Download Felis domesticus papillomavirus, isolated from a skin lesion, is

Journal
of General Virology (2002), 83, 2303–2307. Printed in Great Britain
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SHORT COMMUNICATION
Felis domesticus papillomavirus, isolated from a skin lesion, is
related to canine oral papillomavirus and contains a 1n3 kb
non-coding region between the E2 and L2 open reading
frames
Masanori Terai1 and Robert D. Burk1, 2
1
Department of Microbiology & Immunology1, and Departments of Pediatrics, Obstetrics & Gynecology and Women’s Health, and
Epidemiology & Social Medicine2, Comprehensive Cancer Center, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx,
NY 10461, USA
We have characterized the complete genome (8300
bp) of an isolate of Felis domesticus papillomavirus
(FdPV) from a domestic cat with cutaneous papillomatosis. A BLAST homology search using the
nucleotide sequence of the L1 open reading frame
demonstrated that the FdPV genome was most
closely related to canine oral papillomavirus
(COPV). A 384 bp non-coding region (NCR) was
found between the end of L1 and the beginning of
E6, and a 1n3 kbp NCR was located between the end
of E2 and the beginning of L2. Phylogenetic
analysis placed FdPV in the E3 clade with COPV.
Both viruses contain the atypical second NCR, which
has no homology with sequences in existing databases.
Papillomaviruses (PVs) are a heterogeneous group of highly
species-specific DNA viruses with closed-circular doublestranded DNA genomes about 8 kbp in size. PVs cause benign
and malignant proliferative lesions of squamous and mucosal
epithelial surfaces in a wide range of animal species (Sundberg
et al., 1996). Many domestic and native species of mammals can
be infected by one or more species-specific PVs (Sundberg et
al., 1996).
To understand better the evolution and genetic determinants of PV pathogenicity, comparative molecular analysis
of a wide variety of PV genomes is required. PVs have been
detected in a variety of Felidae, including domestic cats (for
review, see Sundberg et al., 2000). Domestic cats have been in
close proximity to humans for centuries. Thus, they present an
opportunity for horizontal transmission. To explore the origins
Author for correspondence : Robert Burk.
Fax j1 718 430 8975. e-mail burk!aecom.yu.edu
The complete nucleotide sequence of FdPV is available in GenBank,
accession number AF377865.
0001-8530 # 2002 SGM
and molecular characteristics of the cutaneous papillomavirus
that infects domestic cats (Felis domesticus), we determined the
complete nucleotide sequence of an isolate of F. domesticus
papillomavirus (FdPV).
An FdPV genome isolated from a domestic cat with
cutaneous papillomatosis was cloned in the EcoRI site of
pUC18 (Carney et al., 1990 ; Sundberg et al., 2000). The
recombinant FdPV genome was amplified and purified (Qiagen
Plasmid Mini Kit). To determine the nucleotide sequence, the
cloned FdPV DNA was directly sequenced, initially with
primers selected from the vector sequence and thereafter with
additional primers designed by sequence walking. Sequencing
was performed in the Einstein DNA sequencing core facility.
The overlapping sequences were assembled manually and
confirmed by sequencing the complementary strand. Several
additional primers were designed and used to clarify sequence
ambiguities. Once assembled, the sequence was analysed for
homology with other PVs using  software (Altschul et al.,
1997). The same software was used for protein sequence
comparisons. Phylogenetic trees were created using PV
sequences available from GenBank and The Human Papillomaviruses 1997 Compendium Online (http :\\hpv-web.lanl.
gov\stdgen\virus\hpv\compendium\htdocs\HTMLIFILES\
HPVcompintro4.htmlFcomp97). Phylogenetic trees were derived from individual open reading frames (ORFs) and noncoding regions (NCRs) to determine the relationship of FdPV
to the available PV sequences using public domain software
(Higgins & Sharp, 1988).
A  homology search using the nucleotide sequence of
the FdPV L1 ORF revealed that it was most closely related
to canine oral papillomavirus (COPV) (86 % identity). The
assembled sequence of the viral genome had a total size of
8300 bp with a GjC content of 46n12 %. Examination of the
FdPV sequence for potential genes showed a typical complement of PV ORFs, including overlaps between E1 and E2 and
between L2 and L1, and the inclusion of an E4 ORF within E2.
There was no initiation codon in the E4 ORF. The predicted
ORFs are summarized in Table 1(a). The presence of an E5
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M. Terai and R. D. Burk
Table 1. Characterization of the FdPV genome
(a) Location of predicted FdPV ORFs and size of putative proteins
ORF
Start position
First ATG
Stop codon
Length of
protein-coding
sequence (bp)
8271
378
673
2432
3030
4840
6267
1
414
691
2456
417
701
2514
3601
3365
6402
7916
414
285
1821
1143
333
1527
1500
E6
E7
E1
E2
E4
L2
L1
4873
6414
No. of amino
acids
Predicted
molecular mass
of protein (kDa)
138
95
607
381
111
509
500
15n2
10n4
68n8
43n0
12n7
54n9
57n0
(b) Identity (%) of FdPV nucleotide and amino acid sequences compared with COPV
FdPV
COPV
Nucleotide sequence
Amino acid sequence
E6
E7
E1
E2
E4
NCR-2
L2
L1
NCR-1
50n4
40n4
60n9
53n1
62n0
60n5
60n0
50n5
57n9
32n7
46n7
k
55n5
59n4
69n7
75n3
53n8
k
(c) Divergence between the E6, E7, E1, E2, E4, L2 and L1 genes of FdPV and COPV
Identity with compared
codons* ( %)
E6
E7
E1
E2
E4
L2
L1
Nucleotide substitutions by codon
position† (TOTAL 100 %)
Codons
compared*
aa
nt
1st
2nd
3rd
Codons with a
third position
change† (%)
135
94
594
375
107
504
497
40n7
55n3
63n8
53n1
35n5
60n7
76n5
47n8
58n2
66n1
58n4
52n0
61n0
69n6
33n5
31n4
26n0
32n1
27n3
27n1
22n7
24n5
21n2
21n7
22n9
31n8
18n8
16n8
42n0
47n5
52n3
45n1
40n9
54n1
60n5
65n9
59n6
53n2
56n3
58n9
63n3
55n1
aa, Amino acid sequence identity ; nt, nucleotide sequence identity.
* Not counting gaps and terminal extensions.
† Including silent codons.
ORF situated between the end of the E2 ORF and the start
of the L2 ORF, which is found in some but not all PVs, was
sought by comparison of all FdPV ORFs in this region to the
complete PV database. None of the small FdPV ORFs in this
region showed significant homology with known E5 ORFs.
Table 1(b) shows the degree of identity between putative
FdPV proteins and the analogous proteins of the closest related
PV, COPV. The identity of the amino acid sequence of the L1
ORF between FdPV and COPV was relatively higher (75n3 %)
than other ORFs, with identity to E1 (60n5 %) and L2 (59n4 %)
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also elevated compared with the E7 (53n1 %), E2 (50n5 %) and
E6 (40n4 %) ORFs. This suggests that the E1, L1 and L2 ORFs
are more conserved than the other ORFs, consistent with
their essential role during PV evolution. To investigate the
relationship of the FdPV genome with other PV sequences,
the complete nucleotide sequence of FdPV was aligned
with the corresponding sequences of other closely related PVs.
The resulting phylogenetic tree was calculated based on available full-length sequences (de Villiers, 2001). A representative
tree is shown in Fig. 1. FdPV was placed into the E3 group.
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Feline papillomavirus
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Fig. 2. For legend see page 2306.
Two NCRs were identified in the FdPV genome. Organization and comparison of the FdPV NCRs with the related
COPV NCRs are shown in Fig. 2. The usual NCR (NCR-1),
found between the end of L1 and the beginning of E6, is short
in the FdPV (384 bp) and COPV (360 bp) genomes (see Fig.
2a). In other PVs, the NCR-1 sequence is also called the long
control region (LCR) and upstream regulatory region (URR),
since it contains numerous control signals for DNA replication
and transcription. The FdPV NCR-1 contains three E2 binding
sites (ACCN GGT), although neither the FdPV nor the COPV
'
NCR-1 regions have a discernible E1 binding site (Lu et al.,
1993 ; Sun et al., 1996). Papillomavirus NCRs also contain
multiple binding sites for transcriptional regulatory factors,
such as AP-1 (Chan et al., 1990), NF-1 (Apt et al., 1993), SP-1
(Gloss & Bernard, 1990), transcriptional enhancer factor (TEF)1 (Ishiji et al., 1992) and YY-1 (Dong et al., 1994 ; May et al.,
1994), among others. The FdPV NCR-1 contains a variety of
putative regulatory elements, although the FdPV NCR-1 does
not include a TATA box within the E6\E7 promoter region.
However, multiple SP-1 binding sites were identified that
could serve as the transcription initiation site (Peterson et al.,
1990). The COPV NCR-1 has a TATA box, TEF-1, a poly(A)
signal site and two E2 binding sites. The predicted locations of
these sites within the FdPV and COPV NCR-1 are shown in
Fig. 2(a). Although the FdPV NCR-1 is closely related to the
COPV NCR-1 (53n8 % sequence identity), the organization of
many regulatory elements is altered, as shown in Fig. 2(a).
FdPV and COPV have a unique and longer NCR (NCR-2),
which is located between the end of E2 and the beginning of
L2. The FdPV NCR-2 is 1271 bp in length. The NCR-2
sequence of FdPV was found to be similar to that of COPV
(46n7 % sequence identity). The A\T content of FdPV and
COPV NCR-2 are 60n8 and 64n6 %, whereas those of NCR-1
are 51n8 and 57n0 %, respectively. Except for the relatedness
with COPV, FdPV NCR-1 and the atypical FdPV NCR-2 had
no homology with sequences in the existing databases using
the  software. The NCR-2 region either originated from
an ancient integration event into the PV genome prior to the
split of felines and canines, occurred independently in both
viruses, or was acquired by host switching (Novacek, 2001).
Although most closely related to FdPV, COPV was isolated
(a)
Fig. 1. Phylogenetic tree based on the alignment of the full sequences of
the indicated and related PV genomes categorized in group E (de Villiers,
2001). FdPV, Felis domesticus papillomavirus ; COPV, canine oral
papillomavirus ; HPV1, human papillomavirus type 1 ; HPV63, human
papillomavirus type 63 ; CRPV, cottontail rabbit papillomavirus ; ROPV,
rabbit oral papillomavirus.
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M. Terai and R. D. Burk
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(b)
Fig. 2. Alignment and comparison of the NCR-1 (a) and the NCR-2 (b) sequences of FdPV and the related COPV. NCR sequences and the positions of
multiple binding sites are shown. TATA, TATA box ; TEF-1, transcriptional enhancer factor-1 ; E2, E2 binding domain ; Poly (A), poly(A) signal.
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Feline papillomavirus
from a mucosal lesion, florid oral papillomatosis, and is grouped
into the cutaneous papillomaviruses based on nucleotide
sequence homology (Delius et al., 1994 ; Sundberg et al., 1994).
However, since immunosuppressed dogs will develop cutaneous and oral papillomas (Sundberg et al., 1994), it is
possible that COPV at one time also infected skin. Whether
FdPV can infect mucosal epithelium will require additional
study. Based on epitope differences, there appear to be at least
two distinct PVs that infect domestic cats (Sundberg et al.,
2000). We analysed the divergence between the ORFs of FdPV
and COPV by amino acid and nucleotide sequence differences
as shown in Table 1(c). The nucleotide substitutions of the L1
ORF were predominantly found in the third position ; however,
codons with extensive third position changes were found in all
ORFs.
We have determined the complete nucleotide sequence of
a PV isolated and cloned from a domestic cat with cutaneous
papillomatous lesions. FdPV is most closely related to COPV
by amino acid and nucleotide sequence homology and contains
the novel NCR-2 region. PVs are considered highly speciesspecific and are not thought to cross the species barrier ;
however, there are exceptions in the veterinary literature
(Perrott et al., 2000 ; Sundberg & OhBanion, 1989). Nevertheless, a host switch or horizontal movement of virus from
one species to another cannot be ruled out, although the
divergence between these genomes indicates that they split
long ago. In fact, 53–66 % of all codons had a third position
change, suggesting that the genomes have been saturated by
mutations. Subsequently each genome may have evolved as an
independent entity by genetic drift. The lack of significant
homology with human PVs indicates that recent horizontal
transmission has not occurred. The cloning and characterization of FdPV has the potential to be utilized for development
of an additional animal model of PV infection.
We thank Drs John Sundberg and Marc Van Ranst for providing
reagents and helpful suggestions on the manuscript.
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Received 15 February 2002 ; Accepted 19 April 2002
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