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.

HPV6 variants from malignant tumors with sequence alterations in the regulatory region do not reveal differences in the activities of the oncogene promoters but do contain amino acid exchanges in the E6 and E7 proteins.

Human papillomavirus type 6 (HPV6) causes benign epithelial proliferations of the anogenital and aerodigestive tract, which usually tend to regress spontaneously. The low incidence of HPV6 in carcinomas and the rare progression of the benign tumors has led to the classification of HPV6 as "low-risk" virus. A series of reports, however, described the isolation of HPV6 variants from malignant tumors characterized by sequence rearrangements in the noncoding regulatory region (NCR). It was speculated that these sequence alterations play a role in tumor progression by enhancing the promoter activity and thereby increasing the expression of the viral oncogenes E6 and E7. To elucidate if HPV6 isolates from malignancies do regularly exhibit sequence alterations in the regulatory region we first determined and complied the sequences of the NCRs of a number of isolates from benign and malignant lesions. This analysis revealed in general a high degree of sequence conservation between the individual isolates. Most of the isolates, however, differed, independently of origin, by a major and one or two minor insertions from the prototype HPV6b sequence. When tested in a functional assay these altered NCR sequences did not result in significantly different activities of the promoters responsible for the expression of the E6 and E7 genes. Further analysis of the E6 and E7 coding region revealed a surprisingly high sequence variability within the E6 ORF and allowed the detection of amino acid exchanges unique for isolates from carcinomas.

Identification of a differentiation-inducible promoter in the E7 open reading frame of human papillomavirus type 16 (HPV-16) in raft cultures of a new cell line containing high copy numbers of episomal HPV-16 DNA.

Gene expression of human papillomaviruses (HPV) is tightly linked to differentiation processes within the pluristratified epithelium. To analyze changes in the transcription pattern of HPV-16 during epithelial cell differentiation, we established a permanently growing HPV-16 positive cell line, designated KG, from a vulvar intraepithelial neoplasm. KG cells of early passages harbored multiple copies of the HPV-16 DNA as episomes and were able to form a stratified epithelium in an organotypic raft culture system. Analysis of viral gene expression revealed the known transcription pattern of the early region of HPV-16 with the exception of a so far undefined mRNA class with start sites in the E7 open reading frame. Quantitative analysis of primer extension experiments with RNA from KG cells grown in monolayer and raft culture showed a strong induction of this transcript in differentiated KG cells, whereas the level of the mRNAs initiated at the early promoter P97 remained almost constant. Primer extension analyses with four different primers and direct sequencing of the extension product revealed that the differentiation-inducible transcript initiated at a novel promoter with a major start site around nucleotide position 670 (P670) in the E7 open reading frame of HPV-16. Sequence analysis of cDNAs derived from RNA of KG cells grown in raft culture suggested that the transcripts initiated at P670 have a coding potential for an E1E4 fusion protein and for the E5 protein.

Transcriptional silencer of the human papillomavirus type 8 late promoter interacts alternatively with the viral trans activator E2 or with a cellular factor.

The noncoding region of the highly oncogenic, epidermodysplasia verruciformis-associated human papillomavirus type 8 contains a negative regulatory element (NRE). Quantitative RNase protection analysis confirmed that the NRE sequence acts as a silencer of transcription. A 38-bp sequence upstream of late promoter P7535 down-regulated expression from the homologous P7535 promoter, as well as the heterologous tk gene promoter, independently of its orientation relative to the test promoters. It also reduced gene expression when cloned downstream of the transcription units. Transient expression assays with keratinocytes and fibroblasts of epidermodysplasia verruciformis patients and controls demonstrated that the NRE activity is not cell specific. Gel retardation tests suggested that NRE specifically interacts with only one nuclear factor. Mutational analysis identified three NRE mutants which no longer formed a detectable DNA-protein complex but still repressed transcription, indicating that protein-DNA interaction is not relevant for the silencer function. The NRE contains a binding site of viral trans activator protein E2. It was shown that expression of E2 overrides the inhibitory effect of the NRE sequences. Binding of E2 and that of the cellular factor were mutually exclusive. The bifunctional nature of NRE acting as a silencer and a target site for viral trans activator E2 offers an interesting opportunity to regulate the switch from early to late transcription in the human papillomavirus life cycle.

[Distribution of the labial red furrow patterns]. / Untersuchungen zur Verteilung der Lippenrotfurchenmuster.

Following recording and the construction of models, the patterns of labial furrows obtained of one randomly subjects from Europids , Mongoloids, and Negroids were registered on the basis of various systems of classification. Typification was carried out with the help of a reflected light microscope (10-fold magnification). The mathematico -statistical evaluation of the material was made by contingency analysis. The distribution pattern of labial furrows observed in test persons of the mongolian-type population was found to be clearly differentiated from that seen in representatives of the european-type and negro-type population who showed resembling distribution patterns.