1 Department of Microbiology and Immunology, Albert Einstein Cancer Center, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA
2 Department of Pediatrics, Albert Einstein Cancer Center, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA
3 Department of Obstetrics, Gynecology and Women's Health, Albert Einstein Cancer Center, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA
4 Department of Epidemiology and Population Health, Albert Einstein Cancer Center, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA
5 Division of Invertebrate Zoology, American Museum of Natural History, New York, NY 10024, USA
Correspondence
Robert D. Burk
burk{at}aecom.yu.edu
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ABSTRACT |
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GenBank data deposited: bovine papillomavirus 3, AF486184; bovine papillomavirus 5, AF457465; equine papillomavirus, AF394740; and reindeer papillomavirus, AF443292.
GI numbers and names of the specific PV genomes used for the study can be found as supplementary data at JGV Online.
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INTRODUCTION |
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Historically, PVs had been classified according to their tissue tropism, and this grouping was supported by later phylogenetic analysis of PV sequence data (Chan et al., 1995; de Villiers, 2001
; Myers, 1994
). PV phylogenies typically subdivide into mucosal/genital HPVs, cutaneous EV HPVs and three main animal PV clades: the artiodactyl ruminant PVs, the distant avian PV group and a group containing canine, feline, rabbit and rodent PV types. However, sequence analysis has highlighted some significant exceptions. BPV-3, BPV-4 and BPV-6 do not group with the artiodactyl PVs, but instead form an isolated taxon (Jarrett et al., 1984
), and HPV-1, HPV-41 and HPV-63 are most closely related to the canine and feline PVs, sharing little similarity to HPVs in either the mucosal or the cutaneous groups (Egawa et al., 1993
).
Although most work in the area has focused on HPVs, an ever-increasing number of animal PV isolates have been sequenced. These animal PVs share key clinical features of PV infection with their HPV counterparts, making them valuable models of HPV infection. Comparative analysis of a wide variety of PV genomes can contribute to understanding the molecular basis of PV evolution and pathogenicity.
To study the molecular evolution of PVs, we determined the complete nucleotide sequences of bovine PV type 3 and type 5, equine PV and reindeer (Rangifer tarandus) PV, and compared their sequences with the genomes of 34 other animal and human PVs.
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METHODS |
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Plasmid DNAs were purified according to the manufacture's instructions (Qiagen plasmid mini kit, Qiagen). Each plasmid was directly sequenced, first with primers selected from the vector sequence and later with additional primers designed from sequence walking (Delius & Hofmann, 1994). Sequencing was performed in the Einstein DNA sequencing core facility, and the overlapped sequences were assembled manually. Several additional primers were designed and used to clarify sequence ambiguities.
Sequence analysis.
Using GenBank's taxonomy browser, 4011 protein sequences and 1983 nucleotide sequences (some redundant) of papillomaviruses were identified with a batch entrez query on the keywords Papillomavirus and Papillomaviridae (Wheeler et al., 2003). These sequences were downloaded and indexed as local BLAST databases of PV protein and nucleotide sequences. A set of human papillomavirus genomes representative of the various HPV genera was selected for inclusion with all available animal PV genomes. The GI numbers and names of the specific PV genomes used for this study can be found in the supplementary information.
Protein (BLASTP) and nucleotide (tBLASTN) homology searches were performed for all translated open reading frames in the newly sequenced BPV-3, BPV-5, EQPV and RPV (Altschul et al., 1997). BLASTP scores an amino acid sequence against a standard protein database and identifies similar sequences, but is limited in that it restricts searches to a translated set of ORFs, some of which may be frame-shifted due to sequencing error. ORFs are also occasionally misidentified or unrecognized in the annotated record. A tBLASTN search clarifies these uncertainties by querying a protein against all six potential reading frame translations in a nucleotide database.
Standard BLAST parameters were used for most analyses, including filters for non-informative sequence (seg), composition based statistics, a word size of three and the BLOSUM62 scoring matrix. For small ORFs (1530 amino acids in length), the BLAST searches were modified by removing the filter, turning off composition-based statistics, using a word size of two and employing the PAM30 scoring matrix. A 1e5 significance cut off was chosen for all queries. The searches were implemented locally using PERL scripted queries to the local PV databases. The analysis relied heavily on the open-source BioPerl (Stajich et al., 2002) modules available at www.bioperl.org.
Multiple sequence alignments of PV clusters were performed using CLUSTALW with the gap cost 10·0 and the GONNET cost matrix (Higgins & Sharp, 1988). Concatenated E6, E7, E1, E2, L2 and L1 translated open reading frames constitute an alignment of largely conserved PV elements. E4, E5, E6 and E7 protein alignments were also individually generated for those PVs from Fig. 1
that contain them. Genealogical relationships were reconstructed using equal weighted characters. To ensure adequate searches in the tree's space, 100 random addition heuristic searches and TBR (tree bisection and reconnection) swapping were employed in PAUP* version 4.10 (Swofford, 1998
). Alignment gaps were coded as missing before parsimony and neighbour-joining trees were reconstructed. To assess robustness, 100 bootstrap and 100 jackknife replicates were performed for both the parsimony and distance analyses. The resampling approaches yielded essentially identical results, so only bootstrap results are reported here. Bayesian trees were constructed with the Markov Chain Monte Carlo technique in MRBAYES (Huelsenbeck & Ronquist, 2001
) over 10 000 generations with sampling every 100 generations.
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RESULTS AND DISCUSSION |
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BPV-3, BPV-5, EQPV and RPV all have the typical complement of E1, E2, L2 and L1 genes comparable in size and position to other PVs. The canonical E6E7 ORFs are also evident in the newly sequenced PVs except BPV-3, which instead contains an E8 ORF with homology to E8 in BPV-4 (69 % amino acid identity) and BPV-6 (Jackson et al., 1991) (66 % amino acid identity). The E8 ORF of BPV-4 has been shown to share functional similarity with BPV-1 E5, although sequence comparisons indicate these are not homologous ORFs. It has been suggested that the functionally similar BPV-4 E8 should be considered as an E5 protein (Morgan & Campo, 2000
). Since the ORFs are not homologues we have continued with the E8 nomenclature. Other small, putative ORFs include BPV-3 L3 and RPV E9, homologous to BPV-4 L3 (Patel et al., 1987
) (41 % amino acid identity) and EEPV E9 (Eriksson et al., 1994
) (45 % amino acid identity), respectively. The atypical and rare ORFs (e.g. BPV-3 E8 and RPV E9) identified here are difficult to confirm or discount using sequence analysis alone. If an unusual ORF such as RPV E9 exhibits little similarity to any other PV nucleotide or protein sequence, it may be either spurious or novel, and will require identification of the protein in vivo for validation. Even convincing homology to a putative related PV is not definitive evidence that an ORF is actually expressed.
PV core ORFs: E1, E2, L2 and L1
To probe evolutionary relationships between BPV-3, BPV-5, EQPV, RPV and other animal PV genomes, the core ORFs (E1, E2, L1 and L2), common to all PVs, and the E6 and E7 oncogenes (where available) were compiled and aligned with the oncogenic and core ORFs of 34 representative PV genomes. The major (L1) and minor (L2) structural proteins contribute to the formation of the viral capsid, and the replication proteins (E1 and E2) interact with cellular polymerases and primases, stimulating viral genome replication (Scheffner et al., 1994). The proteins encoded by these core ORFs are essential to the structural integrity and biochemical viability of every PV sequenced to date. The resulting phylogenetic tree (Fig. 2
) subdivides into five major clades.
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Though the topology of the tree is at least partially host-species driven, the fact that bovine species are found in two independent clades, defined most clearly by their differences in pathology, demonstrates that any discussion of PV evolution should also consider disease phenotype. This is especially important in the case of BPV-5, where two disease phenotypes, normally tied to deep monophyletic groups, result from infection by a single PV.
E4 and E5
E4 and E5, unlike the core ORFs, are not essential to PV function, but E5 in particular has been established as one of the main factors in host-cell transformation (Petti & Ray, 2000). E4 has also been shown to effect transformation by modulating the cell division cycle (Nakahara et al., 2002
), but its full biological activities are still being explored. The mechanisms of E5 driven transformation in rodent and human fibroblasts have, however, been largely elucidated. E5 activates platelet-derived growth factor (PDGF)
-receptor tyrosine kinase in a ligand-independent fashion. BPV-1 E5 proteins have been shown to bridge two molecules of transmembrane PDGF receptors, resulting in receptor dimerization, activation and recruitment of cell signalling and proliferative proteins (DiMaio & Mattoon, 2001
). In addition, BPV-1 E5 binds to the 16 kDa transmembrane subunit of vacuolar H+-ATPase (Goldstein et al., 1991
), impairing the acidification of the Golgi apparatus and other intracellular organelles. A number of growth regulatory proteins, including PDGF
, are processed in the Golgi, suggesting that the ability of BPV E5 to influence intra-organelle pH may be a factor in transformation. The mucosal HPVs also encode a small, hydrophobic protein superficially resembling the artiodactyl PV E5, but the E5 of these two PV lineages are not homologues and are probably the result of convergent evolution.
Though E4 and E5 are not ubiquitous among animal PVs, among the artiodactyl PVs (clade 4), both ORFs are largely conserved. An alignment of the artiodactyl PV E5 ORFs shows very close similarity (39 % amino acid identity across shared alignment positions), indicating that the E5PDGF -receptor interaction and H+-ATPase binding are probably shared mechanisms of transformation among the PVs of clade 4. Since the pathological hallmark of this clade is the development of fibropapillomas, the underlying genetic basis of fibroblast transformation is thought to involve the E5 transforming factor (Munger & Howley, 2002
). Once again, however, BPV-5 is a glaring exception. As the clade's outlier, it lacks observable E4 and E5 ORFs, but still retains the clade's overall pathology, albeit in somewhat mixed fashion (Bloch & Breen, 1997
). Consequently, the mechanism of fibropapilloma development is probably reinforced by the E5 ORF, but not solely contingent on its presence.
E6 and E7
E6 can be identified in every PV genome except BPV-3, BPV-4 and the two avian PVs, FPV and PePV. Instead, the bovine species contain E8 (Jackson et al., 1991), and the avian species contain a novel ORF with no significant homology to the rest of the PV proteome (Terai et al., 2002
). The remaining PV genomes exhibit E6 sequence homology and share four distinct C-X-X-C motifs, conserved residues that seem to be essential structures in the formation of a multimerized complex. With four cysteine sulphur ligands, two C-X-X-C motifs can sequester a zinc ion in a tetrahedral configuration (Grossman & Laimins, 1989
). E6 proteins complex the tumour suppressor protein, p53, a key factor in flagging cell growth in differentiated or damaged cells. E6 therefore has anti-apoptotic activity, and interferes with the anti-proliferative signalling system of differentiated cells (Mietz et al., 1992
). In PVs that lack E6, like BPV-3, an E8 transforming protein induces the anchorage independent growth of the infected cells and suppresses contact inhibition (O'Brien et al., 1999
), but does so independent of a p53 binding mechanism and appears to have functional similarity to BPV-1 E5, as indicated above (Morgan & Campo, 2000
).
The E7 oncogene also contributes to a PV's interference with the host cell-cycle and cellular differentiation. But where E6 binds p53, the canonical E7 binds pRB, the retinoblastoma protein, preventing its interaction with transcription factor E2F-1, resulting in activation of E2F responsive genes, such as those involved in cell replication (Munger et al., 2001). The hallmark of the pRB-binding domain is an invariate L-X-C-X-E motif shared by most E7 ORFs, including members of clades 1, 2, 3 and 5 (Chan et al., 2001
; Dahiya et al., 2000
; Dick & Dyson, 2002
). An alignment emphasizing pRB-binding conservation among representative PVs is shown in Fig. 3
(a). Note that in addition to the pRB-binding domain, these ORFs all contain two conserved C-X-X-C motifs separated by 2830 amino acids. Clade 4 also exhibits similar dicysteine motifs, separated by 29 amino acids, but the alignment is peculiar in that there is no evidence for the pRB-binding signature. Instead, a novel set of amino acids with a pattern of conserved proline and leucine residues is retained in all these genomes (Fig. 3b
). Since both bird PV genomes (PePV, FPV) contain evidence for a pRB-binding domain (Terai et al., 2002
), the existence of the novel E7 motif in clade 4 appears to have arisen in the common ancestor of clade 4's members, an early divergence from a pRB-binding progenitor. Remarkably, the distribution of papillomas and fibropapillomas almost exactly mirrors the distribution of pRB-binding motif loss. Every animal PV known to cause fibropapillomas also lacks the pRB-binding motif, including the distant EQPV, whose lesion types, like BPV-5, tend to be somewhat mixed (Hamada et al., 1990
). This correlation is not an artefact of gaps in the character matrix underlying the phylogeny in Fig. 2
, because gaps fixed into the CLUSTAL alignments are coded as missing and are therefore neutral with respect to the phylogenetic analysis.
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Still, the correlation between the lack of pRB-binding and development of fibropapillomas is a finding, thus far, restricted to animals, and is most conspicuous among the artiodactyl PVs in clade 4. As demonstrated, E4 and E5 are well conserved in the clade, but because BPV-5 lacks these ORFs, we conclude that the motif shift in E7 sequences of the artiodactyl papillomaviruses may be partially responsible for their unique pathology on infection. Pairwise correlated changes tests examining the association between pRB, E5 and fibropapilloma lesions in Fig. 2 reveal significant correlation between the three characters (pRBFP, P<0·001; pRBE5, P<0·05; E5FP, P<0·05). This correlation extends beyond the phylogenetic topology and pathology. In BPV-1 fibropapillomas, it has been shown that E5 and E7 co-localize within the cytoplasm of undifferentiated basal epithelial cells, and that this co-expression is the basis of cooperative transformation between E5 and an E7 lacking the pRB-binding motif (Bohl et al., 2001
).
In the absence of E5, however, fibroblast transformation may be mediated by cooperation between E6 and E7 ORFs (Neary & DiMaio, 1989). Nevertheless, why a lack of E5 in genomes without E7 pRB-binding activity correlates with dual papilloma/fibropapilloma pathology remains enigmatic. The basal position of BPV-5 in clade 4 reflects the retention of ancestral character states in its protein sequences. BPV-5 may be considered a viral missing link between papilloma-causing animal PVs, and the more derived fibropapilloma-causing clade 4 viruses that contain E5, but have lost the pRB-binding motif. Along with BPV-5, EQPV shares FP/P heterogeneity, lack of pRB-binding, and lack of E5, supporting the idea that the ancestral state shared these characteristics. We expect that other, as yet undiscovered, artiodactyl PVs will have proteins that also display the transitional nature of BPV-5 and EQPV.
Nevertheless, only when the presence of E5 is coupled to the absence of an E7 pRB-binding domain, does PV infection trigger the exclusive development of fibropapillomas. We suggest that the manifestation of fibropapillomas is not attributable to E5 alone, and that an adaptive shift in a single E7 motif that evolved early in the PV phylogeny is also instrumental, providing a distinct marker for risk of fibropapilloma development traced to the sequence level.
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ACKNOWLEDGEMENTS |
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Received 27 October 2003;
accepted 22 January 2004.