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Short Communication |
a Vasiljevi
1
1 Department of Medical Microbiology, Malmö University Hospital, Lund University, Sweden
2 Infectious Diseases Laboratories, Institute of Medical and Veterinary Science, Adelaide, Australia
3 Division for Tumorvirus Characterization, Deutsches Krebsforschungszentrum, Heidelberg, Germany
4 The DNA Tumorvirus Laboratory, Institute of Molecular Pathology, University of Copenhagen, Denmark
Correspondence
Ola Forslund
Ola.forslund{at}med.lu.se
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the complete genome sequences of HPV93 and HPV96 reported in this paper are AY382778 and AY382779, respectively.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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). HPV types infecting cutaneous tissue are the focus of investigation as possible carcinogenic agents of non-melanoma skin cancer (NMSC). The NMSCs basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) are the most frequently occurring malignancies in Caucasian populations (Kiviat, 1999). Exposure to UV radiation is the major risk factor for development of NMSC, but increased risk is also observed among those with immunosuppressive treatment and fair skin type (Alam & Ratner, 2001). Cutaneous HPV infection is associated with NMSC in patients with the rare, hereditary disease epidermodysplasia verruciformis (EV) (Majewski & Jablonska, 1995; Orth, 1986) and about 20 distinct HPV types are found in lesions of these patients (Pfister & Ter Schegget, 1997). These HPV types can also be detected in lesions of immunocompetent patients as well as in healthy skin, making it difficult to assess the pathological significance of these viruses (Antonsson et al., 2000). However, HPV has been proposed as a co-factor for UV radiation in the development of NMSC (Akgul et al., 2006; Harwood et al., 1999).
To date, over 100 HPV types have been characterized, but recently, a sensitive method, FAP PCR (Forslund et al., 1999), has detected about 130 additional putative cutaneous HPV types (Antonsson et al., 2000, 2003a, b; Antonsson & Hansson, 2002; Forslund et al., 1999, 2003b, 2004). Except for HPV92 (Forslund et al., 2003a), only subgenomic sequences are known of these putative HPV types. In this study, the complete genome sequences of two novel types, HPV93 and HPV96, were obtained by generating overlapping amplicons. HPV93 was described previously as FAIMVS6, originally identified in an actinic keratosis (AK) on the dorsum of the hand of an immunocompetent 82-year-old male (Forslund et al., 2003b). HPV96 was described previously as FA47 (Antonsson et al., 2003b) and was, in the current study, detected in a SCC in situ on the upper chest of an immunocompetent 75-year-old male (Forslund et al., 2003b).
Briefly, the overlapping amplicons of HPV93 were generated by using the following primer pairs: FAIMVS4.5.6F, 5'-ATATGTCTGTTTATAACCCGGAAA-3' (DNA Technology A/S), with EVE7, 5'-GTRRCYTSTTTHCCAATCAT-3' (with high identity to the 5' region of the E7 open reading frame (ORF) of the genus Betapapillomavirus), and IMVS6L1.579F, 5'-CATTCCTGGTGAACAAATAGAC-3', with IMVS6L1R, 5'-GCCTCTACAGGCCCAAACTAACC-3'. The overlapping amplicons of HPV96 were generated by using the following primer pairs: E1-1732F, 5'-CTTACTGACCAAAGCTGG-3' (with target site in the E1 ORF of the genus Betapapillomavirus), with FA47.114R, 5'-AGGATTTACCACTGACATGTC-3', and FA47.3339F, 5'-GGGCCTTTAGGGTACACTTACCGG-3', with FA47.82R, 5'-CTCCCCCTCGTCTTCTTGGTCAC-3'. The PCRs, cloning and sequencing of both types were performed as described previously (Forslund et al., 2003a).
Immunoprecipitation was performed to analyse the retinoblastoma protein (pRb)-binding ability of E7 of HPV93 and HPV96, as well as the recently described type HPV92 (Forslund et al., 2003a), which is the only representative of species 4. The E7 genes of HPV16 (positive control), 92, 93 and 96 were immunotagged with FLAG at the 3' end and inserted into pcDNA 3.1 (Invitrogen). U2OS H4tet Vp16 cells (an osteosarcoma cell line cultured in Dulbecco’s modified Eagle’s medium, 10 % fetal bovine serum, 2 mM L-glutamine and 1 % penicillin–streptomycin) in 10 cm dishes (3x106 cells per dish) were transfected with 8 µg of each vector and Lipofectamine plus (Invitrogen). Forty-eight hours post-transfection, cells were washed once with buffer A (10 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 340 mM sucrose, 10 % glycerol, 0.1 % Triton X-100, protease and phosphatase inhibitors, approx. pH 7.9), twice with PBS and then incubated on ice in buffer A. Cells were dispersed by centrifugation at 20 000 g and the proteins were isolated with GammaBind Plus Sepharose (Amersham Biosciences). For analysis of proteins in the total lysate, 50 µl supernatant was removed. The lysate (800 µl) was immunoprecipitated with 40 µl pre-washed ANTI-FLAG M2 agarose affinity beads and FLAG peptide. The samples were separated by SDS-PAGE (12 % gels) and blotted onto a Hybond nylon membrane (Amersham Biosciences). E7 and pRb were detected by using ANTI-FLAG M2 and monoclonal anti-pRb 1F8 (a gift from Professor J. Lukas at The Danish Cancer Society, Copenhagen, Denmark), respectively. The protein bands were visualized by using the ECL (enhanced chemiluminescence) system (Amersham Biosciences).
An additional aim of the study was to investigate the prevalence and viral load of HPV92, 93 and 96 in skin biopsies collected from 269 immunocompetent patients attending Swedish hospitals. The sample series included AK (n=52), seborrhoeic keratosis (SK; n=47), BCC (n=118) and SCC (n=52). After stripping (Forslund et al., 2004), a biopsy was taken from the lesion and from the adjacent healthy skin of the same patient; hence, in total, 538 samples were included. The mean age of the patients was 77±1 years and the sex distribution was 55 % men and 45 % women for BCC, AK and SK, and 58 % men and 42 % women for SCC. The DNA from each biopsy was extracted by using a phenol-free method (Forslund et al., 1999). All samples were run in real-time PCR with HPV type-specific primers and probes, designed using Primer Express 2.0 (Applied Biosystems). Calculations of viral copy numbers for standard curves were based on spectrophotometric measurement of purified viral DNA from plasmids containing the complete genomes of HPV92, 93 and 96, and standard curves were established as described previously (Hazard et al., 2006). For quantification of HPV92, 25 µl PCR mixture contained 2.5 µl template (patient sample diluted 1 : 2 in TE buffer), 1x GeneAmp PCR Buffer II (Roche), 0.2 mM each dNTP (Roche Diagnostics), 3.5 mM MgCl2 (Applied Biosystems), 0.2 µM each primer (HPV92F’, 5'-TCTGTTTATAATCCAGACAAGGAAAGG-3', and HPV92R’, 5'-GATGACCTGTGGTGCCAACAC-3'), 0.04 µM L1 probe (5'-FAM-ATTGGAAATAGGGCGAGGGCAGCC-TAMRA-3') and 0.625 U AmpliTaq Gold polymerase (Applied Biosystems). The 25 µl reaction for HPV93 differed in the concentration of MgCl2 (4 mM), the L1 probe (0.05 µM 5'-FAM-ACTGGGCATCCATTATTTAATAAGGTAAATGATACAGAAA-TAMRA-3'), the primers (0.3 µM each of HPV93F’, 5'-GTTTGCATTAGCTGATATGTCTGTTTATAA-3', and HPV93R’, 5'-GTTTTGTCTATCATCAGTAGAAAATGC-3') and the polymerase (1.25 U), whilst the 25 µl reaction for HPV96 differed only in the concentration of the E1 probe (0.2 µM 5'-FAM-TCTTACCATCCAAGCACAATCTCTAACAATTTTTGCTT-TAMRA-3') and the primers (0.3 µM each of HPV96F’, 5'-GCTCGCGCGTTTTTAGCT-3', and HPV96R’, 5'-TAGACATATATCTCATTTCACCTCGTTTGT-3'). The PCRs were run in a GeneAmp 5700 SDS (Applied Biosystems) with 2 min at 50 °C, 10 min at 95 °C and then 50 cycles (45 cycles for HPV96) of 15 s at 95 °C and 1 min at 60 °C (HPV92 and 96) or 55 °C (HPV93). A sample positive in at least two of three PCR runs was considered positive, but was also confirmed by sequencing. Mean viral copy number and coefficient of variation were calculated for each sample. To determine the viral loads of HPV93 and 96, the quantity of the 
-globin gene was analysed with PCO3/PCO4 primers (Saiki et al., 1985) and SYBR green in PCR (Forslund et al., 2003a), and for samples positive for HPV92, the quantity of the -globin gene was measured with a probe and the number of cells was calculated as described previously (Hazard et al., 2006).
The complete genome of HPV93 (GenBank accession no. AY382778
[GenBank]
) comprised 7450 bp with a G+C content of 40 mol%. HPV93 was categorized phylogenetically into the genus Betapapillomavirus species 1 (de Villiers et al., 2004), and the L1 ORF showed highest identity to HPV type 24 (79 %).
The complete genome of HPV96 (GenBank accession no. AY382779
[GenBank]
) consisted of 7438 bp with a G+C content of 40 mol%. HPV96 represents the first HPV type within the genus Betapapillomavirus species 5 (de Villiers et al., 2004), with an L1 ORF showing highest identity to HPV type 92 (71 %). Notably, HPV96 was detectable in both perilesional skin and SCC, but the complete genome could only be obtained from perilesional skin, indicating a lower viral load in the tumour.
The genome organization of HPV93 and 96, with seven ORFs and lack of the E5 ORF, resembled that of other HPV types in the same genus (Table 1). The upper regulatory regions (URRs), containing cis-responsive elements between the L1 and E6 ORFs that govern gene expression and replication, of HPV93 and 96 consisted of 397 and 399 bp, respectively, which is within the expected range for the genus Betapapillomavirus (Fuchs & Pfister, 1990). Within the URRs of both types, a TATA box (TATAA) was identified, as well as putative binding sites for transcription factors E2 (four sites in HPV93 and six sites in HPV96), NF-1 (four in HPV93 and seven in HPV93) and AP-1 (one in each HPV type), by the use of the SIGNAL SCAN software (Prestridge, 1991). The pattern of binding sites showed similarity to those of other HPV types within the same genus (O'Connor et al., 1995).
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). The E6 ORFs of both types display two translation initiation codons, of which the second was predicted to be the utilized start codon [Neural Network Promoter Predictor, version 2.2 (http://www.fruitfly.org/seq_tools/promoter.html)] (Table 1
). Putative polyadenylation signals (AATAAA) were identified in HPV93 at position 4167 for the early and at position 7200 for the late mRNA; in HPV96, the corresponding signals were at positions 4370 and 7419.
The E7 proteins of HPV93 and HPV96 contained one conserved zinc-binding domain and the consensus pRb-binding motif (LxCxE) (Radulescu et al., 1995). Downstream of the pRb-binding motif of E7, potential casein kinase II (CKII) phosphorylation sites (Marin et al., 1986) were observed at threonines (T) (HPV93 E7, LNCEEELPTEQDTEEE; HPV96 E7, LHCDEELTEEQSENLSESTVAE). Within the E1 protein, a putative phosphorylation site of a serine (S), conserved for all papillomaviruses (Lentz, 2002), was located at amino acid position 577 for HPV93 and at position 582 for HPV96.
In the immunoprecipation assay, the HPV16 E7 was identified as a 25 kDa protein and E7 of HPV92, 93 and 96 as approximately 14 kDa proteins (Fig. 1). Membranes incubated with pRb-specific antibodies showed that pRb was not pulled down in the absence of E7, but was pulled down together with E7 of all four HPV types (Fig. 1). pRb was visible in total cell lysates and was concentrated after immunoprecipitation with E7 (Fig. 1). This demonstrates that HPV92, 93 and 96 E7 possesses the ability to bind pRb. The pRb-binding ability for betapapillomavirus species 1 (represented by HPV93) and, for the first time, for HPVs of betapapillomavirus species 4 (HPV92) and betapapillomavirus species 5 (HPV96) is in agreement with that of betapapillomavirus species 1 and 2 (HPV5, 8, 20 and 38), mupapillomavirus species 1 (HPV1) and alphapapillomavirus species 2 (HPV10) (Caldeira et al., 2003; Schmitt et al., 1994; Yamashita et al., 1993). However, transformation properties of E7, as well as those of E6, of HPV92, 93 and 96 are eligible to be followed up by studies in human keratinocytes.
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. None of the investigated types were detected in SK. The low prevalences detected here are in contrast to previously reported data [18 % (7/38) for HPV92, 11 % (4/38) for HPV93 and 5 % (2/38) for HPV96] among lesions and healthy skin of Australian immunocompetent patients (Forslund et al., 2003b). Possible explanations for the higher prevalence include a smaller study population, higher levels of sun exposure and the fact that no stripping of the skin was performed before collection of biopsies. In the SCC sample with the double infection, the highest viral load was detected for HPV93 with one viral copy per 45 cells, and also the lowest viral load for HPV92 with one viral copy per 10 325 cells (Table 2). The same patient also had the only HPV-positive healthy skin biopsy, with a viral load for HPV93 of one copy per 2987 cells. These results were not unexpected, as low viral loads in tumours have also been reported by others (Bens et al., 1998; Meyer et al., 2001; Weissenborn et al., 2005). In a previous study (Weissenborn et al., 2005), AK appeared to harbour higher levels (one HPV copy per <50 cells) of virus than SCC (one HPV copy per <500 cells), but this was not confirmed in our study. In cervical cancers, there is typically at least one viral copy per cell (Munoz, 2000). In contrast, only low amounts of HPV are detected in NMSC and, so far, no HPV has been detected in cell lines established from cutaneous tumours (Proby et al., 2000; Purdie et al., 1993), which suggests that HPV is not required for maintenance of the malignant phenotype. Thus, it is tempting to speculate that cutaneous HPV might be involved in early steps of development of skin cancer by inhibiting apoptosis in response to UV radiation and by allowing proliferation of genetically unstable cells. The speculation is supported by the facts that E7 binds pRb and that the E6 protein affects the pro-apoptotic Bak protein and the XRCC1 protein (required for repair of DNA single-strand breaks and for genetic stability) (Iftner et al., 2002; Jackson & Storey, 2000).
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ACKNOWLEDGEMENTS |
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Received 1 November 2006;
accepted 5 January 2007.
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