearly associated with sexual activity and smoking habits. Out of 367 Papanicolaou smears only six were abnormal with Pap IIID (MN II) and no obvious vaginal lesion was detected. CONCLUSIONS: Vaginal HPV prevalence and HPV types in previously virgin women after creation of a neovagina are not different from the acquisition of cervical infections in the general population and is clearly associated with sexual activity and with smoking habits. However, abnormal Papanicolaou smears are rarely seen. TWEETABLE ABSTRACT: Vaginal HPV prevalence after creation of a neovagina is similar to that on the cervix in the general population.

The contribution of SP100 to cottontail rabbit papillomavirus transcription and replication.

SP100 proteins are components of nuclear domain 10 structures and have been implicated as inhibitors of human papillomavirus (HPV) replication. In this study, we have addressed the role of SP100 in tumour formation by the cottontail rabbit (Sylvilagus floridanus) papillomavirus (CRPV or SfPV1) in a rabbit model. Tissue culture studies using rabbit keratinocyte lines indicated that rabbit SP100 is an interferon-beta-inducible gene similar to its human counterpart. Stable knockdown of SP100 by shRNA in a cell line harbouring CRPV genomes resulted in a decrease of viral early transcripts. In contrast, infection of domestic rabbits with recombinant CRPV genomes expressing short hairpin (sh)RNAs directed against SP100 did not reveal changes in tumour formation rate, tumour size or early viral transcript levels. However, late viral transcript levels and viral genome copies were consistently lower in CRPV/shSP100-induced tumours than in the control, but these differences did not reach statistical significance. In summary, this study suggests that rabbit SP100 is not an inhibitor but an activator of CRPV replication and transcription.

Detection of mutation-specific epidermal growth factor receptor (E746-A750del) and lack of detection of human papillomavirus in oral squamous cell carcinoma.

The clinical impact of epidermal growth factor receptor (EGFR) (E746-A750del) mutation and human papillomavirus (HPV) in oral squamous cell carcinoma (OSCC) is unclear. EGFR (E746-A750del) expression was analyzed in OSCC specimens (n=161) by immunohistochemistry. The expression results were correlated with clinical characteristics and impact on survival. Using INNO-LiPA Extra, high-risk HPV types were genotyped and analyzed in 211 OSCC specimens. Positive EGFR (E746-A750del) expression (n=40/161, 25%) was not associated with any clinicopathological characteristics, prognostic factors, social habits (smoking, alcohol consumption), or tumour-specific survival. HPV16 DNA was detected in three out of 211 samples (HPV16-positive: n=3/211, 1.4%). This study shows that mutation-specific EGFR (E746-A750del) expression and HPV do not appear to be relevant to the survival of patients with OSCC.

The Aptima HPV assay fulfills the cross-sectional clinical and reproducibility criteria of international guidelines for human papillomavirus test requirements for cervical screening.

The Aptima HPV assay (Hologic Gen-Probe, San Diego, CA) is an FDA-approved assay for detecting human papillomavirus (HPV) E6/E7 mRNA from 14 high-risk HPV types. This study evaluated the clinical performance of the Aptima HPV assay for cervical intraepithelial neoplasia of grade 2 or worse (CIN2+), relative to the high-risk HPV GP5+/GP6+ PCR, in a cross-sectional clinical equivalence analysis using the noninferiority score test with cervical samples from population-based screening, i.e., 69 cervical scraping samples from women with CIN2+ and 843 from women without evidence of CIN2+. In addition, intralaboratory reproducibility over time and interlaboratory agreement of the Aptima HPV assay results were assessed with another set of 548 cervical samples. The Aptima HPV assay showed a clinical sensitivity for CIN2+ of 94.2% (95% confidence interval [CI], 85.5 to 97.8%) and a clinical specificity for CIN2+ of 94.5% (95% CI, 92.8 to 95.9%); by comparison, these figures were 97.1% (95% CI, 89.1 to 99.3%) (67/69 samples) and 93.6% (95% CI, 91.7 to 95.0%) (785/839 samples), respectively, for GP5+/GP6+ PCR. The clinical sensitivity and specificity of the Aptima HPV assay were noninferior to those of GP5+/GP6+ PCR (P = 0.039 and 0.00016, respectively). In addition, high reproducibility of the Aptima HPV assay, as reflected by the intralaboratory reproducibility over time of 96.0% (95% CI, 94.4 to 97.3%) (526/548 samples; kappa = 0.89) and interlaboratory agreement of 96.7% (95% CI, 95.4 to 98.1%) (531/548 samples; kappa = 0.91), was found. Altogether, these data show that the Aptima HPV assay meets the cross-sectional clinical and reproducibility criteria of the international guidelines for HPV test requirements for cervical screening. Longitudinal data are needed to ensure that the long-term negative predictive value of this mRNA assay is similar to those of validated HPV DNA tests.

Parity as a cofactor for high-grade cervical disease among women with persistent human papillomavirus infection: a 13-year follow-up.

BACKGROUND: Several environmental factors have been associated with increased risks for cervical cancer. We examined whether reproductive history, contraceptive use, or sexual behaviour increase the risk for cervical intraepithelial neoplasia grade 3 or worse (CIN3+) among women with persistent human papillomavirus (HPV) infection. METHODS: A population-based cohort of women participated in a personal interview and underwent a gynaecological examination at which cervical specimens were obtained for HPV DNA testing. Follow-up information (~13 years) on cervical lesions was obtained from the Danish Pathology Data Bank. Women who had a high-risk HPV infection comprised the overall study population (n=1353). A subgroup of women with persistent high-risk HPV infection (n=312) was identified. Hazard ratios (HRs) for a diagnosis of CIN3+ and the corresponding 95% confidence intervals (CIs) were calculated. RESULTS: Women with persistent HPV infection who had given birth had a significantly increased risk for CIN3+ (HR=1.78; 95% CI: 1.07-2.94). No association was found with pregnancy, use of intrauterine devices, or sexual behaviour. Based on small numbers, women with persistent HPV infection had a decreased risk for CIN3+ with any use of oral contraceptives (HR=0.54; 95% CI: 0.29-1.00). CONCLUSION: Childbirth increases the risk for subsequent CIN3+ among women with persistent HPV infection.

HPV16 is associated with younger age in women with cervical intraepithelial neoplasia grade 2 and 3.

OBJECTIVE: To evaluate if women with HPV16 positive CIN2 and CIN3 are diagnosed at a younger age. METHODS: We conducted a population-based cohort study including more than 40,000 women having a liquid based cervical cytology sample taken as part of routine screening. HPV analysis was performed using Hybrid Capture 2 and LiPAv2. The study population was linked to the Danish Pathology Data Bank to retrieve information on subsequent cervical histology. We included HR HPV positive CIN2/3 samples, comprising 173 CIN2 and 467 CIN3 lesions. Due to a high number of multiple concurrent HPV infections, the causative HPV type was assigned to a hierarchically group. RESULTS: In CIN3, the estimated proportion of lesions positive for HPV16 was 68.1% among women aged 20 years and decreased to 38.9% among women aged 50 years. A decrease in HPV16 positivity with increasing age was also observed in CIN2. In a multinomial logistic regression analysis, young age was strongly associated with HPV16 positivity in CIN3 lesions (OR=0.46 per 10 year increase in age, 95% CI: 0.32-0.65). The proportion of HPV16 and/or 18 positive lesions among women diagnosed with CIN2 and CIN3 below 30 years of age was 44% and 75%, respectively. CONCLUSIONS: HPV16 positivity was significantly associated with younger age at diagnosis of CIN3. In a population vaccinated against HPV16 and 18, we will experience a shift to older ages in cervical precancerous lesions. These findings may imply that cervical cancer screening programs could start at an older age in HPV vaccinated populations.

Primary cervical cancer truly negative for high-risk human papillomavirus is a rare but distinct entity that can affect virgins and young adolescents.

Cancer of the uterine cervix is almost exclusively associated with human papillomavirus (HPV). Carcinogenesis is slow, the minimal time from initial HPV infection to invasive carcinoma seems to be less than ten years. In order to identify rapid onset cervical cancer, we carried out a retrospective re-analysis of an extended cohort of patients with invasive cervical cancer, and reviewed cases identified within the cancer registry of Lower Saxony or using Medline or ISI data. No instances of a rapid-onset cancer or true HPV-DNA negative cancer were found among our hospital cohort of 178 women with primary cancer of the uterine cervix. Registry data identified four out of 5,878 patients who were diagnosed with primary cervical cancer at 14 to 20 years of age. They were classified as clear-cell and endometriod adenocarcinoma and tested persistently negative for high-risk HPV-DNA. Fourteen more cases of cervical cancer in virgins and very young women were identified by a Medline search, mostly with unknown histologic type or rare subtypes of adenocarcinoma. In conclusion, rare adenocarcinoma of the uterine cervix may represent an entity unrelated to HPV, thus explaining instances of rapid onset cervical cancer.

Inclusion of HPV testing in routine cervical cancer screening for women above 29 years in Germany: results for 8466 patients.

In a prospective cohort study 8466 women attending routine cervical cancer screening were recruited. Colposcopy was performed on women with any degree of atypia on cytology and/or a positive high-risk human papillomavirus (HPV)-DNA test (HC2; Hybrid Capture 2((c))), and for a randomly selected sample of 3.4% women with negative findings on both. Quality control included reviews of cytology, histology, colposcopy images and retesting of samples with polymerase chain reaction. Test diagnostic performances were based on 7908 women who had complete baseline and follow-up results. Routine histology identified 86 women with high-grade cervical intraepithelial neoplasia (CIN2+), which was confirmed by review histology in only 46 cases. Sensitivity of routine cytology for the detection of CIN2+ was 43.5%, with a specificity, positive predictive value (PPV), negative predictive value (NPV) of 98.0, 11.4 and 99.7%, respectively. Sensitivity of the HC2 test for the detection of CIN2+ was 97.8%, with a specificity, PPV and NPV, of 95.3, 10.9 and 100%, respectively. No high-grade neoplasia was detected in the randomly selected control group. A negative HPV-test result, even in combination with a positive Papanicolaou (Pap) result, virtually excluded any risk of underlying high-grade disease, but this was not the case for a negative Pap result. These data show that HPV testing is of value for the detection or exclusion of prevalent CIN in a routine cervical cancer-screening setting and could be used for further risk classification of women for follow-up management.

Economic evaluation of human papillomavirus screening in Germany.

Cytology-based screening programs for cervical cancer have been effective in reducing cancer incidence and preventing premature deaths worldwide. However, there is concern about the relatively low sensitivity of current screening procedures. Although the causal association between infection with certain high-risk types of human papilloma virus (HPV) and the development of cervical cancer has been clearly established, testing for the major risk factor is not part of current screening practice. We created a tree decision model over time to evaluate different policy choices for implementing a population-based screening program. Results of the economic analysis indicate that testing with any implemented HPV DNA testing (stand alone or in combination with the Papanicolaou smear) is superior to cytology and measures presently in use. Additional costs per life-years gained cannot be reported because the HPV branches had fewer discounted overall costs (euro 222 million vs. euro 82 and euro 76 million, respectively), and they saved more life years (19,599 vs. 19,163 and 903, respectively) then the smear alternative. Any HPV DNA testing is preferable over the current state of the art performed in Germany. This is true not only for economic reasons but also for life-years gained. Therefore HPV DNA testing must become an essential component to back up the relatively weak sensitivity of the standard procedure.

Current methods of testing for human papillomavirus.

Human papillomavirus DNA testing is gaining wider acceptance, yet the majority of testing is still undertaken in the research setting using protocols that are unsuitable for clinical diagnostic laboratories. Large-scale clinical human papillomavirus DNA testing is likely to be introduced as an adjunct to cervical cytology, so the cytology laboratory is an appropriate place for it to be undertaken. This poses a challenge for any human papillomavirus DNA test since it will be performed by technicians not specifically trained in virology or molecular biology, and will need to produce consistently reliable results in this setting. This is only realistically possible with a standardized and quality-controlled, commercially produced human papillomavirus DNA test. There is currently only one such commercially available assay, although there are a number of research-based tests that could logically lead to additional commercial products. The technological basis of these assays, together with available performance data, is reviewed in this chapter and the clinical utility of the tests is evaluated.

The E8 domain confers a novel long-distance transcriptional repression activity on the E8E2C protein of high-risk human papillomavirus type 31.

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 E8E2C fusion protein which were due to conserved residues W6 and K7 of the E8 domain. In contrast to E2, E8E2C 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 E8E2C to inhibit the HPV6a P2 promoter and minimal-promoter constructs containing E2 binding sites. Thus, E8E2C may represent the master repressor of viral gene expression during a high-risk HPV infection, and changes in the activity of E8E2C might contribute to the progression of high-risk HPV-induced lesions.

Synthesis of viral DNA and late capsid protein L1 in parabasal spinous cell layers of naturally occurring benign warts infected with human papillomavirus type 1.

We investigated human papillomavirus type 1 (HPV1)-specific transcription, viral DNA replication, and viral protein expression in naturally occurring benign tumors by in situ hybridization, 5-bromodeoxyuridine (BrdU) incorporation, and immunohistochemistry and obtained results different from other HPV-infected benign tumors characterized so far. Moderate amounts of transcripts with a putative coding potential for E6/E7, E1, and E2 were demonstrated from the first subrabasal cell layer throughout the stratum spinosum and granulosum. In addition very large amounts of E4 and L1 transcripts were present in the same epithelial layers. This finding was substantiated by the demonstration of L1 and E4 protein already in the bottom-most spinous cell layer. Furthermore massive amplification of the viral DNA as measured by BrdU incorporation and different methods of in situ hybridization took place in the lowest 5 to 10 suprabasal cell layers. These findings are in contrast to the assumption that late gene expression and viral DNA synthesis are restricted to the more differentiated cell layers of the epithelium and point to differences in the regulation of the vegetative life cycle between different papillomavirus types.

The E8E2C protein, a negative regulator of viral transcription and replication, is required for extrachromosomal maintenance of human papillomavirus type 31 in keratinocytes.

The viral E2 protein is a major regulator of papillomavirus DNA replication. An important way to influence viral replication is through modulation of the activity of the E2 protein. This could occur through the action of truncated E2 proteins, called E2 repressors, whose role in the replication cycle of human papillomaviruses (HPVs) has not been determined. In this study, using cell lines that contain episomal copies of the "high-risk" HPV type 31 (HPV31), we have identified viral transcripts with a splice from nucleotide (nt) 1296 to 3295. These transcripts are similar to RNAs from other animal and human papillomaviruses and have the potential to fuse a small open reading frame (E8) to the C terminus of E2, resulting in an E8E2C fusion protein. E8E2C transcripts were present throughout the complete replication cycle of HPV31. A genetic analysis of E8E2C in the context of the HPV31 genome revealed that mutation of the single ATG of the E8 gene, introduction of a stop codon downstream of the ATG, or disruption of the splice donor site at nt 1296 led to a dramatic 30- to 40-fold increase in the transient DNA replication levels in both normal and immortalized human keratinocytes. High-level expression of E8E2C from heterologous vectors was found to inhibit E1-E2-dependent DNA replication of an HPV31 origin of replication construct as well as to interfere with E2's ability to transactivate reporter gene constructs. In addition, HPV31 E8E2C strongly repressed the basal activity of the major viral early promoter P97 independent of E2. E8E2C may therefore exert its negative effect on viral DNA replication through modulating E2's ability to enhance E1-dependent DNA replication as well as by regulating viral gene expression. Surprisingly, HPV31 genomes that were unable to express E8E2C could not be maintained extrachromosomally in human keratinocytes in long-term assays despite high transient DNA replication levels. This suggests that the E8E2C protein may play a role in copy number control as well as in the stable maintenance of HPV episomes.

Human papillomavirus testing in primary screening for cervical cancer of human immunodeficiency virus-infected women, 1990-1998.

OBJECTIVE: Women infected with the human immunodeficiency virus (HIV) have an increased risk of cervical neoplasia while the value of cytologic screening is limited due to a high prevalence of inflammatory disease. The study was conducted to determine whether testing for human papillomavirus (HPV) DNA could improve primary screening for cervical cancer of these patients. METHODS: One hundred thirty-eight HIV-infected women were examined between 1990 and 1998. Ninety-four patients with a total of 279 women-years were eligible for incidence evaluation. Colposcopy, cytology, and HPV DNA testing with the hybrid capture I assay were performed at each visit. RESULTS: Seventeen cases of high-grade cervical neoplasia were diagnosed at study entry and 13 developed CIN II or CIN III during follow-up. The hybrid capture I assay detected 94.1% of prevalent and 100% of incident high-grade neoplasia, while the corresponding sensitivity of Pap smears using CIN I or worse as the referral criteria was 82.3% for prevalent and 69.2% for incident high-grade neoplasia. Eleven of 13 patients who progressed to histologically confirmed CIN II/III tested positive for HPV DNA at study entry compared with 5/13 women presenting with any degree of cytologic atypia at recruitment. The Pap smears of 36/94 women remained normal throughout the study while 54/94 patients remained negative for high-risk HPV types. CONCLUSION: Hybrid capture I identified high-grade cervical neoplasia more accurately than the Pap smear and appeared to be beneficial for primary cervical cancer screening in HIV-infected women.

Induction of E6/E7 expression in cottontail rabbit papillomavirus latency following UV activation.

Latent human papillomavirus (HPV) infections are widespread in the genital and respiratory tracts and are a source of recurrent disease. This study used a cottontail rabbit papillomavirus (CRPV) model to determine the presence of E1, E6, and E7 transcripts in latent infection and to determine the temporal change in transcripts following UV activation. We found E1 transcripts in all latently infected sites but no detectable E6 and E7 transcripts, consistent with our earlier studies of HPV6/11 latency. These results suggest that this transcription pattern is broadly characteristic of latent papillomavirus infections. E6/E7 transcripts were detectable within 1 week of irradiation, with maximal induction (approximately 40% of sites) at 2 weeks postirradiation. Papillomas were induced in approximately 26% of irradiated sites after a 3- to 5-week lag. Sites that did not form papillomas by 3 months after irradiation were CRPV DNA positive but E6/E7 RNA negative. Thus, only a subset of latent infections can be induced to express E6/E7 transcripts and form papillomas. We propose that CRPV can be used to study the molecular processes regulating papillomavirus activation.

Cell-type-specific separate regulation of the E6 and E7 promoters of human papillomavirus type 6a by the viral transcription factor E2.

Gene expression of human papillomaviruses (HPV) is tightly controlled by cellular factors and by the virally encoded E2 protein through binding to distinct sites within the regulatory noncoding region. While for the high-risk genital papillomaviruses a single promoter drives the expression of all early genes, a second promoter present in the E6 open reading frame of the low-risk HPV type 6 (HPV6) would allow an independent regulation of E6 and E7 oncogene expression. In this report, we provide the first evidence that E2 regulates both early promoters of HPV6 separately and we show that promoter usage as well as E2 regulation is cell type dependent. Among the different epithelial cell lines tested, only RTS3b cells allowed an expression pattern similar to that observed in naturally infected benign condylomas. While the E6 promoter was repressed by E2 to 50% of its basal activity, the E7 promoter was simultaneously stimulated up to fivefold. Activation of the E7 promoter was mediated predominantly by the binding of E2 to the most promoter-distal E2 binding site. Repression of the E6 promoter depended on the presence of two intact promoter-proximal binding sites. Mutation of both of these repressor binding sites reversed the effect of E2 on the E6 promoter from repression to activation. In contrast, in HT3 cells we observed an E2-mediated activation of the E6 promoter in the context of the wild-type noncoding region. This indicated that repression of the E6 promoter by binding of E2 to both promoter-proximal binding sites did not function in the cellular environment provided by HT3 cells. These data suggest that the separate regulation of the E6 and E7 promoters of HPV6 is mediated through successive occupation of binding sites with different affinities for E2 depending on the intracellular concentration of E2 and on the cellular environment provided by the infected cell.