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

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

This study provides a first characterisation of β-HPV life-cycle events in tumours abscised from EV patients (the human model of β-HPV-induced skin cancer), and shows how changes in E4 expression patterns relate to disease severity. β-HPV life-cycle has also been reconstructed in organotypic raft cultures created using EV-derived keratinocytes. In EV lesions and raft cultures, abundant cytoplasmic E4 expression was detectable in differentiating cells along with viral genome amplification as reported for other HPV types. E4 expression was also seen in PCNA-positive basal cells in some EV skin cancers as well as in tumours from HPV8CER (Complete Early Region) transgenic mice. In these lesions, E4 staining extended throughout the full thickness of the epithelium and was apparent in the markedly atypical cells. The loss of such staining at the tumour border suggests a distinct type of E4 dysregulation that may be exploited as a marker of viral expression during β-HPV-associated skin cancer progression.

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

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

Human papillomavirus 8 (HPV8) is involved in skin cancer development in epidermodysplasia verruciformis patients. Transgenic mice expressing HPV8 early genes (HPV8-CER) developed papillomas, dysplasias and squamous cell carcinomas. UVA/B-irradiation and mechanical wounding of HPV8-CER mouse skin led to prompt papilloma induction in about 3weeks. The aim of this study was to analyze the kinetics and level of transgene expression in response to skin irritations. Transgene expression was already enhanced 1 to 2days after UVA/B-irradiation or tape-stripping and maintained during papilloma development. The enhanced transgene expression could be assigned to UVB and not to UVA. Papilloma development was thus always paralleled by an increased transgene expression irrespective of the type of skin irritation. A knock-down of E6 mRNA by tattooing HPV8-E6-specific siRNA led to a delay and a lower incidence of papilloma development. This indicates that the early increase of viral oncogene expression is crucial for induction of papillomatosis.

Human papillomavirus 8 (HPV8) is a representative of Epidermodysplasia verruciformis (EV)-associated viruses. Transient assays in the human skin keratinocyte cell line RTS3b have shown that its replication depends in trans on expression of the viral proteins E1 and E2, similarly to other HPVs. Using deletion mutants and cloned subfragments of the noncoding region (NCR) of HPV8 we identified a 65-bp sequence in the 3′ part of the NCR to be necessary and sufficient to support replication in cis. The origin of replication (ori) of HPV8 is composed of the sequence motifs “CCAAC” (nt 57–73) and M29 (nt 84–112), which are highly conserved among the majority of EV HPVs. Analysis of M29 revealed an unconventional binding site of the E2 protein and an overlapping DNA recognition site of the tumor suppressor protein p53. Both these factors competitively bind to M29. In transient replication assays p53 acted as a potent inhibitor of ori activity, most probably in a DNA-binding-dependent fashion. The minimal ori sequences are also functionally critical for the E6 oncogene promoter P175. In contrast to its effect on replication, p53 stimulated promoter activity depending on its interaction with M29. Our observations suggest that p53 is involved in controlling the balance between DNA replication and gene expression of HPV8.

Out of 50 papilloma virus isolates from individual human warts (verrucae vulgares and verrucae plantares) 36 were analyzed for cleavage pattern of their DNA after restriction enzyme digestion with the endonucleases EcoRl, BamHI, Hind II, Hind III, Hpa II, and Hae III. Furthermore, the electrophoretic mobility of virion proteins from some of the same as well as from the additional isolates was studied by SDS-polyacrylamide gel electrophoresis. Four different cleavage patterns were observed: While three individual isolates (HPV 1, HPV 2, HPV 3) had many cleavage sites in common, and differed only in a few sites, a fourth one (HPV 4) was found to be entirely different. The electrophoretic mobility of proteins of HPV 4 was also observed to differ from those of HPV 1–3. cRNA transcribed either from HPV 1 or HPV 4 did not hybridize with the heterologous isolate. Rabbit antiserum reacting highly against HPV 1 did not react with HPV 4 proteins in complement fixation tests. We thus conclude that several human papilloma viruses exist: While HPV 1–3 are closely related isolates, HPV 4 represents a new human papilloma virus, profoundly different from the previous ones as far as base composition and antigenicity are concerned. Preliminary data suggest that the latter type occurs in approximately 20% of verrucae vulgares with low virus production, whereas HPV 1, representative for about 70% of these papillomas, predominates in warts with high virus yields.

A role for the beta genus HPVs in keratinocyte carcinoma (KC) remains to be established. In this article we examine the potential role of the beta HPVs in cancer revealed by the epidemiology associating these viruses with KC and supported by oncogenic properties of the beta HPV proteins. Unlike the cancer associated alpha genus HPVs, in which transcriptionally active viral genomes are invariably found associated with the cancers, that is not the case for the beta genus HPVs and keratinocyte carcinomas. Thus a role for the beta HPVs in KC would necessarily be in the carcinogenesis initiation and not in the maintenance of the tumor.

The protein compositions of full and empty particles of human papilloma viruses 1, 2, and 3 were compared by SDS-gel electrophoresis. The protein patterns revealed a considerable variation in the relative concentrations of three major proteins (VP2, 3, and 4) in individual preparations. In some cases, "heavy" and "light" full particles prepared from the same wart showed a similar variability in the concentrations of VP2, 3, and 4. The different protein patterns were interpreted as resulting from conversion of VP2 into VP3 and 4. The molecular relationship of these three proteins was confirmed by BrCN cleavage which led to corresponding oligopeptides. Electron micrographs of empty particles revealed that the only detectable protein components VP3 and 4 are present in typical capsomeres.

Papillomavirus DNA from an oral papilloma of a beagle was characterized by cleavage with the restriction enzymes BamHI, EcoRI, HindII, and HaeIII. A 32P-labeled probe hybridized to Hind fragments A, B, and C of HPV 1 DNA. A serological relationship to HPV 1 could be demonstrated by using a rabbit antiserum against SDS-disrupted HPV 1.

Papillomavirus-specific transcription was investigated in the absence of virus particle production in BPV 1-induced hamster tumors. The analysis revealed a single virus-specific polyadenylated transcript of 1300-base length, which was mapped on the genome of BPV 1. Hybridization with 32P-labeled probes confined the transcribed region to two HpaII fragments, which are adjacent to the BamHI cleavage site within the 1.4 × 106 dalton BamHI/EcoRI fragment. Related DNA sequences could be detected within the genomes of HPV 1 HPV 4 using relaxed hybridization conditions.

BPV 1-induced hamster tumors were used as model system to investigate the state of viral DNA in non-virus-producing tumors arising from papillomavirus infection. DNAs from a fibroma and three fibrosarcomas were analyzed for BPV 1 DNA by the Southern blot technique. The vast majority of viral DNA persists in a nonintegrated form, since: (i) supercoiled BPV-DNA can be purified by CsCl-ethidium bromide equilibrium centrifugation, (ii) restriction endonucleases, which do not cut viral DNA, do not change the DNA pattern in comparison to uncleaved samples, and (iii) digests of BPV-DNA with restriction enzymes Hpa I, Hpa II, and Hha I, respectively, lead to identical patterns with DNA from hamster tumors and from warts. Less than one genome equivalent per cell could persist in an integrated form. Virus-specific, high-molecular-weight DNA in the tumors may be interpreted as oligomeric or catenated DNA. Cleavage analysis of tumor DNA with the restriction enzymes Hpa II and Hha I, both sensitive to DNA methylation, provided no evidence for methylation of BPV 1 DNA in hamster tumors.

Human papillomavirus (HPV) 6 usually induces tumors of the genital, oral, or laryngeal mucosa. An HPV 6-related DNA of 8.2 kb was detected in an extrachromosomal state in atypically located condylomas of the mamilla and was molecularly cloned. The identity of the cloned HPV DNA and the viral DNA in the biopsy was confirmed by comparative Pst l cleavage analysis, which showed typical HPV 6 DNA fragment patterns except for 0.2-kb larger B fragments. Sequencing revealed an exact 236-bp duplication encompassing nucleotides 7681 to 7896 of HPV 6b. This tandem repeat is just upstream from the putative early promoter and contains a 20-bp insertion at position 7720, which constitutes an enhancer element described by R. F. Rando, W. D. Lancaster, P. Han, and C. Lopez ( Virology 155, 545–556, 1986). A Hin fl- Pst l fragment containing the whole duplication was cloned into an enhancer-dependent CAT expression vector and led to three- to sevenfold increased CAT activity when compared with the monomeric sequence in C127 cells and BPV1 transformed C127 cells. This indicates that the duplication within the HPV 6 isolate from the mamilla may influence early gene expression and possibly tissue tropism.