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Recently, several novel human polyomaviruses (HPyVs) have been detected. HPyV6, 7, 9 and 10 are not associated with any disease so far. Trichodysplasia spinulosa (TS)-associated polyomavirus (TSPyV) can cause the rare skin disease TS. We have evaluated cutaneous DNA prevalence and viral loads of five HPyVs in HIV-infected men compared to healthy male controls. 449 forehead swabs were analysed by HPyV-specific real-time PCR. HPyV6, HPyV7, TSPyV and HPyV10 were found significantly more frequently on the skin of 210 HIV-infected compared to 239 HIV-negative men (HPyV6, 39.0 vs 27.6 %; HPyV7, 21.0 vs 13.4 %; TSPyV, 3.8 vs 0.8 %; HPyV10, 9.3 vs 3.4 %; P

It has been demonstrated previously that E7 oncogene expression of human papillomavirus (HPV) type 8 in keratinocytes induces cell invasion and accelerated differentiation. Looking for cellular genes deregulated by HPV-8 E7, lipocalin-2 was identified as being upregulated in these cells by cDNA microarray analysis. Lipocalin-2 is known to be overexpressed in many human cancers and is implicated in the regulation of cell proliferation, differentiation and apoptosis. In this study, increased levels of lipocalin-2 were observed in extracts from HPV-8 E7-positive keratinocytes and from keratinocytes expressing E7 of HPV-1, -4, -5, -15, -20 and -38, but not of HPV-16. Similar results were obtained when measuring secreted lipocalin-2 in the supernatants of the cell cultures. Lipocalin-2 expression was associated with cell differentiation in keratinocyte monolayers and in organotypic skin cultures. It was found in the uppermost layers of HPV-5, -8, -15, -16, -20 and 38 E7-expressing but not low-risk HPV-1 and -4 E7-expressing keratinocytes. Immunohistochemical staining of HPV-positive and -negative human skin squamous cell carcinomas (SCCs) revealed lipocalin-2 expression mostly in differentiated, filaggrin-positive areas of 15 out of 17 HPV-positive and three out of nine HPV-negative SCCs. These data indicate that lipocalin-2 expression correlates with HPV positivity of cutaneous SCCs. © 2011 SGM.

Cutaneous human papillomavirus type 8 (HPV8) is carcinogenic in patients with epidermodysplasia verruciformis. Transgenic mice with the complete early region (CER) of HPV8 spontaneously developed papillomas, dysplasia and squamous cell carcinomas of the skin. To characterize the role of individual early genes in carcinogenesis, the E6 and E6/E7 genes were expressed separately in transgenic mice. Nearly all HPV8-E6-positive mice spontaneously developed multifocal tumours, characterized by papillomatosis, hyperkeratosis and varying degrees of epidermal dysplasia. In 6 % of the cases, the tumours became malignant, comparable with HPV8-CER mice. Thus, in the murine epidermis, E6 is the major oncogene necessary and sufficient to induce spontaneous tumour development up to the level of squamous cell carcinoma. To evaluate the synergistic effects of UV light and wound healing, the skin of HPV8 mice was irradiated with UVA/UVB light or wounded with punch biopsies. These treatments induced papillomatosis in HPV8-CER and -E6 mice within 3 weeks. Irradiation with UVA alone did not induce papillomatosis and UVB alone had a weaker effect than UVA/UVB, indicating a synergistic role of UVA in UVB-induced papillomatosis. An HPV8 infection persisting over decades in interaction with sun burns and wound healing processes may be a relevant cause of skin cancer in humans.

The long control region (LCR) and the E2 protein of human papillomaviruses (HPV) are the most important viral factors regulating transcription of the viral oncogenes E6 and E7. Sequence variation within these genomic regions may have an impact on the oncogenic potential of the virus. Sequence variation in the LCR and in the E2 gene of human papillomavirus type 16 (HPV-16) isolates originating from cervical cancer patients from East Hungary was studied. In 30 samples, sequencing and/or single-strand conformation polymorphism analysis revealed variants belonging to the European variant lineage of HPV-16. These variants differed from the reference European clone only slightly in their E2 and LCR sequences. Three samples represented variants belonging to the Asian-American group. These differed from the published reference sequence at several positions in the LCR and E2 regions. Compared to the reference clone, the LCR clones of the European isolates showed very similar transcriptional activities, while that of an Asian-American isolate had approximately 1.7-fold increased activity. Most of the increased activity of the Asian-American isolate could be ascribed to nucleotide changes found at the 3' end of the LCR (nt 7660-7890). The transcriptional transactivation potentials of the HPV-16 E2 isolates differed only slightly from each other, and the differences seemed to be independent of the taxonomic position of the isolates.

Evidence of the functional significance of two naturally occurring mutations at nt 40 or 41 in the Sp1 motif in the promoter proximal segment of the upstream regulatory region (URR) of human papillomavirus (HPV) type 18 is presented. In electrophoretic mobility shift assays, Sp1 protein bound more efficiently to the Sp1 mutant motifs than to the prototype; while in both HeLa and HT3 cells, luciferase activity controlled by the mutant URRs was upregulated 2- and 3-fold, or 4- and 6-fold, in comparison with the prototype URR or HeLa cell-derived URR respectively. The HeLa URR represents a more appropriate baseline for promoter activity, containing a series of point mutations representative of most HPV-18 cancer isolates, including one in the Yin Yang 1 (YY1) site at the P105 promoter. The effect of the Sp1 mutations was found to be largely maintained in the context of the HeLa URR containing the prototype YY1 site.

Human papillomavirus (HPV) DNA, originally isolated from patients suffering from the skin disease epidermodysplasia verruciformis (EV), and a growing number of related sequences have recently been detected in a high percentage of benign and malignant skin lesions of both immunosuppressed and immunocompetent people. HPV L1 DNA fragments (374-389 bp long) from a solar keratosis and a squamous cell carcinoma (SCC) of a renal transplant recipient were amplified, cloned and sequenced. In 54 clones, six different HPV sequences were identified. One of these six corresponded to the known type HPV-8 and two (RTRX3 and RTRX7) have been described previously in cutaneous lesions of immunosuppressed patients. The remaining three sequences were different from all known HPV types: an HPV-9-related sequence (77.4% identity), an RTRX2-related sequence (82.6% identity), and an HPV-22-related sequence (83.7% identity). These three sequences, representing putatively new HPV types, were named RTRX8, RTRX9 and RTRX10, respectively. RTRX7 was found in the majority of clones from both lesions. The complete genome of RTRX7 (7731 bp) was cloned as six overlapping subgenomic fragments, generated by nested PCR with DNA extracts from the SCC. RTRX7 showed a genome organization typical of HPVs associated with EV. The L1 DNA sequence differed by 15% from the corresponding region of its closest known relative, HPV-12; thus, RTRX7 can be regarded as a new HPV type. RTRX7 DNA could not be detected by Southern blot hybridization with the homologous probe, indicating that the DNA concentration was below one copy per 10 cells in the investigated SCC.

The non-coding region (NCR) of the epidermodysplasia verruciformis (EV)-associated human papillomavirus 8 (HPV-8) has been investigated for sequence-specific DNA-protein interactions with nuclear proteins from epithelial HeLa cells. Using DNase I protection analysis 18 footprints could be found within the HPV-8 NCR, covering altogether over 60% of both DNA strands. Several footprints coincided with the known binding sites of transcription factors (NF1, AP1, octamer and PEA3 consensus sequences); the other displayed no obvious similarities in this regard. The overall distribution of sequences involved in DNA-protein interactions differed clearly from the binding patterns reported for other HPVs. Parts of the two binding sites for the viral trans-activator protein E2 were shown to interact with non-E2 factors. The EV-specific NCR motifs M33, M29 and A/T box were all involved in protein binding. By comparing the footprints within the respective motifs of the closely related types HPV-8 and -19, quantitative and qualitative differences were detected for M33 and the A/T box.

Human papillomavirus type 8 (HPV-8) is one aetiological agent of macular and flat wart-like lesions in patients with epidermodysplasia verruciformis and appears to be closely linked to skin carcinogenesis. A 1.2 kb region of the genome, which was previously shown to contain a viral E2-dependent enhancer, was progressively shortened from both ends with Bal 31. The resulting fragments were tested for their ability to stimulate chloramphenicol acetyltransferase (CAT) expression from the simian virus 40 (SV 40) promoter. This analysis showed a complex interaction between cis-active, positive and negative control elements located throughout the non-coding region and the flanking reading frames. Two separate positively acting sequences significantly stimulated expression only in cooperation with a third region, which led to 12-fold, E2-dependent enhancement on its own. A major negative element was not only active in the context of HPV-8 sequences, but also down-regulated SV40 enhancer-promoter-driven CAT expression when cloned downstream of the transcription unit. It acted at the transcriptional levels as shown by RNase protection assays and can therefore be regarded as a cis-acting silencer of transcription.

Purified lipopolysaccharide (LPS) from the bacteriocin sensitive strain Rhizobium lupini i6-2 was shown to neutralize the killing activity of the bacteriocin. In the electron microscopical preparation the phage tail-like bacteriocin appears to be adsorbed to the LPS; the tail sheath is contracted and the fibres are oriented towards the LPS ribbon. In contrast, no interaction was observed between the bacteriocin and the LPS of two resistant strains of Rhizobium (16-2/Ii and 16-3). The inactivation of the bacteriocin by LPS depends on salt concentration, pH, and temperature. The receptor activity of LPS was destroyed by mild acid hydrolysis and by treatment with deoxycholate, which indicates that the micellar structure of the LPS is necessary for bacteriocin adsorption. The chemical composition of the 16-2 LPS was compared to that of the LPS of two resistant strains. In the case of 16-2/ii LPS minor modifications suffice to confer resistance against the bacteriocin.