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Recombination and gene duplication in the evolutionary diversification of P1 proteins in the family Potyviridae

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

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
Genome structure and sequence are notably conserved between members of the family Potyviridae. However, some genomic regions of these viruses, such as that encoding the P1 protein, show strikingly high variability. In this study, some partially conserved motifs were identified upstream of the quite well-conserved protease domain located near the P1 C terminus. The irregular distribution of these motifs suggests that the potyviral P1 proteins have undergone complex evolutionary diversification. Evidence was found of recombination events in the P1 N-terminal region, similar to those reported in potyviruses of the bean common mosaic virus subgroup, but also affecting other potyviruses. Moreover, intergeneric recombination events affecting potyviruses and ipomoviruses were also observed. Evidence that these recombination events could be linked to host adaptation is provided. Specific sequence features and differences in net charge help to classify the P1 proteins of members of the family Potyviridae into two groups: those from potyviruses and rymoviruses and those from tritimoviruses. The ipomovirus Cucumber vein yellowing virus has two P1 copies arranged in tandem, the most N-terminal one being of the potyvirus type and the other being of the tritimovirus type. These findings suggest that both recombination and gene duplication have contributed to P1 evolution and helped to facilitate successful adaptation of members of the family Potyviridae to a wide range of host species.

Amino acid alignments of potyviral and rymoviral P1 protein domains are available as supplementary figures in JGV Online.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
The Potyviridae is the largest family of plant viruses, containing over 100 different species in six genera (López-Moya & García, 1999; 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.

	METHODS

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Table 1 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.

	RESULTS AND DISCUSSION

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General and specific features of potyviral P1 proteins
The P1 gene, together with the coding sequence for the N-proximal region of the capsid protein (CP), shows the greatest variability in size and sequence within the potyvirus genome (Adams et al., 2005b). 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-(x_28–31)-G-x-S-G-(x_10–21)-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/YS. However, exceptions are found at all positions except for the Phe/Tyr located immediately before the cleavage point (Adams et al., 2005a; Fig. 1).

View larger version (103K): [in this window] [in a new window]   Fig. 1. Features conserved in P1 proteins of the family Potyviridae. Asterisks indicate the catalytic residues H, D/E and S, as well as other residues that are invariable in potyviral and rymoviral P1s. Dashes represent gaps. The most conserved residues are in boxes of particular colours according to chemical similarities. Distances to the N-terminal end of the protein or to the previous conserved motif (in aa) are indicated upstream of each sequence. 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.

A highly conserved motif with the consensus sequence (N-terminal end) (x_4–6)-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.

View larger version (45K): [in this window] [in a new window]   Fig. 2. Recombination events at the P1 coding sequence involved in the generation of BCMV-related viruses. (a) Partial amino acid alignment around the putative recombination sites of BCMV-Y and WMV. Boxed amino acids are identical in BCMV-Y and at least one of the other aligned sequences (grey boxes), in SMV and at least one of the other aligned sequences (diagonally hatched boxes) or in BCMV-Y and SMV (black boxes). The position of the first residue in each row is indicated on the left and the number of amino acids downstream of the aligned fragment in each P1 protein is shown in parentheses at the end of the sequence. (b) Partial amino acid alignment around the putative recombination sites of BCMNV NL-3 K and BCMV-R. Amino acids in black boxes are identical in SYSV and at least one other aligned sequence; residues in grey boxes are identical in BCMNV NL-3 K and either BCMV-R or BCMNV NL-3 D, but are not conserved in SYSV. The first residue of the aligned P1 fragments and the number of downstream amino acids (in parentheses) are indicated on the left and right sides of the sequence, respectively. In (a) and (b), dashes represent gaps and bars indicate the putative recombination sites, which coincide with those detected by using GARD analysis of the whole P1 nucleotide sequences (see text for details). (c) Evolutionary pathway proposed for BCMV-related viruses. Boxes represent the P1 proteins and the different shadings indicate the viruses that might supply the different regions. The recombination event involving potyviruses outside the BCMV subgroup is shown with a dashed arrow.

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.

View larger version (53K): [in this window] [in a new window]   Fig. 3. Recombination events involved in the generation of the Pinellia isolate of SMV (SMV-P; GenBank accession no. AJ507388) and KoMV. (a) Partial amino acid alignment around the putative recombination site of SMV-P. Boxed amino acids are identical in SMV-P and the SMV severe strain (SMV; GenBank accession no. AJ312439) (diagonally hatched boxes), in SMV-P and DsMV (grey boxes) and in the three viruses (black boxes). The first residue of each row is indicated on the left and the number of amino acids downstream of the aligned fragment of each potyviral polyprotein is shown in parentheses at the end of the sequence. The border between P1 and HCPro is also indicated. (b) Partial amino acid alignment around the putative recombination site of KoMV. Amino acids in grey boxes are identical between KoMV and DsMV. The position of the first residue of each row is indicated on the left and the number of amino acids downstream of the aligned fragment of each P1 protein is shown in parentheses at the end of the sequence. In (a) and (b), dashes represent gaps and bars indicate the putative recombination sites, which coincide with those detected by using GARD analysis of the whole P1 nucleotide sequences (see text for details). (c) Schematic representation of RNA recombination events. Boxes represent the P1 proteins and the different shadings indicate the viruses that supplied the different regions. Recombination events involving potyviruses outside the BCMV subgroup are indicated by dashed arrows.

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.

View larger version (19K): [in this window] [in a new window]   Fig. 4. Partial amino acid alignment of ipomoviral and tritimoviral P1s. Boxed amino acids are identical or chemically similar between the two ipomoviral sequences (diagonally hatched boxes), between the three tritimoviral sequences (grey boxes) and between at least four of the aligned sequences (black boxes). Dashes represent gaps. The position of the first residue of the aligned P1 fragments and the number of downstream amino acids (in parentheses) are indicated on the left and right sides of the sequence, respectively. P1b of CVYV is considered to begin at aa 526. Cys and His residues that probably compose a zinc-finger structure are shown with asterisks.

View larger version (83K): [in this window] [in a new window]   Fig. 5. Partial amino acid alignment of the P1 proteins of the ipomoviruses CVYV (P1a+P1b) and SPMMV and the potyviruses PRSV and SPFMV. Boxed amino acids are identical or chemically similar between PRSV and CVYV (grey boxes), between SPMMV and SPFMV (diagonally hatched boxes) and between the four aligned sequences (black boxes). Dashes represent gaps. The position of the first residue of each row is indicated on the left and the number of amino acids downstream of the aligned fragment of each P1 protein is shown in parentheses at the end of the sequence. The catalytic triad is shown with asterisks and the cleavage sites that separate P1 from HCPro in PRSV and SPFMV, and P1a and P1b in CVYV are indicated by an arrow. The region of the putative recombination site involving SPFMV and SPMMV, which coincided with that detected by using GARD analysis of the whole P1 nucleotide sequences, is underlined (see text for details). (b) Schematic representation of RNA recombination events that may have produced the existing viral sequences and the putative parental lineages that are involved.

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.

View larger version (13K): [in this window] [in a new window]   Fig. 6. (a, b) Two alternative models for evolution of the P1–HCPro region in the family Potyviridae. Boxes represent the P1 and HCPro proteins. The conserved protease domains and the divergent P1 N-terminal domains are represented by solid and framed shading, respectively. Green and blue scissors mark serine protease and cysteine protease domains, respectively. Different colours illustrate different evolutionary lineages.

	ACKNOWLEDGEMENTS

 
We would like to thank M. J. Adams for his advice on identification of the CVYV internal P1a cleavage site. J. Castresana and D. Posada are acknowledged for guidance on selection of bioinformatic tools. This work was supported by grants BIO2004-02687 from the Spanish MEC, CPE03-022-C5 from INIA and SP22-CT-2004 from the European Union to J. A. G. and by grant AGL2004-00704 from the Spanish MEC to J. J. L.-M. Consorci CSIC-IRTA receives support from the Centre de Referència en Biotecnologia (CeRBa) of the Generalitat de Catalunya. A. V. was a recipient of an I3P fellowship from CSIC-Fondo Social Europeo.

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Received 20 July 2006; accepted 28 November 2006.

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