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1 Centro Nacional de Biotecnología-CSIC, Campus Universidad Autónoma de Madrid, 28049 Madrid, Spain
2 Laboratori de Genètica Molecular Vegetal, Consorci CSIC-IRTA, IBMB, Jordi Girona 18–26, 08034 Barcelona, Spain
Correspondence
Juan José López-Moya
jlmgmy{at}ibmb.csic.es
Juan Antonio García
jagarcia{at}cnb.uam.es
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Amino acid alignments of potyviral and rymoviral P1 protein domains are available as supplementary figures in JGV Online.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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; Berger et al., 2000; http://www.dpvweb.net/notes/showfamily.php?family=Potyviridae). The largest genus, Potyvirus, which contains >100 species, and four additional genera, Rymovirus, Macluravirus, Ipomovirus and Tritimovirus, have genomes that consist of a sole single-stranded RNA molecule of messenger polarity. The sixth genus, Bymovirus, contains viruses with bipartite genomes. The genomic RNA of monopartite members of the family Potyviridae is translated into a polyprotein that is processed proteolytically to yield a conserved set of ten mature proteins. The two bymovirus genomic RNAs are also translated into polyproteins; the polyprotein encoded by RNA 1 includes eight proteins that share homology with those encoded by the 3' region of viral genomes from the other genera. RNA 2 encodes two proteins that are quite distinct from those found in the 5' region of monopartite viruses (Adams et al., 2005b).
Overall similarity of the polyproteins of viruses of the family Potyviridae is rather high, with levels of amino acid identity ranging from 42 to 56 % in different species of the same genus and from 25 to 33 % in viruses from different genera (Adams et al., 2005b). However, conservation of individual mature proteins varies. P1, the first protein of the polyprotein, is the most divergent with regard to both length and amino acid sequence (Adams et al., 2005b). It is a serine protease that self-cleaves at its C terminus (Verchot et al., 1991) and acts as an accessory factor for genome amplification (Verchot & Carrington, 1995). The role of P1 in potyvirus infection is still unknown; however, there is some indication that P1 can strengthen the ability of HCPro to suppress RNA silencing (Kasschau & Carrington, 1998; Rajamäki et al., 2005; Valli et al., 2006) and enhance the pathogenicity of heterologous plant viruses in synergistic interactions (Pruss et al., 1997).
Recombination is one of the main forces driving plant virus evolution (García-Arenal et al., 2003; Roossinck, 2003). Although frequent in many virus groups, recombination events are especially common in potyviruses (Chare & Holmes, 2006). Indeed, both intraspecies (Cervera et al., 1993; Bousalem et al., 2000; Glais et al., 2002; Glasa et al., 2004; Krause-Sakate et al., 2004; Moreno et al., 2004; Tan et al., 2004; Zhong et al., 2005) and interspecies (Desbiez & Lecoq, 2004; Larsen et al., 2005; Ali et al., 2006) recombination events are involved in potyviral evolution, some of which affect the P1 sequence (Glais et al., 2002; Desbiez & Lecoq, 2004; Tan et al., 2004; Larsen et al., 2005; Ali et al., 2006).
In this study, we performed an extensive computational analysis of P1 proteins from 53 virus species of four genera in the family Potyviridae. Our results suggest that not only intraspecies and intragenus, but also intergenus, recombination within the P1 gene contributed to potyvirus evolution. P1 gene duplication is also shown to contribute to P1 diversification.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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lists monopartite members of the family Potyviridae for which full-length genomic sequences were available in international databases as of July 2006. In most cases, a single sequence for each virus species was used for the analysis (labelled in bold). The EditSeq program of the DNASTAR Lasergene7 software suite, as well as online facilities at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov), were used for BLAST similarity searches and comparisons of expectation (e) values that represent the statistical significance of matches. The EditSeq program was also used for isoelectric point (pI) calculations. Sequence alignments were carried out by using the DNASTAR MegAlign program and refined by manual editing. Aligned sequences were used to build an unweighted pair-group method arithmetic mean (UPGMA) tree with 1000 bootstrap replicates by using the MEGA (version 3.1) program.
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) known as GARD (Genetic Algorithms for Recombination Detection), available at http://www.datamonkey.org, was used to search for evidence of recombination break points in selected nucleotide alignments of P1 sequences. In brief, the nucleotide sequences encoding P1 and HCPro proteins in which recombination events were suspected after amino acid alignment and visual comparison were aligned by using CLUSTAL_W, displayed in PHYLIP format and analysed. In each case, the nucleotide-substitution model was selected automatically before being applied to the recombination-site analysis. The algorithm scans alignments for evidence of discordant phylogenetic signals. The results showed evidence of recombination break points with score improvements in information-based criteria, such as the small sample-corrected Akaike information criterion (c-AIC). The predicted break points were located in the alignments according to their scores, and the confirmatory values were evaluated after considering their proximity to previously suspected recombination sites. |
RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). However, well-conserved motifs can be identified within the C-proximal protease domain of all potyvirus P1s. The consensus sequence of these motifs, including the active site, is H-(x8)-D/E-(x28–31)-G-x-S-G-(x10–21)-I/V-I/V-R-G (residues of the catalytic triad are in bold; Fig. 1). His and Glu are separated by nine residues in the dasheen mosaic virus (DsMV) P1 protein. The cleavage site between P1 and the next protein, HCPro, is also well conserved. It is 22–28 aa downstream of the strictly conserved RG dipeptide and has as its consensus sequence I/V/L/M-x-H/E/Q-F/Y
S. However, exceptions are found at all positions except for the Phe/Tyr located immediately before the cleavage point (Adams et al., 2005a; Fig. 1).
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). In our study, many branches of the tree generated by the computer program were not well supported by the bootstrapping data, such that only the previously described bean common mosaic virus (BCMV) (Ward & Shukla, 1991), sugarcane mosaic virus (SCMV) (Shukla et al., 1992) and potato virus Y (PVY) (Spetz et al., 2003) subgroups could be recognized. As a result, we looked for conserved motifs in the extremely variable N-terminal domains by visually analysing alignments that were derived from members of known groups, and searched for equivalent motifs in P1 sequences from other viruses. This strategy allowed us to identify several motifs, as discussed below (Fig. 1
). A highly conserved motif with the consensus sequence (N-terminal end) (x4–6)-n-I-m-F-G-S/T-F-e-C-k-L was detected in members of the PVY subgroup (residues in upper- and lower-case letters were found in at least five and three or four species, respectively; see Supplementary Fig. S1, available in JGV Online). Careful inspection detected similar motifs with a more relaxed consensus sequence around a conserved Gly in most potyvirus species and in three rymoviruses (Supplementary Fig. S1). However, we were unable to identify this signature in viruses from the SCMV subgroup. Interestingly, although this motif was located primarily near the N terminus of the protein, in some viruses it was located more internally.
Another ubiquitous motif was detected 11–21 aa upstream of the catalytic His (see Supplementary Fig. S2, available in JGV Online). This motif is characterized by a Glu residue preceded by one hydrophobic residue (mainly Val) and followed by another two hydrophobic amino acids, Gly, and between two and five basic amino acids (Lys or Arg) in the next five positions. However, the consensus sequence of this motif is very relaxed and none of its residues are conserved in all potyvirus sequences.
We detected other motifs that were conserved in smaller sets of potyviruses. Two sequential conserved motifs separated by 9 or 10 aa were placed 57–58 aa upstream of the His in the catalytic triad of viral P1 proteins from the PVY subgroup. Consensus sequences of the two motifs were P-s/y-I/v-V/i-S/t-x-I-s/t-I/v-A/g-G-G-x2-p-S and p-l/i-h/n-k/t-T-P-S/r-x-K/r-x-k (residues in upper-case letters were found in at least five of the six subgroup species; see Supplementary Fig. S3, available in JGV Online). The two motifs were detected at the same distance from the protease domain in five potyviruses that did not belong to the PVY subgroup: Lettuce mosaic virus (LMV), Sweet potato feathery mottle virus (SPFMV), Turnip mosaic virus (TuMV), Plum pox virus (PPV) and Japanese yam mosaic virus (JYMV). Although these viruses did not cluster together in the phylogenetic tree of the P1 protease domains, probably because of low resolution outside the three main potyvirus subgroups (data not shown), all of them were linked closely within complete phylogenetic trees (e.g. Adams et al., 2005b; Petrzik & Franova, 2006). In contrast, these motifs could not be identified in Scallion mosaic virus (ScaMV), a close relative of TuMV at the full-genome scale. Although the first of these motifs was well conserved in Lily mottle virus (LMoV) and still recognizable in Tobacco vein mottling virus (TVMV), the second motif was not visible in these two viruses, suggesting that P1 evolved irregularly (see Supplementary Fig. S3, available in JGV Online).
A distinctive motif of viral P1 proteins from the BCMV subgroup was found 92–96 aa upstream of the catalytic His (between 94 and 252 aa from the N terminus of the protein; see Supplementary Fig. S4, available in JGV Online). It has the consensus sequence E-E-e-a-F-L-a-G-x-Y-e (residues in upper-case letters were found in at least nine of the 12 subgroup species). More degenerate forms of this motif were located at the same distance from the protease domain in viral P1 proteins from the PVY subgroup, as well as in Chilli veinal mottle virus, Peanut mottle virus (PeMoV), Beet mosaic virus (BtMV), TVMV, LMoV, PPV and Yam mosaic virus (YMV). No simple phylogenetic relationships justify the presence or absence of this motif. Interestingly, Thunberg fritillary virus, the only potyvirus that shares with BCMV subgroup members and their closest relatives, BtMV and PeMoV, the peculiarity of having a Glu instead of an Asp within the P1 catalytic triad (Fig. 1), lacked this conserved motif (see Supplementary Fig. S4, available in JGV Online).
All of these results suggest that the potyviral P1 gene has undergone extensive and uneven evolutionary diversification that has not always paralleled the evolution of the complete genome.
Recombination events in potyviral P1 evolution
To investigate the suspected frequent recombination affecting the P1 gene of potyviruses, we decided to select a few examples in which the recombination events could be inferred easily by protein sequence comparison and to confirm those cases by using bioinformatic approaches. Published evaluations of the available methods of recombination detection were considered in order to select the most satisfactory (Posada, 2002; Kosakovsky Pond et al., 2006).
Sequence alignment of the BCMV subgroup viruses suggests that Watermelon mosaic virus (WMV) may have resulted from a recombination event in the P1 genes of BCMV and a soybean mosaic virus (SMV)-related potyvirus (Desbiez & Lecoq, 2004). The presumed crossover region of WMV is shown in Fig. 2(a). We performed further sequence alignment analysis and included potyviruses outside the BCMV subgroup. The BCMV-derived region of WMV included sequences that are very similar to sequences from the completely unrelated potyvirus Papaya leaf distortion mosaic virus (PLDMV) (Fig. 2a). Interestingly, BCMV/PLDMV similarity ended upstream of the BCMV/SMV recombination site of WMV (Fig. 2), suggesting that the BCMV-related parent of WMV was indeed a recombinant virus.
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for alignments that detected the recombinant origin of WMV originated from BCMV Y (GenBank accession no. AJ312438
[GenBank]
). The P1 sequences from other BCMV isolates, such as the previously named Peanut stripe virus (GenBank accession nos AY968606
[GenBank]
, U34972
[GenBank]
and U05771
[GenBank]
), also included the PLDMV-related sequence; however, this fragment was not present in the P1 of the BCMV isolates BCMV-R or RU-1, or the isolate previously identified as Blackeye cowpea mosaic virus (GenBank accession nos AJ312437
[GenBank]
, AY863025
[GenBank]
and AY575773
[GenBank]
, respectively). A BLAST search using the N-terminal region of these P1 sequences as a query revealed that they were related very closely to the equivalent region in the NL-3 K isolate of Bean common mosaic necrosis virus (BCMNV; GenBank accession no. AY864314
[GenBank]
), but not to other BCMNV isolates (NL-3 D; GenBank accession nos AY138897
[GenBank]
, AY282577
[GenBank]
and U19287
[GenBank]
). This supports a previous report that found that BCMNV NL-3 K was a natural recombinant derived from BCMNV and BCMV (Larsen et al., 2005). Interestingly, the P1 region that was similar in some BCMV and BCMNV isolates also showed notable similarity to the N-terminal region of Shallot yellow stripe virus (SYSV), a potyvirus that does not belong to the BCMV subgroup. The sequence similarity between SYSV and the BCMV-R- and BCMNV NL-3 K-like isolates was much lower than the similarity between PLDMV and the WMV and BCMV Y-like isolates (Fig. 2), suggesting that the recombination event involving the SYSV relative occurred much earlier than recombination involving PLDMV. In addition, the observation that the SYSV-related regions of the BCMV-R- and BCMNV NL-3 K-like isolates were more similar to each other than to SYSV (Fig. 2b) suggests that these isolates did not derive from independent recombination events involving SYSV, but were evolutionarily sequential. This agrees with the suggestion that BCMNV NL-3 K is derived from recombination between BCMV RU1 and BCMNV NL-3 D (Larsen et al., 2005).
For further confirmation of these visually detectable putative recombination events, corresponding GARD analyses (Kosakovsky Pond et al., 2006) were performed. We began by testing the SMV, BCMV-Y and WMV sequences. The analysis showed a high score (c-AIC score improvement of 58.2) for a single break point that coincided with the previously described recombination event (Desbiez & Lecoq, 2004). A similar analysis performed with PLDMV, BCMV-R and BCMV-Y sequences located another single recombination site (c-AIC score improvement of 61.1) slightly downstream of the break point predicted from the protein alignment (Fig. 2). Finally, the SYSV, BCMV-R, BCMNV NL-3 D and BCMNV NL-3 K sequences were analysed by using GARD tests for multiple recombination, showing two break points corresponding to positions 445 (aa 102) and 508 (aa 123) in the BCMNV NL-3 K sequence (Fig. 2). The neighbour-joining trees that were derived from automatic analysis of the corresponding fragments between the recombination sites supported the expected relationships (data not shown).
After considering all of the recombination events, a potential evolutionary pathway was designed for these potyviruses (Fig. 2c). An early recombination event between the SYSV ancestor and another potyvirus would have produced the BCMV-R precursor. Recombination between BCMV-R-type isolates and PLDMV or a BCMNV NL-3 D-type isolate would have produced the BCMV-Y-type and BCMNV NL-3 K-type isolates, respectively. Finally, WMV would be the result of a third round of recombination between a BCMV-Y-type isolate and SMV.
Interestingly, the P1 protein of another BCMV subgroup potyvirus, East Asian passiflora virus (EAPV), like the P1 proteins of BCMV-Y-type isolates and WMV, has a PLDMV-related domain. However, in contrast with WMV P1, EAPV P1 does not share close sequence similarity with SMV P1 and it is not evident whether EAPV is derived from a BCMV-Y-type recombinant (by linear evolution or recombination with an unidentified potyvirus) or from an independent recombination event involving PLDMV (data not shown). GARD analysis of EAPV, BCMV-Y and PLDMV confirmed a putative recombination break point approximately 15 residues upstream of the region where recombination was detected in PLDMV, BCMV-R and BCMV-Y, although with a lower score (
c-AIC of 12.2).
As WMV has a wider host range than SMV, it is suggested that the N-terminal region of P1, the primary feature that distinguishes between these two viruses, is especially relevant for host–virus interaction (Desbiez & Lecoq, 2004). A role for this genomic region in pathogenicity is also supported by the disparate symptoms caused by the BCMNV NL-3 K- and BCMNV NL-3 D-type isolates. However, in this instance, differences within the P1 N terminus do not appear to affect virus host range (Larsen et al., 2005). Further support for the importance of P1 in host-range definition is provided by the finding that one Pinellia ternata potyvirus is related closely to SMV, with the exception of the P1 gene, which resembled the P1 of another BCMV subgroup member, DsMV (Chen et al., 2004). Sequence alignment analysis suggested that this virus might have derived from a recombination event that occurred at a point close to the P1/HCPro junction (Fig. 3a). The DsMV/SMV recombinant (SMV-P) differs from typical SMV isolates in its ability to infect Pinellia, but maintains the ability to infect some soybean cultivars (Chen et al., 2004). Interestingly, we have observed DsMV-related sequences in the N-terminal region of the P1 gene from the potyvirus Konjak mosaic virus (KoMV), whose genomic sequence was reported recently (Nishiguchi et al., 2006) (Fig. 3b). Corresponding GARD analysis of the aligned nucleotide sequences of SMV, SMV-P, DsMV and KoMV supports evidence for multiple recombination break points, with the P1/HCPro site having the highest score (Fig. 3a), followed by the upstream recombination site predicted from the protein alignment (Fig. 3b). KoMV does not belong to the BCMV subgroup and is related most closely to YMV (Nishiguchi et al., 2006). Both KoMV and DsMV are shown to infect different species of the family Araceae (Lesemann & Winter, 2002), which also includes Pinellia, the natural host of the DsMV/SMV recombinant. These results further support a role for the N terminus of P1 in potyvirus host-range selection.
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) (data not shown). In contrast, tritimoviral P1s constituted an independent branch in the tree, which is consistent with previous phylogenetic reports of the family Potyviridae (Adams et al., 2005b). In addition, some features clearly distinguished tritimovirus P1s from rymovirus and potyvirus P1s. The distance between the His and the Asp or Glu residues in the catalytic triad of potyvirus and rymovirus P1s is 8 aa in 49 of the 50 species analysed and 9 aa in DsMV (Fig. 1). In contrast, these residues were separated by only 7 aa in P1s from the three tritimoviruses whose complete genome sequences have been reported (Fig. 1). Moreover, there is a conserved motif between the Ser residue of the catalytic triad and the autocleavage site, which contains an invariable dipeptide Arg–Gly in all potyviral and rymoviral P1s. However, the Arg residue was replaced by Gln or Met in tritimoviral P1s (Fig. 1).
The most conspicuous difference between the two types of P1 was their pI. Despite having extreme sequence divergence, a universal feature of potyvirus and rymovirus P1s is their high pI, which is >10 in 20 viral species, between 9 and 10 in 27 species and between 8.4 and 8.9 in the remaining three species. In contrast, the pI of the P1 protein of Brome streak mosaic virus was 6.0 and the pI of those of both Wheat streak mosaic virus (WSMV) and Oat necrotic mottle virus was 7.4 (Fig. 1). This difference probably reflects not only a large phylogenetic distance, but also some functional divergence.
P1 duplication in ipomoviruses
As mentioned above, potyvirus P1 proteins show a huge size divergence, ranging from the 211 aa of ScaMV to the 664 aa of SPFMV. However, the size of the ipomovirus cucumber vein yellowing virus (CVYV) P1 reported by Janssen et al. (2005) was 843 aa, which is notably higher. These authors identified a P1-like protease domain near the C-terminal end of the protein, with a catalytic triad formed by His 746, Asp 754 and Ser 789, and a putative cleavage site between Tyr 843 and Cys 844. However, another P1-like protease domain was recognized, with a catalytic triad formed by His 442, Asp 451 and Ser 484, and a presumed scissile bond between Tyr 525 and Thr 526 (Fig. 1). Cleavage at this site would produce two mature proteins, P1a and P1b, of 525 and 318 aa, respectively. When the two putative P1 protease domains were included in the phylogenetic analysis, the P1a domain clustered with the potyviral and rymoviral P1s and the P1b domain was related more closely to the tritimoviral P1s (not shown). The assignment of CVYV P1a and P1b to each P1 type was supported strongly by the following facts: (i) the His and Asp residues of the catalytic triad were separated by eight and seven residues in P1a and P1b, respectively (Fig. 1), (ii) the Arg–Gly dipeptide was present in the conserved domain downstream of the catalytic Ser in P1a, whereas Gln–Gly was the dipeptide present at the equivalent position in P1b (Fig. 1), and (iii) P1a was a basic protein of pI 8.5, whereas P1b had a pI of 5.1 (Fig. 1). Preliminary evidence indicating that the internal protease domain is functional and that cleavage takes place to yield P1a and P1b was obtained recently by transient expression of the complete TAP-tagged CVYV P1a–P1b region in plants (Valli et al., 2006).
The P1 protein of Sweet potato mild mottle virus (SPMMV), the other ipomovirus whose genomic sequence has been published, consisted of 743 aa. Sequence analysis revealed a single protease domain at the C terminus of the protein. This domain clustered with the tritimovirus P1s and the CVYV P1b in phylogenetic analysis. Moreover, the SPMMV P1 had hallmarks of being a tritimovirus-like P1 (Fig. 1): (i) the first two residues of the catalytic triad were not separated by 8 aa, (ii) the Arg that precedes the invariable Gly of the conserved motif located downstream of the Ser of the catalytic triad was absent and (iii) the protein had a low pI (5.4).
Sequence conservation upstream of the protease domain of tritimo-like P1s was rather poor. However, there was a conserved Cys-rich domain that resembled a zinc finger (Fig. 4). The third of four Cys residues that compose the putative zinc finger was replaced by His in the P1 proteins of the tritimoviruses analysed. Zinc finger-like sequences are not a general feature of potyviral P1s. However, Cys and His residues that may form part of zinc-finger structures were detected in several potyviruses: PLDMV/BCMV-Y/EAPV/WMV, SYSV/BCMV-R/BCMNV NL-3 K, OYDV/Pea seed-borne mosaic virus, Leek yellow stripe virus/LMV and Papaya ringspot virus (PRSV)/SPFMV (Figs 2a and 5; Supplementary Fig. S5, available in JGV Online). The functional relevance of these putative zinc fingers remains unknown.
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; Rajamäki et al., 2005; Valli et al., 2006). The ipomovirus CVYV lacks an HCPro gene and its tritimo-like P1b protein appears to compensate for this defect, as it has been shown to have RNA silencing-suppression activity similar to that observed in the potyviral HCPro (Valli et al., 2006). This is conclusive evidence for the occurrence of functional diversification between poty-like and tritimo-like P1s. It would be interesting to assess whether the tritimovirus and ipomovirus SPMMV P1 proteins have RNA silencing-suppression activity. This is likely, considering that an HCPro-deficient mutant of the tritimovirus WSMV is able to establish a productive systemic infection (Stenger et al., 2005).
Evidence for intergenus recombination between ipomovirus and potyvirus P1s
Sequence alignment of the two ipomoviral P1 proteins showed that the only similarity was present between the last 300 aa. Thus, we performed a BLAST analysis of the remaining sequences. The P1a protease domain of CVYV showed clear homology to the P1 protease domains of potyviruses and rymoviruses. Interestingly, sequence similarity to CVYV P1a extended upstream to the N terminus of the P1 protein from a single potyvirus species, PRSV, with an e value of 4.8e–15 in the BLAST search (Fig. 5b; data not shown). Moreover, the N terminus of the ipomovirus SPMMV P1 was related very closely to the potyvirus SPFMV P1 (e value of 4.3e–39). High similarity between the SPMMV and SPFMV P1s ended approximately 183 aa upstream of the His in the SPFMV catalytic triad and 58 aa upstream of the SPMMV region that is similar to CVYV (Fig. 5); no significant similarity was detected for these 58 aa in any other proteins. Low similarity was also detected between the N-terminal regions of PRSV and SPFMV (e value of 0.082), suggesting that these sequences may share a common ancestor. Interestingly, the four sequences shared four conserved cysteines (Fig. 5) that resembled the zinc finger-like motif at the N-terminal region of the tritimo-like P1s (Fig. 4). This would support a model in which the common ancestor of the N-terminal regions of the potyviruses PRSV and SPFMV, the ipomoviruses CVYV and SPMMV and the tritimovirus-like P1 derived from a preceding P1 duplication. In this scenario, SPMMV would derive from an ancient ipomovirus that harboured two copies of the P1 gene, by deletion of the protease domain of the first copy and the first amino acids of the second copy, and the SPFMV P1 would have resulted from a recombination event between the ipomovirus SPMMV and an unknown potyvirus (Fig. 5). Given the high similarity between the homologous SPMMV and SPFMV sequences, this putative recombination event appears to have occurred relatively recently. Similarly, PRSV P1 would have resulted from a recombination event between the ipomovirus CVYV, which retains both P1 copies, and an unknown potyvirus (Fig. 5). In this second case, the recombination event could have occurred much earlier, such that the recombination site would not be recognized easily. Attempts to apply automated tools of recombination detection to these sequences were unsuccessful because of the intrinsic difficulty of aligning sequences with so much divergence (data not shown). However, when a GARD analysis was applied to the PRSV, SPMMV and SPFMV P1 nucleotide sequences that were arranged according to the amino acid alignment shown in Fig. 5, a single break point was detected at position 1276 (P1 aa 379) in the SPMMV sequence (c-AIC score improvement of 76.6). The high score obtained for this recombination site not only confirmed the presumed break point between SPMMV and SPFMV, but also justified the reliability of the alignment.
The sequence shared by the potyvirus SPFMV and the ipomovirus SPMMV suggests strongly that the N-terminal region of their P1s is important for fitness within their common sweet potato host. In this respect, it is important to note that SPFMV and SPMMV are able to coinfect sweet potato (Mukasa et al., 2006), which can facilitate recombination events that result in more well-adapted viruses. Evidence for a relationship between sequence homology in the N-terminal region of P1 and common host adaptation is less compelling for PRSV and CVYV. However, whilst Carica papaya is the nominal host of PRSV, this virus can also infect cucurbits, the only host of CVYV, and previous studies suggest that the papaya-infecting variants of PRSV may have been derived from cucurbit-infecting ancestors (Bateson et al., 2002).
Concluding remarks
Our understanding of plant virus evolution has improved because of renewed interest in the subject, caused in part because plant viruses serve as excellent model systems (reviewed by García-Arenal et al., 2003; Roossinck, 2003). Some evidence for virus coevolution with their hosts and vectors is available, providing information on virus origin and evolutionary history (Lovisolo et al., 2003). Regarding the family Potyviridae, data reported here illustrate the extensive evolutionary divergence of the P1 region in members of this family. In an intuitive scenario, the ancestor of the genus Potyvirus would have a single P1 gene (Fig. 6a). Duplication of a potyvirus P1 gene with a zinc finger-like motif at its N terminus would have produced the ancestor of the ipomovirus and tritimovirus P1s. Each of the P1 copies would have evolved independently and adopted different functions. In the evolutionary branch containing the ipomovirus SPMMV and tritimoviruses, the function of the first protease domain would have been superfluous, and would have been partially or totally eliminated. In the branch containing the ipomovirus CVYV, the functions assumed by the two P1s would have allowed the virus to dispense with the HCPro gene. However, as the first protease domain of CVYV resembles the single protease domain of potyviruses more closely than that of tritimoviruses, P1 duplication and divergence of the two resulting P1 copies could have taken place before the potyvirus, ipomovirus and tritimovirus evolutionary pathways split. According to this hypothesis, potyviruses would have derived from a deletion of the second P1 protease domain (Fig. 6b). Importantly, gene duplication might have facilitated the functional diversification of P1, although, during the course of evolution, some of these functions may have become dispensable in some lineages. Of course, the models presented in Fig. 6 are simplistic and further research will be required to unravel the details of the evolutionary history of potyviruses.
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). However, more recent studies tended to include rymoviruses in the genus Potyvirus (Adams et al., 2005b). Our data do not resolve this issue, but demonstrate that P1 gene recombination between members of different genera in the family Potyviridae is not unusual (Fig. 5) and that both intra- and intergenus recombination can play an important role in host adaptation (Figs 3, 5). Most recombination events described here affect P1 sequences upstream of the protease domain, implying that it could be the N-terminal region of the protein that interacts with the plant host. It would be interesting to assess whether other potyviral P1 activities, such as proteolytic processing and enhancement of RNA silencing suppression, are related functionally to their hypothesized role in host adaptation. This would help to shed light on our understanding of the constraints and driving forces of potyviral evolution.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Received 20 July 2006;
accepted 28 November 2006.
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Motifs that can be recognized in each P1 sequence: 1, motif FG (see Supplementary Fig. S1, available in JGV Online); 2, motif FLxG (Supplementary Fig. S4); 3, motif ISIxGG (Supplementary Fig. S3); 4, motif TPS (Supplementary Fig. S3); 5, motif VELI (Supplementary Fig. S2). Motifs weakly conserved are in italics. 
It appears that a frameshift has occurred in the reading of the sequence between just upstream of the Asp of the catalytic triad and the beginning of the conserved motif RG.
