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.

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.

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.

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.

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.

Human papillomavirus type 6 in squamous cell carcinoma of the bladder and cervix.

Human papillomavirus (HPV) types 16 and 18 are the most frequent genotypes identified in genital malignancies, while HPV types 6 and 11 are found predominantly in condylomas and low-grade dysplasias. It is thought that HPV types 16 and 18 represent high-risk genotypes, while HPV types 6 and 11 rarely, if ever, participate in the development of malignant tumors. In a series of over 300 invasive tumors of the lower genital tract analyzed for the presence of HPV three have been found to contain HPV type 6 DNA: two invasive squamous cell carcinomas of the cervix and one squamous cell carcinoma of the bladder. Human papillomavirus type 6 was the only HPV type detected in these tumor DNAs by Southern blot hybridization and by the polymerase chain reaction using both consensus and type-specific primers. In situ hybridization using whole genomic RNA probes localized viral DNA to tumor cells. Although extensive virologic and epidemiologic studies conducted in the last decade indicate that HPV types 16 and 18 are more likely to be associated with high-grade dysplasias and invasive cancer, HPV type 6 may not be as innocuous as previously supposed.

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.

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.

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 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.

A comparative analysis of the interactions of the E6 proteins from cutaneous and genital papillomaviruses with p53 and E6AP in correlation to their transforming potential.

Common necessity for all papillomaviruses is to induce DNA synthesis in quiescent cells. This is commonly achieved by the E7 gene product, which interferes with the function of members of the retinoblastoma family controlling transition from the G1-phase to the S-phase of the cell cycle. Uncontrolled entry into S-phase activates, however, negative growth control signals which have to be bypassed to achieve production of progeny viruses. In addition to inherent activities of the E7 protein, high risk genital types encode an E6 protein that overcomes p53-mediated G1-arrest and apoptosis in concert with the cellular factor E6AP by targeting p53 for the enhanced ubiquitin-dependent degradation. The key question, which of these functions of genital E6 and E7 proteins is responsible for the carcinogenic phenotype, is still not completely answered. In contrast to high risk genital types no immortalizing or transforming activities have been found for the E7 proteins of the high risk cutaneous HPV8 and 47. On the other hand the ability of the E6 protein to transform established rodent fibroblasts seems to be a property shared by high risk genital and cutaneous types. To examine the existence of a common E6-mediated transforming pathway for both virus groups we compared the properties of the cutaneous E6 proteins with already known functions of E6 proteins of genital viruses. For this we analyzed the E6 proteins of low nak and high risk cutaneous and genital papillomaviruses with respect to cell transformation, to their abilities to bind, degradate, and influence the activity of human p53, and to bind E6AP. The results of our study demonstrate a clear lack of interaction between the transforming E6 proteins of HPV1 and HPV8 and both cellular proteins p53 and E6AP. In contrast, we found E6AP-independent binding of HPV16 E6 and HPV6 E6 to p53, although both proteins were different in their transforming potential. Of all four proteins investigated, only HPV16 E6 was able to bind to p53 and E6AP and to induce degradation of the p53 protein in the reticulocyte system. When we investigated in frame deletion mutants of the E6 protein of HPV16 for their abilities to bind to p53 or E6AP, degradate, and inhibit the transactivation function of p53 and to transform rodent fibroblasts, no correlation between the different activities could be found. Mutants still able to bind p53 and E6AP lacked transforming ability and other mutants that were transformation-competent were deficient in p53 and E6AP bindings.

The E7 protein of human papillomavirus 8 is a nonphosphorylated protein of 17 kDa and can be generated by two different mechanisms.

The E7 protein of human papillomavirus (HPV) 8 shows no in vitro transforming activity, in contrast to E7 of the genital HPVs, but seems to be involved in the control of viral DNA replication. To determine whether functional differences between the E7 proteins of HPV16 and HPV8 were reflected in differences in their biochemical properties, we characterized the E7 protein of HPV8 expressed from the late simian virus 40 promoter in COS 7 cells. An E7-specific antiserum was obtained by immunizing rabbits with a beta-gal-E7 fusion protein containing all of the E7 polypeptide. This antiserum specifically precipitated from [35S]cysteine but not from 32PO4-labeled transiently transfected COS 7 cells a protein with a low mobility of 17 kDa in SDS-PAGE. The high sedimentation rate in nondenaturing glycerol gradients pointed to an interaction with other proteins or to the existence of E7 oligomers. An association with the retinoblastoma protein could, however, be excluded. The 5' ends of HPV8 transcripts derived from the E6-E7 region in G418 selected rodent cells, which were mapped by nuclease S1 digestion, are located upstream of ORFs E6 and E7, respectively. No evidence for an E6*I-like mRNA was found. In COS 7 cells transfected with a plasmid expressing the E6-E7 region of HPV8 under the control of the late SV40 promoter, however, the E7 protein was translated from a polycistronic mRNA potentially encoding E6 and E7. These data indicate that the E7 protein of HPV8 may be expressed in two ways: (i) through translation of an E7-specific mRNA, as with HPV6 and HPV11, or (ii) through internal initiation of translation at the ATG of E7.

Involvement of human papillomavirus type 8 genes E6 and E7 in transformation and replication.

We investigated the transforming activity of human papillomavirus type 8 (HPV8) by expressing all early open reading frames from a heterologous promoter in different rodent fibroblast lines. Morphological transformation was observed only in G418-selected mouse C127 and Rat 1 cells containing an intact E6-coding region. E6 of HPV8 did not transform NIH 3T3 cells as did E6 of bovine papillomavirus type 1. C127 cells transformed by E6 were anchorage independent and had a reduced serum requirement but did not form tumors in nude mice. E7 of HPV8 showed no transforming potential, in contrast to E7 of HPV18 and HPV16. It was, however, able to complement an E7 mutant of bovine papillomavirus type 1 with a defect in high-copy-number DNA maintenance. The data indicate that the expression of the HPV8 E6 open reading frame is sufficient to induce morphological transformation in rodent fibroblasts, whereas E7 is involved in the replication of the viral DNA.

The predominant mRNA class in HPV16-infected genital neoplasias does not encode the E6 or the E7 protein.

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.

Epidermodysplasia verruciformis-associated human papillomavirus 8: genomic sequence and comparative analysis.

Human papillomavirus (HPV) 8 induces skin tumors which are at high risk for malignant conversion. The nucleotide sequence of HPV8 has been determined and compared to sequences of papillomaviruses with different oncogenic potential. The general organization of the HPV8 genome is similar to that of other types. Highly conserved, genus-specific sequences were found in open reading frames (ORFs) E1, E2, and L1. In ORFs E6, E7, and L2, HPV8 is more distantly related, but it was possible to differentiate subgenera in which HPV8 belonged to the HPV1-cottontail rabbit papillomavirus group. Sequences within ORF E4 and part of ORF L2 are rather type specific. HPV8 stands out by several unique features: the considerably reduced size of the noncoding region (397 base pairs), with a seemingly low potential for forming complex secondary structures; a cluster of putative promoter elements in the 3' half of ORF E1; an RNA polymerase III promoter-like sequence close to the C terminus of ORF E2; and of particular interest, the homology between the putative protein encoded by ORF E4 and the Epstein-Barr virus nuclear antigen 2 protein, which may reflect similar mechanisms in virus-mediated transformation.