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1 Laboratory of Clinical and Epidemiological Virology, Rega Institute for Medical Research, University of Leuven, Belgium
2 The James Graham Brown Cancer Center, University of Louisville, KY, USA
3 The Jackson Laboratory, Bar Harbor, ME, USA
Correspondence
Marc Van Ranst
marc.vanranst{at}uz.kuleuven.ac.be
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Present address: Department of Microbiology and Immunology, Albert Einstein Cancer Center, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA. 
The GenBank/EMBL/DDBJ accession number for the nucleotide sequence of the MmPV-1 genome is DQ269468.
GenBank accession numbers for all sequences used in this study are available in Supplementary Table S1 in JGV Online.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Sundberg, 1987; Tachezy et al., 2002b; Terai et al., 2002). In these animals, PVs cause a broad spectrum of genotype-specific lesions with proliferation of the stratified squamous epithelium of the skin or the mucosa (de Villiers et al., 2004; Van Ranst et al., 1992a). Carcinogenic PVs are directly responsible for virtually all cases of cervical squamous carcinoma, and an association between raised antibody titres against certain cutaneotropic PVs and non-melanotic proliferative diseases of the skin, particularly solar keratosis and squamous carcinoma, has been observed (Jenson et al., 2001). However, study of these tumorigenic PVs has been hampered by the species-specific nature of PVs and the absence of a good in vitro culture system and well-defined small animal in vivo model.
Papillomatosis has been described in several species within the order Rodentia such as hamsters, guinea pigs, laboratory rats, mice, gerbils and American porcupines (Rector et al., 2005; Sundberg, 1987). The only rodent PVs that have been completely characterized at the genomic level were isolated from an African multimammate rat (Mastomys natalensis; MnPV), a Syrian golden hamster (Mesocricetus auratus; HaOPV) and a north-American porcupine (Erethizon dorsatum; EdPV-1) (Iwasaki et al., 1997; Rector et al., 2005; Tan et al., 1994). Three papers report on the cloning and partial genetic characterization of the European harvest mouse (Micromys minutus) PV (O'Banion et al., 1988; Sundberg et al., 1988; Van Ranst et al., 1992b) and partial sequence data of this MmPV were submitted to GenBank with accession nos AH002399
[GenBank]
and X65200
[GenBank]
.
In this study we report the complete genomic sequence of MmPV and its analysis. It contains 7393 bp and shows the seven classical PV open reading frames (ORFs) and non-coding region (NCR). Phylogenetic analysis places MmPV with HaOPV in the genus Pipapillomavirus (de Villiers et al., 2004). The availability of the complete MmPV genome might provide researchers with an opportunity to study PV carcinogenesis in a homologous mouse model.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Cloning and sequencing of the MmPV genome.
The cloning of the MmPV genome was described earlier (O'Banion et al., 1988). Briefly, the phenol/chloroform-extracted DNA was digested with the single-cutting restriction enzyme EcoRI and cloned in a pUC18 vector. This vector was used to transform TB-1 cells. Hybridization with a bovine PV (BPV)-2 probe confirmed that a selected clone was papillomaviral-DNA positive. The first partial sequences were obtained with the M13 consensus primer set. Subsequent sequencing was performed using primer walking. Chromatogram sequencing files were inspected with Chromas 2.2 (Technelysium), and contigs were compiled using SeqMan II (DNASTAR).
DNA and protein sequence analysis.
The putative ORFs were predicted with the ORF Finder tool on the NCBI server of the National Institutes of Health (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). The molecular mass of the putative proteins was calculated using the ExPASy Compute pI/Mw tool (http://www.expasy.org/tools/pi_tool.html). Multiple nucleotide sequence alignments were constructed in DAMBE version 4.2.7 (Xia & Xie, 2001). The sequences of MmPV and 55 non-human animal and human PVs were first aligned at the amino acid level using CLUSTAL W (Thompson et al., 1994). This alignment was then used as a template to align the nucleotide sequences. The alignment was corrected manually in GeneDoc version 2.6.002 (http://www.psc.edu/biomed/genedoc). The unambiguously alignable regions of the E1, E2, L2 and L1 ORFs were pasted together in one concatenated alignment which was used to construct a neighbour-joining phylogenetic tree in MEGA version 2.1 (Kumar et al., 2001).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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shows a diagram of the MmPV genomic organization and exact locations of the ORFs. With all ORFs located on the same strand, the genomic organization is comparable to all known PVs.
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The E1 ORF encodes the largest MmPV protein (604 aa), which contains the conserved ATP-binding site for the ATP-dependent helicase (GPPDTGKS) in its carboxy-terminal part (Titolo et al., 1999). Although a leucine-zipper domain is more commonly found in mucosal ‘low risk’ HPVs, the E2 protein of MmPV contains a leucine-zipper domain (L-X6-L-X6-L-X6-L). We also identified a leucine-zipper domain in the E2 protein of the mucosal HaOPV, supporting the phylogenetic relationship between the two viruses. In HaOPV, the second leucine residue is replaced by a valine. Because both leucine and valine have hydrophobic side groups, this substitution should not pose any problems for the functionality of the leucine-zipper domain. Completely contained within the E2 gene, but read in another frame, we identified a putative E4 ORF with the typical high proline content (15 proline residues out of 121 aa). MmPV does not contain an identifiable E5 or E8 ORF.
The late region encodes the major (L1) and minor (L2) capsid protein genes. Both L1 and L2 contain a series of arginine and lysine residues at their carboxy-terminal end, which are likely to function as nuclear localization signals.
The classic NCR between the stop codon of L1 and the start codon of E6 contains 557 bp (nt 6846–3). The NCR contains several regulators of the papillomavirus replication; this NCR usually contains an E1 recognition site flanked by two E2-binding sites. This conformation allows for the binding of an E1/E2 complex in order to activate the origin of replication. An E1-binding site (E1BS, TGATTGTTGCCAACTAT) is present at nt 7328–7344. The E1BS is flanked by an E2-binding site (E2BS) at nt 7294–7305 (ACCG-N4-CGGT) and a putative modified E2-binding site (E2BS*) at nt 7373–7384 (GACG-N4-CGGT). Since the E2BS and the E2BS* are equidistant from the E1BS (22 and 28 bp, respectively), both sites are probably functionally important. Two more E2BS* are located at position 7142–7153 (AACG-N4-CGGT) and 7220–7231 (ACCT-N4-CGGT). At its 5' end, the NCR also contains a polyadenylation site (TATAAAA, nt 7389–2), upstream of a CA dinucleotide (nt 7357) and the G/T cluster, necessary for the processing of the L1 and L2 capsid mRNA transcript (Birnstiel et al., 1985). In the 3' end, a TATA box of the E6 promoter is present at nt 6894–6908 (TGCTGCTATATATAT). The NCR also contains a tandem repeat of 14 bp (CTATGTACTGTGAA) separated by 11 bp (nt 6945–6983). The putative function of this repeat is unknown, but might play a role in replication.
Sequence similarity to other papillomaviruses
Table 1 shows the pairwise sequence similarity between MmPV and the other rodent PVs (HaOPV, EdPV-1 and MnPV), the benign cutaneous human papillomavirus (HPV)-1a, HPV-4 and HPV-5, representing the gamma- and betapapillomavirus genera, respectively, and the prototype high-risk mucosal HPV-16. Since a probe based on the BPV-2 genome was used to confirm the presence of PV DNA (O'Banion et al., 1988), BPV-2 was also included (Table 1). The L1 sequences of MmPV and HaOPV share 77.4 % sequence similarity at the nucleotide level. According to the recently published definitions, this places MmPV together with HaOPV in species 1 of the genus Pipapillomavirus (de Villiers et al., 2004). Interestingly, the L1 ORF of MmPV and HPV-4 share 61 % nucleotide similarity. This prompted us to compare the L1 ORF of HaOPV, MmPV’s closest relative (Fig. 2), to that of HPV-4. Our analysis showed 62 and 63 % similarity at the nucleotide and amino acid level, respectively. This suggests that, according to our analysis, HaOPV and MmPV should be classified in species 6 of the genus Gammapapillomavirus. However, because MmPV and HaOPV infect rodents, while the members of the genus Gammapapillomavirus infect humans, we believe that the current classification into Pipapillomavirus and Gammapapillomavirus should be maintained.
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). For this, nucleotide sequence alignments, based on the corresponding amino acid alignments, were constructed separately for the different ORFs. Regions where an unambiguous alignment could be obtained were included in one combined alignment of 2097 nt. The resulting neighbour-joining phylogenetic tree clusters the PVs in the different genera, described in the new classification of PVs (de Villiers et al., 2004), the additional rho and sigma genera, (Rector et al., 2004, 2005), and an additional, as yet unassigned genus which holds the recently characterized Capra hircus papillomavirus (ChPV-1) (Van Doorslaer et al., 2006). This phylogenetic tree clusters MmPV together with another rodent PV, isolated from a Syrian golden hamster (Mesocricetus auratus), another member of the suborder Sciurognathi (Iwasaki et al., 1997) in the genus Pipapillomavirus. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Van Ranst et al., 1995), the need for direct contact for transmission and the worldwide distribution of PVs in mammals and birds, co-evolution and co-speciation of PVs with their respective host species was suggested (Sundberg et al., 1997). In order for this hypothesis to hold, PVs of closely related host species should be closely related themselves and cluster together in the PV phylogenetic tree, with dating of PV divergence coinciding with the host-species divergence (Fahrenholz’s rule; Hafner & Nadler, 1988). In the PV phylogenetic tree, MmPV clusters with HaOPV in the genus Pipapillomavirus. Both hosts Micromys minutus and Mesocricetus auratus are members of the family Muridae, in the suborder Sciurognathi. Under the assumption of co-phylogenetic descent, we used a 2097 nt concatenated alignment (phylogenetic analysis) and the published PV mutation rate of 0.73 to 1.20x10–8 (Tachezy et al., 2002a) to calculate that MmPV and HaOPV diverged about 16–23 million years (MYR) ago. This coincides with what has been reported as the divergence time of the rodent hosts (20.6 to 26.9 MYR ago) (Steppan et al., 2004).
Although the first prophylactic species- and type-specific (polyvalent) vaccines are used successfully to protect several non-human animals against PV infections (Lin et al., 1992; Pilacinski et al., 1986; Suzich et al., 1995) and the commercialization of an HPV vaccine is being prepared (Shaw, 2005), there is still a long road to travel before human and animal PV infections are eradicated (Franco & Harper, 2005). A number of non-human animal models played a ground-breaking role in our understanding of papillomaviral disease (Campo, 2002). Although animal models, such as the BPV, the cottontail rabbit PV, the canine PV and the rhesus monkey PV, have been used with promising results as homologous test systems (Campo, 2002; Harvey et al., 1998; Jahan-Parwar et al., 2003; Peh et al., 2002), the experimental use of these animals has its own specific problems (e.g. cost, technical support and ethical questions).
The development of a successful therapeutic vaccine against cervical cancer depends on understanding the pathogenesis of cervical cancer. This research has been hampered by the lack of an accessible animal model for PV infections. The difficulty in creating a good animal model lies in the species-specific nature of PVs. Rodents or other non-human mammals can therefore not be readily infected with an HPV type. This problem has been circumvented by using heterologous test systems. Most heterologous test systems use fast-growing transplantable tumours, e.g. the use of BPV-2 to induce cutaneous fibrosarcomas in hamsters (Moar et al., 1981). The malignancies observed in human cervical cancer patients usually have a much slower progression, which might lead to immune suppression and/or immune tolerance. Heterologous test systems suffer the extra drawback of not using a host species’ specific own PV, which makes extrapolation to other animals (including humans) even harder. So far, no PVs have been isolated from inbred laboratory mice. The availability of a mouse model might prove essential to our understanding of PV pathogenesis. Not only are mice the most commonly used laboratory animals, but extensive research materials and immunological and molecular biological data related to mice are also available. With the availability of the complete genomic sequence of this wild mouse species PV, it should become possible for researchers to use this virus in a homologous test system, thus speeding up the development of both vaccines and antiviral drugs. Although MmPV causes cancer in its natural host, thus making it a good target for a model system, its natural host might prove to be a problem. Micromys minutus are wild rodents, which require special permission from the US Department of Agriculture (USDA) to be used in the laboratory in the US. Even with this special permission, these rodents will not breed outside their natural habitat. A transgenic mouse model which expresses the MmPV oncogenes in their epidermis should mimic what happens upon regular infection with MmPV, thus allowing us to exploit the well-studied mouse genetics and immunology to study the molecular pathways leading to squamous cell carcinoma.
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ACKNOWLEDGEMENTS |
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Received 2 October 2006;
accepted 20 December 2006.
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