Journal of General Virology (2005), 86, 2029–2033
Short
Communication
DOI 10.1099/vir.0.80874-0
Isolation and cloning of the raccoon
(Procyon lotor) papillomavirus type 1 by using
degenerate papillomavirus-specific primers
Annabel Rector,1 Koenraad Van Doorslaer,1 Mads Bertelsen,2,33
Ian K. Barker,2 Rolf-Arne Olberg,2,34 Philippe Lemey,1 John P. Sundberg4
and Marc Van Ranst1
1
Laboratory of Clinical and Epidemiological Virology, Rega Institute for Medical Research,
University of Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
Correspondence
Marc Van Ranst
[email protected]
2
Department of Pathobiology, Ontario Veterinary College, University of Guelph, Ontario N1G
2W1, Canada
3
Toronto Zoo, Ontario M1B 5K7, Canada
4
The Jackson Laboratory, Bar Harbor, ME 04609-1500, USA
Received 3 January 2005
Accepted 18 March 2005
Partial sequences of a novel papillomavirus were amplified from a cutaneous lesion biopsy of a
raccoon (Procyon lotor), by using PCR with degenerate papillomavirus-specific primers. The
Procyon lotor papillomavirus type 1 (PlPV-1) DNA was amplified with long template PCR in two
overlapping fragments, together encompassing the entire genome, and the complete PlPV-1
genomic sequence was determined. The PlPV-1 genome consists of 8170 bp, and contains the
typical papillomaviral open reading frames, encoding five early proteins and two late capsid
proteins. Besides the classical non-coding region (NCR1) between the end of L1 and the
start of E6, PlPV-1 contains an additional non-coding region (NCR2) of 1065 bp between the
early and late protein region, which has previously also been described for the canine oral
papillomavirus (COPV) and the Felis domesticus papillomavirus (FdPV-1). Phylogenetic
analysis places PlPV-1 together with COPV and FdPV-1 in a monophyletic branch which
encompasses the Lambda papillomavirus genus.
The Papillomaviridae are a large family of species-specific
viruses that cause proliferations of the stratified squamous
epithelium of the skin or the mucosa in a wide variety of
host species (Sundberg et al., 2001). Over 90 human papillomavirus (HPV) types have been completely sequenced
(de Villiers et al., 2004), and these cause a wide spectrum
of genotype-specific lesions (Van Ranst et al., 1992). Only
a limited number of non-human PV genotypes have been
fully genetically characterized so far (Sundberg et al., 1997,
2001; de Villiers et al., 2004), but these cover a broad range
of host species, and it is likely that most mammalian and
avian species carry their own set of species-specific PV types.
Papillomatosis has been documented in a number of
carnivores, mainly Canidae and Felidae (Sundberg, 1987;
Sundberg et al., 2000). In 1994, the genomic sequence of
3Present address: Copenhagen Zoo, Copenhagen, Denmark.
4Present address: Kristiansand Zoo, Kristiansand, Norway.
The GenBank/EMBL/DDBJ accession number of the sequence
reported in this paper is AY763115.
Supplementary material is available in JGV Online.
0008-0874 G 2005 SGM
the canine oral PV (COPV), associated with oropharyngeal
papillomatosis in dogs, coyotes and wolves, was characterized (Delius et al., 1994). COPV is the largest of all known
PV genomes, and contains a unique second non-coding
region (NCR2) between the early and late protein region.
Recently, a second carnivore PV genome, containing a similar NCR2, was isolated from a cutaneous lesion of a Persian
cat (Felis domesticus PV type 1, FdPV-1) (Tachezy et al.,
2002a). COPV and FdPV-1 share a high degree of sequence
similarity, and belong to the genus Lambda PV.
Papillomavirus infection has been reported in raccoons
(Procyon lotor), small carnivores that are widely distributed
throughout North America. In one study, 2 of 53 wild
trapped raccoons showed proliferative skin lesions that
stained positive for PV group-specific antigens (Hamir et al.,
1995). Since dogs and raccoons both belong to the suborder Caniformia of the Carnivora, investigation of the
relationship between the canine COPV and the raccoon PV
would be interesting to test the hypothesis that PVs have
co-evolved with their host species.
We report here the complete genomic sequence and
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A. Rector and others
phylogenetic position of the first raccoon PV (Procyon lotor
papillomavirus type 1, PlPV-1), isolated from papillomatous skin lesions of an adult free-ranging raccoon that
was trapped and euthanized at the Toronto Zoo (Ontario,
Canada). This animal showed numerous small dermal
proliferations on the palmar surface of both front feet.
Histological examination of these skin lesions showed
numerous small exophytic epidermal nodules, characterized
by epithelial dysplasia, hyperplasia and orthokeratotic
hyperkeratosis. Koilocytes, with ballooning degeneration
and amphophilic intranuclear inclusion bodies, were
present in the stratum granulosum. Total genomic DNA
was extracted from a lesion biopsy as described previously
(Tachezy et al., 2002a), and amplicons suggestive of
PV-specific amplification were generated by PCR using
degenerate PV-specific primers (Supplementary material in
JGV Online). The PCR products were sequenced with the
same primers as used for PCR. Similarity searches, performed with the National Center for Biotechnology Information (NCBI) BLAST (Basic local alignment search tool)
server on GenBank DNA database release 132.0 (Altschul
et al., 1990), showed that partial L1 and E1 sequences of a
novel PV were amplified. Primers for long template PCR
were chosen in these partial E1 and L1 sequences in order
to amplify the complete genome of the raccoon PlPV-1 in
two overlapping long PCR fragments: RAC1 of approximately 3?7 kb, amplified with forward and reverse primers
RAC-longF1 (59-TATGGGCTGTTCGTGGTTTAGAGATTGGTCGTGG-39) and RAC-longR1 (59-CTGCGAGTGGCTGCAACCAGAACTGACTCTTGC-39), and fragment
RAC2 of approximately 6?2 kb, amplified with primers
RAC-longF2 (59-GGCTAGAGCTGAGGCAGGTAAGACACTGTTGC-39) and RAC-longR2 (59-CTACATGTCTCATGTAGCACCTATAGTCTGCAGGG-39). Long template
PCR was performed with the Expand Long Template PCR
system (Roche Diagnostics). The RAC1 and RAC2 PCR
products were purified on a 0?8 % agarose gel with crystal
violet staining, and isolated from the gel by using SNAP
purification columns (TOPO XL PCR Cloning kit; Invitrogen). The fragments were ligated into a pCR-XL-TOPO
vector, followed by transformation into One Shot TOP10
competent cells (TOPO XL PCR Cloning Kit; Invitrogen).
The bacteria were selectively grown on Luria–Broth agar
plates containing 50 mg kanamycin ml21, and one clone
containing the 3?7 kb RAC1 PCR fragment and one containing the 6?2 kb PCR fragment were selected. The complete nucleotide sequences of the cloned RAC1 and RAC2
PCR products were determined by primer-walking sequencing, starting from the universal primers in the pCRXL-TOPO vector, and using 32 primers to cover the two
fragments on both strands. Sequencing was performed on an
ABI Prism 3100 Genetic Analyser (Perkin-Elmer Applied
Biosystems), chromatogram sequencing files were inspected
with Chromas 2.2 (Technelysium), and contigs were prepared using SeqMan II (DNASTAR). The complete nucleotide
sequence of the PlPV-1 genome consists of 8170 bp, and was
deposited in GenBank under the accession no. AY763115.
PlPV-1 has the fourth largest PV genome, after COPV
(8607 bp; GenBank accession no. NC_001619), deer DPV
(8374 bp; NC_001523) and FdPV-1 (8300 bp; AF480454),
all of which contain two non-coding regions (Delius et al.,
1994; Groff & Lancaster, 1985; Tachezy et al., 2002a). PlPV-1
contains the seven classical PV major ORFs, encoding five
early (E) proteins E1, E2, E4, E6 and E7, and two late (L)
capsid proteins L1 and L2. The exact location of the PlPV-1
ORFs and the molecular mass of the predicted proteins
are indicated in Fig. 1. The position of the first nucleotide of
the PlPV-1 genome was fixed corresponding to the start of
the first major ORF in the early region.
Pairwise nucleotide and amino acid sequence alignments
of the different ORFs and their proteins were performed
with the GAP-program on the Sequence Analysis Server at
Michigan Technological University (http://genome.cs.
mtu.edu/align/align.html), to determine the degree of
similarity of PlPV-1 to the feline FdPV-1, the canine
COPV, the prototype benign cutaneous HPV-1a (GenBank
accession no. NC_001356), the mucosal high-risk HPV-16
(NC_001526) and the bovine fibropapillomavirus BPV-1
(X02346) (Table 1). The PlPV-1 sequence is most similar
to the other PVs from carnivores, COPV and FdPV-1. The
L1 ORF is the most conserved region among PV types, and
PVs that share between 60 and 70 % nucleotide identity in
this region are defined as different species of the same genus.
Since PlPV-1 shares 68 % nucleotide identity with COPV
Fig. 1. Linear representation of the ORFs of the PlPV-1 genome (with the molecular mass in kDa of the predicted proteins
shown in parentheses). Numbers show the nucleotide positions of the start and stop codons. URR, Upstream regulatory
region or non-coding region (NCR1). NCR2, Second non-coding region.
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Journal of General Virology 86
Procyon lotor papillomavirus (PlPV-1)
Table 1. Percentage nucleotide (amino acid) similarity of the
different PlPV-1 ORFs with the ORFs of COPV (GenBank
accession no. NC_001619), FdPV-1 (AF480454), HPV-1a
(NC_001356),
HPV-16
(NC_001526)
and
BPV-1
(X02346)
PlPV-1 ORF
COPV
FdPV-1
HPV-1a
HPV-16
BPV-1
E6
E7
E1
E2
E4
L2
L1
45
59
65
53
36
57
68
53
61
65
54
33
58
63
44
42
58
46
37
49
58
27
37
49
42
26
24
46
31
(39)
(53)
(60)
(45)
(25)
(54)
(71)
(46)
(49)
(63)
(51)
(21)
(57)
(67)
(37)
(35)
(53)
(40)
(14)
(44)
(57)
(22)
(32)
(44)
(34)
NA (NA)
39 (32)
52 (49)
(25)
(20)
(39)
(24)
NA (NA)
38 (24)
55 (47)
NA,
Insufficient similarity between the two sequences to allow
unambiguous alignment.
and 63 % with FdPV-1, it can be classified as the third
species of the genus Lambda.
The putative PlPV-1 E6 protein contains four C-X-X-C
motifs, involved in zinc-binding, whereas the E7 contains
two such motifs. The PlPV-1 E7 also contains the conserved retinoblastoma tumour suppressor binding domain
(DLYCDEHMPSDEEE). The E1 encodes the largest PlPV-1
protein (600 aa), and contains the conserved ATP-binding
site for the ATP-dependent helicase (GPPNTGKS) in its
carboxy-terminal part. In the E2, a slightly modified leucine zipper domain (F-X6-L-X6-L-X6-L) is present, in which
the first leucine residue is replaced by a phenylalanine. Since
these are both neutral and hydrophobic amino acids, this
modification will probably only result in limited structural
changes. The E4 ORF is completely contained within E2, and
in PlPV-1 an E4 start codon was identified, which is not
the case in most other PVs. A high proline content, which is
typical for the E4 ORF, was noted (19 proline residues of
117 aa). The late region contains the major (L1) and minor
(L2) capsid protein genes. Both L1 and L2 contain a series
of arginine and lysine residues at their carboxy terminus,
likely to function as a nuclear localization signal.
The classic non-coding region (NCR1) between the stop
codon of L1 and the start codon of E6 consists of 494 bp in
PlPV-1, from nt 7797 to 120. PVs usually contain an E1
recognition site (E1BS) flanked by two E2-binding sites, for
binding of an E1/E2 complex in order to activate the origin
of replication. In the PlPV-1 NCR1, an E1BS (TTATTGTTGTTAACAAT) is present at nt 10–26. Three typical
E2-binding sites (E2BS) with the consensus sequence
ACC-N6-GGT are present at nt 7972–7983, 8093–8104
and 55–66. Three additional modified E2-binding sites
(E2BS*), one with the sequence AAC-N6-GGT at nt 73–84,
and two with the sequence ACC-N6-GCT, at nt 8147–8158
and 8011–8022, could be identified through comparison of
the NCR1 regions of PlPV-1, COPV and FdPV-1. Since the
putative E2BS* at position 8147–8158 and the E2BS at 55–66
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are equidistant to the E1BS, this E2BS* might be functionally important, although it is possible that the modifications could result in lower affinity binding. In its 59 end, the
NCR1 also contains a polyadenylation site (AATAAA, nt
7889–7894), 20 bp upstream of a CA dinucleotide, and the
G/T cluster, necessary for the processing of the L1 and L2
capsid mRNA transcript. In the 39 end, NCR1 contains a
TATA box of the E6 promoter present at nt 43.
The PlPV-1 genome contains a second non-coding region
(NCR2) of 1065 bp (nt 3666–4730) between the early and
the late protein region. A similar NCR2 has previously only
been characterized in COPV (Delius et al., 1994) and FdPV1 (Tachezy et al., 2002a), both also belonging to the genus
Lambda, and DPV, which belongs to the genus Delta (Groff
& Lancaster, 1985). It is tempting to speculate that the
presence of an NCR2 is a common feature of all Lambda
PVs, and that the NCR2 of all three Lambda PVs could
originate from a DNA sequence (of unknown function and
origin) that was incorporated in their common ancestor.
Since this is a non-protein-coding region, numerous point
mutations, insertions and deletions have accumulated in
this sequence during evolution, and a possible common
origin is no longer recognizable above this background.
A BLAST search with the PlPV-1 NCR2 failed to detect
similarity with the NCR2 of FdPV-1 and COPV, or any
other known sequences in GenBank. In contrast to the
NCR1, the NCR2 contains no recognizable E1BS and E2BS.
There is no classical polyadenylation site for processing
of the early viral mRNA transcripts present in this NCR2,
but several degenerate polyadenylation sites, followed at an
appropriate distance of about 20 bp by a CA dinucleotide,
were detected.
A neighbour-joining phylogenetic tree was constructed,
based on a concatenated E1/E2/L2/L1 nucleotide sequence
alignment of PlPV-1 and 45 type species of the different
PV genera and species. Multiple nucleotide sequence alignments were performed at the amino acid level by using the
CLUSTALW program (Thompson et al., 1994) in the DAMBE
software package version 4.2.7 (Xia & Xie, 2001), after which
the nucleotide sequences were aligned according to the
aligned amino acid sequences. This was done separately for
the different ORFs, and the regions of the genome that
contained unambiguously alignable homologous positions
(four regions in E1, three in E2, four in L2 and seven in L1)
were pasted together in one compiled alignment of 2658 nt.
The resulting neighbour-joining phylogenetic tree (Fig. 2),
which was constructed in MEGA version 2.1 (Kumar et al.,
2001), clusters the PVs in the different genera described in
the new PV classification (de Villiers et al., 2004), and the
additional Rho and Sigma genera (Rector et al., 2004; Rector
et al., 2005). In this tree, PlPV-1 clusters with COPV and
FdPV-1 in the genus Lambda.
With a mutation rate of 0?73–1?261028 nucleotide substitutions per base per year (Van Ranst et al., 1995; Tachezy
et al., 2002a), PVs are very stable viruses that evolve through
slow accumulation of point mutations, and recombination
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A. Rector and others
Fig. 2. Neighbour-joining phylogenetic tree,
based on a concatenated E1/E2/L2/L1
nucleotide sequence alignment of PlPV-1
and 45 other PVs. The PV genera are indicated with their Greek symbols. The numbers at the internal nodes represent the
number of bootstrap probabilities, as determined for 1000 iterations by the neighbourjoining method. Only bootstrap values
greater than 85 % are shown. The scale bar
indicates the genetic distance (nucleotide
substitutions per site).
between different PV types has never been documented.
The observed global distribution of a broad genetic diversity of PVs, together with the viral species specificity, the
stability of their genomes, and the requirement for close
contact for PV transmission, has led to the hypothesis that
PVs are ancient viruses that have co-evolved and cospeciated with their host species during vertebrate evolution
(Van Ranst et al., 1995). In order for this hypothesis of cophylogenetic descent to hold, PVs of closely related host
species should be closely related themselves, with dating of
PV divergence largely coinciding with the host species
divergence. This has previously been confirmed for the
parrot PePV and the chaffinch FcPV (Tachezy et al., 2002b),
for PVs from Artiodactyla (even-toed ungulates), and for
the pygmy chimpanzee PCPV-1, the common chimpanzee
CCVP-1, and human HPV-13 (Van Ranst et al., 1995). The
raccoon PlPV-1 is most closely related to COPV and FdPV1, the only other PVs isolated from carnivores. The second
requirement, that the PVs evolved and speciated in synchrony with their hosts (Fahrenholz’s rule), was examined
by comparing the phylogeny of PlPV-1, COPV and FdPV-1
with the phylogeny of their carnivore host species.
Comparative genomics of the Carnivora indicate monophyly of the order, with an early split into two monophyletic suborders, the Feliformia and the Caniformia. Within
the Caniformia, there exists a well-supported dichotomy
between the canids and arctoids (including the mustelids,
ursids and procyonids) (Flynn & Nedbal, 1998; BinindaEmonds et al., 1999). Based on the co-evolution hypothesis,
2032
stating that the PV evolutionary tree should mirror the host
species phylogeny, it was expected that PlPV-1 would cluster with COPV in a monophyletic branch. A maximumlikelihood phylogenetic analysis of the Lambda PV dataset,
with HPV-1a as the outgroup, did not indicate this scenario
as the best tree, but it could not be statistically rejected
(data not shown). A more expanded dataset, including
novel Lambda PV sequences, could prove to be useful to
fully resolve the Lambda PV phylogeny. The recent understanding of parasite–host co-evolution, however, also argues
that a less strict interpretation of Fahrenholz’s rule is often
desirable, since most host and parasite phylogenies are only
imperfect mirrors. These imperfections could be caused by
host switching between lineages, but possible causes also
include the interplay between co-speciation and other
co-phylogenetic events, such as sorting (extinction, when
parasites are entirely or apparently removed from host
species), duplication (intrahost speciation), and inertia (lack
of parasite speciation) (Paterson & Banks, 2001). Also in the
case of carnivores and their PVs, these effects could be of
influence.
Acknowledgements
We would like to thank the colleagues of the laboratory of Clinical
& Epidemiological Virology, Department of Microbiology & Immunology, Rega Institute for Medical Research, University of Leuven,
Belgium, for helpful comments and discussion. This work was
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Journal of General Virology 86
Procyon lotor papillomavirus (PlPV-1)
supported by a fellowship of the University of Leuven (Leuven,
Belgium) to A. R. and by the Flemish Fonds voor Wetenschappelijk
Onderzoek (FWO-grant G.0288.01).
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