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Short Communication |
1 Electron Microscope Unit, University of Cape Town, Observatory, Cape Town 7925, South Africa
2 Department of Molecular and Cell Biology, University of Cape Town, Observatory, Cape Town 7925, South Africa
3 Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Observatory, Cape Town 7925, South Africa
Correspondence
Darren P. Martin
darren{at}science.uct.ac.za
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ABSTRACT |
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Supplementary material is available in JGV Online.
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MAIN TEXT |
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). Although most human papillomaviruses (HPVs) are benign, a ‘high-risk’ oncogenic subset contains causal agents of various oral and anogenital cancers (Clifford et al., 2003; Muñoz, 2000; Steller, 2002).
The circular, double-stranded DNA genomes of nearly all PVs are approximately 8 kb in size and may, depending on the species, encode three oncogenes (E5, E6 and E7), two replication proteins (E1 and E2), a protein involved in productive infection (E4) and two capsid proteins (L1 and L2). Linkage of genome replication with host cell cycles, coupled with a low replication-error rate, means that the most variable parts of PV genomes are diverging at a rate of approximately 1 % every 40 000–80 000 years (Bernard et al., 1994) – an evolutionary rate around one million times lower than that of most RNA viruses (Halpern, 2000). It has been suggested convincingly that HPVs have been infecting and co-evolving with humans for at least a few hundred thousand years (Bernard, 2005; Calleja-Macias et al., 2005; Chan et al., 1992).
Although various sequence analyses have indicated that PV evolution has occurred in the absence of recent recombination (Chan et al., 1992, 1997; Halpern, 2000), slight differences between internal branches of phylogenetic trees constructed by using different parts of the same PV genomes (Bravo & Alonso, 2004; Schiffman et al., 2005) have raised suspicions that recombination may have occurred, albeit infrequently, between ancestral PV lineages. A rigorous search for evidence of PV recombination using currently available recombination-detection methods (Posada & Crandall, 2001) has, however, been seriously hindered by the technical difficulty of aligning extremely diverse PV sequences accurately. Nevertheless, a recent statistical analysis of topology differences in trees constructed from inferred amino acid sequences encoded by different genes from 
-PVs has provided the most convincing evidence yet of PV recombination (Narechania et al., 2005).
However, an important question remains: do these reported differences in tree topology represent evidence of ancient recombination events or are they artefacts of imperfect sequence alignment and phylogenetic-reconstruction methodologies? Here, we report the combined use of rigorous alignment-accuracy tests and some of the most powerful recombination-detection methods currently available to more conclusively identify and characterize statistically probable recombination events in PV full-genome sequences.
One hundred and five complete PV genome sequences, representing the full scope of currently sequenced PVs (de Villiers et al., 2004) (see Supplementary Table S1, available in JGV Online), were obtained from GenBank. All genomes were ‘linearized’ at the first nucleotide of their L1 genes and aligned by using POA (Grasso & Lee, 2004) (gap open penalty, 12; gap extension penalty, 4). We constructed five multiple sequence alignments: GP0, containing all 105 sequences; GP1, containing -PV sequences; GP2, containing 
-, 
-, 
-, 
-, 
-, 
-, 
-, 
-, µ- and 
-PV sequences; GP3, containing 
-, o-, 
- and 
-PV sequences; and GP4, containing 
-PV sequences. Analysis of these alignments with the RDP (Martin & Rybicki, 2000), GENECONV (Padidam et al., 1999), BOOTSCAN (Martin et al., 2005a), MAXIMUM 
2 (Maynard Smith, 1992), CHIMAERA (Martin et al., 2005b) and SISCAN (Gibbs et al., 2000) recombination-detection methods as implemented in RDP2 (Martin et al., 2005b) indicated that GP0, GP1, GP2, GP3 and GP4 respectively contained evidence of 224, 168, 45, 27 and 65 unique potential recombination signals (see Supplementary Methods for information on how these methods work and RDP project files for detailed analysis results and program settings; all available in JGV Online).
Due to the difficulty of aligning the PV sequences reliably, we thought it likely that many of the apparent recombination signals were in fact alignment artefacts. Artefactual signals were identified by using two tests of alignment consistency in the genome region surrounding each of the potential recombination events. In both tests, sequences that had been used to identify a specific recombination signal were selected from the alignment and realigned in isolation. In the first test, the region of sequence between estimated recombination breakpoints was realigned by using CLUSTAL W (default settings) (Thompson et al., 1994) and the pattern of nucleotide matches and mismatches between the three sequences in the original alignment was compared with that in the new alignment by using a 2 test. A 2 P value of <0.1 constituted a test failure. The second test involved realignment of the three full-length sequences with both POA and CLUSTAL W and reanalysis for recombination with RDP2. Inability to detect evidence for the same recombination signal in either the CLUSTAL W or POA realignments with a Bonferroni P value correction identical to that used for the initial signal detection constituted a test failure. CLUSTAL W and POA apply substantially different alignment algorithms, implying that recombination signals detectable in both POA and CLUSTAL W alignments have occurred in consistently alignable genome regions. Of the 529 potential recombination signals, only ten passed both tests. As three very similar signals were detected in the GP0 and either GP1 or GP2 alignments, these signals together represent evidence of at least seven unique interspecies PV recombination events (Fig. 1).
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) by using two portions of the relevant multiple sequence alignment bounded by the approximated recombination breakpoints for that event (e.g. Fig. 2). We considered ‘movement’ of the identified recombinant sequence(s) between branches of the two trees with an excess of 60 % bootstrap support to constitute good phylogenetic evidence in favour of recombination having occurred during the history of the sequence(s).
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; Fig. 2). Importantly, one of these four events (event 1) has been localized to the same approximate region of species 5, 6 and 7 -PV genomes (Fig. 2) identified previously, using a completely independent set of phylogeny-based statistical tests, as potentially having a recombinant origin (Narechania et al., 2005). However, use of recombination-detection methods that directly identify specific sequences responsible for aberrant phylogenetic signals has enabled us to identify more accurately the portion of the L2 gene exchanged during this recombination event.
Our phylogenetic analyses also revealed that the identified recombination events are probably all ancient, in some cases predating the diversification of major PV groupings (events 1 and 4 in Fig. 1; Fig. 2). This is significant in that it means that viable PV recombinants, once formed, can spread and thrive.
Although we have detected PV recombination and have characterized a number of individual events, we feel that there is limited scope for further analysis. For example, whereas five of the seven detected events have breakpoints within the L2 gene, it is not possible to infer the presence of a L2 recombination hotspot. This is because the absolute need for reliable nucleotide sequence alignment necessarily biases recombination analyses in favour of breakpoint detection in conserved genomic regions such as L2. The extreme genetic diversity amongst PVs also makes it difficult to envision accurate estimation of recombination rates by using model-based population genetic approaches.
Nevertheless, recombination is a potent evolutionary mechanism and evidence of viable PV recombinants is important. In light of our results and those of Narechania et al. (2005), it is of some concern that the global AIDS epidemic has created favourable conditions for the generation and emergence of recombinant HPVs. Whilst the higher incidence of multiple HPV infections in human immunodeficiency virus (HIV) patients (Chaturvedi et al., 2005a, b; Haas et al., 2005; Hameed et al., 2001) is likely to support the generation of recombinants, HIV-induced immune dysfunction may facilitate both short-term survival and eventual adaptation of recombinants to new niches within humans.
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ACKNOWLEDGEMENTS |
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Received 7 February 2006;
accepted 2 May 2006.
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