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Journal of Virology, August 2006, p. 8124-8132, Vol. 80, No. 16
0022-538X/06/$08.00+0     doi:10.1128/JVI.00603-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Insights into the Selective Pressures Restricting Pelargonium Flower Break Virus Genome Variability: Evidence for Host Adaptation

Patricia Rico, Pilar Ivars, Santiago F. Elena, and Carmen Hernández*

Instituto de Biología Molecular y Celular de Plantas, Consejo Superior de Investigaciones Científicas-UPV, 46022 Valencia, Spain

Received 24 March 2006/ Accepted 4 June 2006


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ABSTRACT
 
The molecular diversity of Pelargonium flower break virus (PFBV) was assessed using a collection of isolates from different geographical origins, hosts, and collecting times. The genomic region examined was 1,828 nucleotides (nt) long and comprised the coding sequences for the movement (p7 and p12) and the coat (CP) proteins, as well as flanking segments including the entire 3' untranslated region (3' UTR). Some constraints limiting viral heterogeneity could be inferred from sequence analyses, such as the conservation of the amino acid sequences of p7 and of the shell domain of the CP, the maintenance of a leucine zipper motif in p12, and the preservation of a particular folding in the 3' UTR. A remarkable covariation, involving five specific amino acid sites, was found in the CP of isolates largely propagated in the local lesion host Chenopodium quinoa and in the progeny of a PFBV variant subjected to serial passages in this host. Concomitant with this covariation, up to 30 nucleotide substitutions in a 1,428-nt region of the viral RNA could be attributable to C. quinoa-specific adaptation, representing one of the most outstanding cases of host-driven genome variation for a plant virus. Globally, the results indicate that the selective pressures exerted by the host play a critical role in shaping PFBV populations and that these populations are likely being selected for at both protein and RNA levels.


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INTRODUCTION
 
It is commonly believed that the high mutation rates characteristic of RNA viruses, together with their short replication times and large populations, favor their rapid adaptation to changing situations (12, 13). Despite the high potential for genetic variation of these pathogens, a considerable number of studies on the diversity of plant virus populations have found a remarkable genetic stability (reviewed in reference 21). This stability is usually considered to be the result of selective pressures inherently imposed by genome replication and expression strategies and by virus-vector and virus-host interactions. In addition, random genetic drift due to bottlenecks associated with transmission or colonization events may also limit the eventual accumulation of variation (21, 23, 47). Investigating the factors affecting the diversity levels of viral populations can undoubtedly provide significant clues for the development of efficient and stable control strategies for viral pathogens (22, 27, 49).

Pelargonium flower break virus (PFBV) is one of the most prevalent viruses infecting geraniums (Pelargonium spp.) throughout the world (1, 3, 6, 19, 41, 52). Its most conspicuous symptoms are white flower streaking, chlorotic spotting of leaves, and growth reduction causing significant losses in crop production. PFBV is mainly transmitted by vegetative propagation and mechanical inoculation, although transmissions by irrigation systems and by the western flower thrips Frankiniella occidentalis may also occur (30).

PFBV is a member of the genus Carmovirus (family Tombusviridae) and, like all the species in the group, produces icosahedral virions that encapsidate a linear positive-sense single-stranded RNA (33). The complete nucleotide sequence of PFBV genomic RNA has been recently determined (45). It comprises 3,923 nucleotides (nt) and contains five open reading frames (ORFs) flanked by untranslated regions (UTRs) of 32 nt and 236 nt at the 5' and 3' termini, respectively. The 5'-proximal ORF encodes a 27-kDa protein (p27) and terminates with an amber codon which may be read through into an in-frame p56 ORF to generate an 86-kDa protein (p86) containing the viral RNA-dependent RNA polymerase (RdRp) motifs. Two small ORFs, located in the central part of the viral genome, encode polypeptides of 7 (p7) and 12 (p12) kDa, respectively, which are presumably involved in virus movement, and the 3'-proximal ORF encodes a 37-kDa capsid protein (CP). This genome organization was deduced from a Spanish isolate, and no additional sequences from other isolates have been reported so far, with the exception of those corresponding to the CP gene and flanking sequences of a Dutch isolate and a small portion (408 nt) of the RdRp gene of a Russian isolate (5, 39). Moreover, in contrast with other viral groups for which extensive analysis of sequence variation has been done (see reference 22 and references therein), studies on the genetic heterogeneity of carmoviruses are very scarce, precluding an estimation of the genome stability in this viral genus.

To gain insights into the molecular variability of PFBV, we have determined the sequence of a region of 1,828 nt from 18 PFBV isolates from two distinct Pelargonium spp., five different countries, collected at different times. The genomic portion examined corresponds to approximately the 3' half of the viral genome and comprises the p7, p12, and CP genes together with the 3' UTR. Comparative sequence analyses have allowed identification of conserved elements within the viral RNA and/or the encoded proteins and establishing of phylogenetic relationships between isolates. Interestingly, the information derived from these comparative analyses suggested that prolonged propagation of PFBV in the local lesion host Chenopodium quinoa may drive significant changes in the composition of the viral populations. To confirm this point, the pattern of molecular evolution of a single PFBV variant subjected to serial transfers in this experimental host has been investigated. The results indicate that host plant species may be a major factor in shaping the structure of PFBV populations.


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MATERIALS AND METHODS
 
Viral isolates. Field PFBV isolates were collected at different times from two Pelargonium hosts and distinct geographical locations (Table 1). Sap from the original plants was used to transmit the virus into the propagation host C. quinoa by mechanical inoculation, and the viral population was recovered from this infected material. The isolates were named using the initials of the country of origin followed by a number to distinguish isolates from the same country. In addition, isolates PV-0201 and PV-0202 were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). Hereafter, these isolates will be designed as DSMZ1 and DSMZ2, respectively.


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  		TABLE 1. PFBV isolates used in this work

Reverse transcription, PCR amplification, cloning, and sequencing. Total RNA preparations obtained from infected C. quinoa leaves by phenol extraction and lithium precipitation (57) were used as templates for reverse transcription (RT) reactions with Superscript II-RT (Invitrogen) and primer CH1 (5'-TTCCCGGGCGGGTTAAGGTCTCCATC-3'), complementary to the 3' terminus of the PFBV genome (nt 3903 to 3923) with some additional nucleotides underlined. RT products were PCR amplified with the Expand High Fidelity PCR system (Roche) and two pairs of primers: CH1 in combination with CH2 (5'-ATGGTGGTAATGGGGGTTCTTGGGTTG-3'), homologous to nt 2424 to 2450, and CH7 (5'-GAGTACTGATTGTGCGTTACTCTCTCTGCC-3'), complementary to nt 2570 to 2599, in combination with CH31 (5'-ATCAGGAACGTACAGGTTAGCCCAG-3'), homologous to nt 2096 to 2120. The PFBV genomic regions amplified with these primers covered the p7, p12, and CP genes as well as the 3' UTR. Cycling conditions consisted of an initial denaturizing step at 94°C for 2 min, followed by 35 cycles at 94°C for 30 s, 60 to 65°C (depending on the isolate and primer combination) for 30 s, 68°C for 3 min, and a final elongation step at 68°C for 10 min. The resulting RT-PCR products were separated by electrophoresis in 1% agarose gels, eluted, and cloned into the pGEM-T easy vector (Promega) or the plasmid pCR2.1 (Invitrogen). Two clones were randomly selected for each DNA fragment, and the nucleotide sequence of the inserts was determined with an ABI PRISM DNA sequencer 377 (Perkin-Elmer).

Serial passage experiments. Transcripts were generated in vitro with T7 RNA polymerase (Fermentas) from construct pSP18-IC, an infectious clone of PFBV derived from isolate SP18 (46), and used to mechanically inoculate C. quinoa plants (three leaves per plant, 0.7 µg of transcript per leaf). This initial inoculation constituted passage zero. Subsequent passages (1 through 7) were done at 7-day intervals, using as inocula total RNA preparations from the infected plant material. Eight plants were inoculated in each passage, and 7 g of infected leaves were pooled to prepare the inoculum for the next passage. Plants were maintained under greenhouse conditions (16 h light at 24°C, 8 h dark at 20°C) after inoculation. Total RNA preparations from infected leaves of passages 1, 2, 4, and 7 were used as templates for RT-PCR amplification, cloning, and sequencing of the PFBV genome segment between positions 2424 and 3923.

Sequence analysis. The coding capacity of the nucleotide sequences was determined by the program TRANSLATE (available at the ExPASy proteomics server; http://www.expasy.org/tools/dna.html). Pairwise comparisons of the nucleotide and amino acid sequences were performed using the program LALIGN at the EMBnet server (http://www.ch.embnet.org/software/LALIGN_form.html) (42). Multiple alignments of deduced protein sequences were generated with the program MUSCLE (14). Nucleotide sequences were then aligned, concatenating triplets according to the amino acid sequence alignment using the DAMBE program, version 4.2.7 (59). For each genomic region, the model of nucleotide substitutions that better explained the observed pattern of variability was inferred by a maximum likelihood approach using the FINDMODEL server (http://hcv.lanl.gov/content/hcv-db/findmodel/findmodel.html). Similarly, the model of amino acid substitution that maximized the likelihood of the observations was inferred using the PROTTEST server (http://darwin.uvigo.es/software/prottest_server.html). Neighbor-joining phylogenetic trees, using the most likely models of nucleotide or amino acid substitutions, were obtained using the MEGA package, version 3.1 (31). The statistical reliability of the constructed trees was assessed by the bootstrap method based upon 10,000 pseudoreplicates. The homologous sequences of Carnation mottle virus (CarMV) were used for rooting purposes (accession number CMO309511). To identify specific amino acid sites under selective constraints, the difference between nonsynonymous (dN) and synonymous (dS) substitution rates was estimated for each position in the alignments using a fixed-effects likelihood (FEL) method (29) as implemented in the HYPHY server (http://www.datamonkey.org). A dN – dS value of >0 is taken as evidence for positive or directional selection, whereas values of <0 are a signature for negative or purifying selection (29). Finally, the RNA secondary structure of the PFBV 3' UTR was predicted using MFOLD version 3.1 (36, 63).

Nucleotide sequence accession numbers. The 36 sequences reported in this paper have been deposited in the EMBL database under accession numbers DQ443003 to DQ443012 and DQ443014 to DQ443039.


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RESULTS
 
Analysis of nucleotide variability of PFBV reveals a noticeable genomic conservation. A fragment of 1,828 nt corresponding approximately to the 3' half of the PFBV genomic RNA was retrotranscribed and amplified for each PFBV isolate listed in Table 1. The likelihood of artifactual changes during PCR amplification was minimized by using a thermostable DNA polymerase endowed with proofreading activity. The resulting DNA fragments were cloned, and two clones per isolate were randomly selected and sequenced in both directions. None or no more than one nucleotide difference was found in the overlapping regions of the two cDNAs generated to cover the region examined, and therefore the sequences of these cDNAs were concatenated to facilitate subsequent analyses. Sequences were named for the isolates from which they were derived, followed by a number when more than one clone was included for comparison. Sequences from the previously characterized isolate SP10 (45) were also incorporated into the analyses, adding thus up to 19 isolates (Table 1).

On average, the two nucleotide sequences derived from a given isolate showed identities higher than 99.8% (with a standard error of 0.3%), suggesting that every isolate is composed of a relatively homogeneous mixture of RNA species. Considering the 37 sequence variants included in the study, the number of polymorphic positions in the region analyzed was relatively low (187, representing 10.2% of the positions in the alignment), and the vast majority of these positions (94.6%) had only one other base as an alternative. The percentage of variable positions was 6.3% within the p7 gene, 9.3% within the p12 gene, 10.8% within the CP gene, and 8.4% in the 3' UTR. Mutations were randomly distributed across the four gene regions ({chi}2 = 5.427; 3 df; P = 0.143). Some variations surrounding the ORF start codons were detected, but they did not alter the translation context. In addition, variation was found in the stop codon of the ORF(p7), being UAG for the DSMZ1 and DSMZ2 isolates but UAA in the other cases. A bias for transition mutations was observed (81.5% transitions versus 18.5% transversions), similar to that found in other viral systems (32, 34, 49, 56). Net nucleotide sequence identities between isolates ranged from 96.4% to 99.8%, suggesting a high level of conservation despite their diverse geographical origins.

Insights into the selective constraints operating on coding regions. The direction and intensity of the selective constraints operating in a coding region were estimated by the difference between dN and dS substitution rates. Table 2 shows the average values of dN, dS, and dN – dS (with standard errors) estimated for each genomic region using MEGA. For all three coding regions, dN was significantly smaller than dS (Table 2), supporting the notion that purifying selection has been acting as a major force during the diversification of PFBV isolates. Concurrently, the FEL analysis at the HYPHY server identified several amino acid sites with dS values significantly greater than dN values but not a single case of positive selection. In the case of p7, three negatively selected sites were obtained (D13, E15, and N25). In the case of p12, three amino acids also were detected as targets of negative selection (S56, T70, and Y99). The situation for CP is more complex, and 24 amino acid sites were detected as negatively selected. As reported for carmoviruses and other members of the family Tombusviridae (33), three different structural domains can be distinguished in the PFBV CP: (i) R, the N-terminal internal domain which contains many positively charged residues and interacts with RNA, (ii) S, the shell domain which forms a barrel structure made up of ß strands and constitutes the capsid backbone, and (iii) P, the protruding C-terminal domain. When the average dN – dS value was computed for each of the CP structural domains, several observations came out: (i) all three domains were under significant purifying selection, (ii) the intensity of selection on domain S was approximately 4.2 times stronger (Table 2), suggesting that more functional and/or structural constraints act on this structural domain, and (iii) in agreement with this observation, the number of negatively selected sites estimated by FEL was nonrandomly distributed among the three domains ({chi}2 = 7.603; 2 df; P = 0.022), being more abundant in domain S (18 sites) than in domains R and P (1 and 5 sites, respectively). However, it is fair to call for special caution when considering the statistical significance of the FEL results reported, since none of the sites identified as negatively selected remained significant after the significance level was adjusted, by Bonferroni's sequential method, to account for multiple tests of the same hypothesis.


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  		TABLE 2. Average synonymous and nonsynonymous substitution rates for each coding regiona

Natural diversity in the 3' UTR supports the folding predicted in silico. As indicated above, considerable sequence variation was found in the 3' UTR of PFBV, with nucleotide identity values ranging from 96.2% to 100% between isolates. Due to the noncoding nature of the 3' UTR, it is reasonable to assume that its functionality will be determined by the preservation of structural motifs. To corroborate this hypothesis, the minimum free-energy folding for the 3' UTR of PFBV was predicted with the MFOLD program (Fig. 1). The resulting structure resembled, at least partially, those proposed for related viruses (16, 40, 58, 62). The sequence variability found in this region essentially maintained the predicted conformation, since most mutations were located in single-stranded regions or, when affecting double-stranded regions, they either were compensatory or were located at the base of loops or stems (Fig. 1). The only exceptions were the changes U3776C and C3821A, which would disrupt base pairs at the middle of a stem, but these mutations were found only in one and two clones, respectively. All mutations but one, an A inserted at position 3831 in one of the SP10 clones, corresponded to nucleotide substitutions, with the transitions being more frequent than the transversions (14 transitions versus 5 transversions), as in the coding regions.


Figure 1
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  		FIG. 1. MFOLD-predicted RNA secondary structure of the 3' UTR of PFBV showing the distribution of polymorphic positions. The most stable folding for the reference sequence, corresponding to isolate SP10 (45), is depicted, although it is consistent for every sequence variant characterized. Nucleotide substitutions are indicated by arrows, and the only insertion detected is preceded by a plus sign. Numbers denote positions in the PFBV genomic RNA. The nucleotides of the putative replication silencer of PFBV, able to establish a base-pairing interaction with the last 3' nucleotides, are white in black circles.

PFBV proteins show different degrees of sequence conservation. The comparison of the p7 movement protein sequences of the 19 isolates presented an extremely high level of conservation, with only two isolates showing amino acid changes at three different positions (S19P in isolate SP18 and I6T and V55I in isolate KN5). The percentage of polymorphic positions was slightly higher in the case of p12 (13 out of a total of 106 amino acid residues, representing 12.3%). The polymorphism was the result of amino acid substitutions except for a deletion of the last amino acid residue in isolates SP1 to SP4 (as a consequence of the transition C2643U, which converted the codon CAA, specifying Q106, into a stop codon). The protein sequence was totally conserved within isolates, with the only exception being isolate KN3 (differences among KN3 variants are shown in Fig. 2), whereas amino acid identities between isolates ranged from 95.3% to 100%. Curiously, most of the changes among isolates were located in the N-terminal region of the protein (Fig. 2) despite its coding sequence overlaps in a different frame with that of p7, suggesting that the latter one is under stricter selective constraints.


Figure 2
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  		FIG. 2. Alignment of partial amino acid sequences of p12 from different isolates of PFBV. The reference sequence derived from isolate SP10 (45) is shown at the top of the figure. Those residues conserved in all isolates are indicated by dots. Only the two sequences from isolate KN3 presented amino acid variations in the regions shown here, and both have been included in the alignment. The L residues at position d of the heptads potentially involved in leucine zipper formation are shown in underlined bold characters, and positions a and g of the first heptad are indicated with lowercase letters on the top. Numbers above the reference sequence correspond to positions of the residues in the complete protein.

A peculiar trait of PFBV p12 is the presence of a putative leucine zipper motif which is absent in related proteins and that is predicted to be four heptads in length (45). Four of the amino acid changes detected from sequence comparison among isolates affected positions of the first (position e), third (positions a and c), and fourth (position f) heptads but corresponded to conservative substitutions between either nonpolar (changes I61M and L71M) or polar (changes S73T and E83Q) amino acids (Fig. 2). Strikingly, the amino acid substitution S53L found in isolate KN5 would extend the number of heptads from four to five (Fig. 2). It is also worth highlighting the strict preservation of key L residues at position d of the heptads (38), despite the considerable nucleotide variation detected in the corresponding codons. For example, three and four different codons were found specifying L at position d of the second and third heptads, respectively. Concurrently, FEL analysis found positions d of these two heptads to be under negative selection, with the estimates being only marginally nonsignificant (P = 0.079 and P = 0.075, respectively).

Concerning the CP, amino acid identities ranged from 97.4% to 100% among isolates. In agreement with the above results on negatively selected sites across CP domains, the percentages of polymorphic positions were considerably higher in the R (15.9%) and P domains (11.4%) than in the S domain (5.8%), although this difference was only marginally nonsignificant ({chi}2 = 5.916; 2 df; P = 0.052). Remarkably, a covariation between amino acid positions 39 and 49, embedded within the R domain, and amino acid positions 248, 271, and 284, included within the P domain, was found (Fig. 3). Specifically, the changes A39V and S49P correlated with the changes H248Y, M271I, and V284I, suggesting the existence of tertiary interactions between the two regions of the molecule. The 19 isolates included in this study could be classified into two main phenetic groups according to the specific pattern of amino acids shown at the above positions, a proposal supported by phylogenetic analysis (see below).


Figure 3
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  		FIG. 3. Schematic representation of the PFBV CP showing the location of the amino acid changes found among viral isolates. The three domains of the protein (R, S, and P) are outlined with white boxes and the small amino acid arm that connects the R and S domains with a gray box. The amino acid changes with respect to the CP of the reference sequence of isolate SP10 (45) are indicated. The mutations giving rise to a five site-specific amino acid covariation are shaded.

Phylogenetic analyses display two main groups of PFBV isolates. Phylogenetic trees were constructed with both nucleotide and amino acid sequences for the three genomic regions independently as well as concatenating them. The neighbor-joining tree showing the highest bootstrap support values was found for CP when the evolution of amino acid sequences was modeled by using the empirical JTT substitution matrix (26). The resulting phylogram shows a significant monophyletic clustering for the two DSMZ isolates (hereafter group II), with a bootstrap P value of 0.98, whereas the rest of the isolates may have a polyphyletic origin (hereafter group I) (Fig. 4). The biological relevance of this classification into two groups is grounded in the aforementioned covariation found at different positions of the R and P domains of the protein (Fig. 3). Since group I embraced isolates of different geographical origins collected through the last 5 years, while group II was formed exclusively by two isolates collected about 15 years ago (Table 1), the results may suggest significant variation in the genetic composition of isolates separated in time. Alternatively, the two DMSZ isolates have been maintained by serial propagation onto C. quinoa, in contrast with the rest of isolates, and the observed genetic variation could be attributable to host-specific selection, a factor that may result in a decrease or a bias in diversity within a viral population (20). Indeed, if host-specific selection operated on the branch leading to the DSMZ isolates, then acceleration in the rate of nonsynonymous substitutions should be expected for this branch, whereas the rate of synonymous substitutions would remain the same. To test these predictions, two Tajima's relative ratio tests (54) were computed using MEGA. In both tests, isolates GER1 and CZ1 (as outgroup) were compared with isolate DSMZ1 (alternative comparisons gave identical results; data not shown). Nine synonymous substitutions occurred in the DSMZ1 lineage and seven in the GER1, with the difference among rates of synonymous substitutions being not significant ({chi}2 = 0.25; 1 df; P = 0.617). Five nonsynonymous substitutions occurred in the DSMZ1 lineage and none in the GER1, with the difference being significant in this case ({chi}2 = 5.00; 1 df; P = 0.025). In conclusion, the isolates serially passed into C. quinoa experienced an accelerated accumulation of nonsynonymous changes, as expected by host-imposed positive selection. Relevant to the issue of host-driven adaptation is the observation that the four PFBV isolates recovered from Pelargonium peltatum (SP1 to SP4) consistently clustered together, forming a subgroup within group I (Fig. 4, bootstrap P = 0.88), reinforcing the possibility of the genetic composition of PFBV populations being highly controlled by virus-host interactions, although this observation should be taken with caution, since the P. peltatum isolates were collected within a small area and, therefore, other factors besides host adaptation could contribute to their high level of similarity.


Figure 4
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  		FIG. 4. Neighbor-joining phylogenetic tree inferred from CP amino acid sequences derived from 19 isolates of PFBV (see Table 1). The numbers at the nodes indicate the number of times out of 10,000 trees that this grouping occurred after bootstrapping of the data; only values of >70% are shown. The root corresponds with the location of CarMV.

Host adaptation is responsible for the major genomic changes detected among the two groups of PFBV isolates. To investigate whether the significant segregation of the DSMZ isolates in Fig. 4 could be the result of host-specific adaptation, as suggested by the above analysis, an infectious PFBV clone derived from isolate SP18, a representative of group I, was used to study CP sequence variations throughout serial passages onto C. quinoa. This clone, named pSP18-IC, was obtained by fusing a full-length PFBV cDNA to the T7 RNA polymerase promoter (46). The RNAs synthesized from pSP18-IC by in vitro transcription were used to establish an initial infection in C. quinoa which was subjected to serial transfers in the same host. Partial cDNA clones encompassing nucleotides 2424 to 3923 of the PFBV genome were generated from the progenies of passages 1, 2, 4, and 7 by RT-PCR amplification and vector ligation. Two clones from each passage were randomly selected and fully sequenced. Analysis of the CP progeny sequences showed the progressive appearance of the covariation among the five site-specific amino acid positions characteristic of the DSMZ isolates (Table 3). In passage 1, two of the five specific mutations (H248Y and V284I, located in the P domain of the CP) were already found in one of the clones of the progeny viruses, and one more passage was enough for detecting the covariation of the five specific amino acid positions. Whereas in these first two passages, a mixture of parental SP18 and mutated sequences was found, the five-amino-acid covariation was present in the two clones of passage 4 and remained apparently unchanged up to passage 7. Some punctual amino acid changes were also observed in other regions of the CP in several clones, but they were not maintained in subsequent passages with the exception of the substitution V3A. However, A was detected at position 3 in all isolates but SP18, SP12, and SP20 (data not shown), and therefore a host effect on the fixation of this change seems unlikely.


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  		TABLE 3. Analysis of PFBV CP sequence variations through serial passages in C. quinoaa

The nucleotide substitutions that gave rise to the specific amino acid changes were the same for all clones checked and, moreover, exactly matched those present in the DSMZ variants (Table 3). Indeed, a detailed inspection of the progeny nucleotide sequences revealed that, apart from the mutations leading to the CP amino acid covariation, up to 30 additional nucleotide substitutions characteristic of the DSMZ isolates were fixed through the passages. The nucleotide changes were located both in coding regions and in the 3' UTR (Fig. 5), and although not all of them were exclusive to DSMZ isolates, such a specific combination of mutations was found only in these isolates. The mutations included even the change UAA -> UAG in the stop codon of the ORF(p7) mentioned above. Globally, the results indicated that specific and strong selection processes for certain PFBV variants are taking place in C. quinoa and, moreover, that the viral populations are being selected at both protein and RNA levels.


Figure 5
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  		FIG. 5. Condensed alignment showing the PFBV nucleotide sequence variations through serial passages in C. quinoa in the genomic region encompassing nt 2424 to 3923. SP18 corresponds to the parental sequence derived from the infectious PFBV clone pSP18-IC (46), and DSMZ represents any of the sequence variants characterized from isolates DSMZ1 and DSMZ2. Only those nucleotide changes fixed through the passages are shown. The numbers at the top indicate the positions in the genomic RNA, and the lines at the bottom demarcate the regions to which the residues belong. The asterisk marks the nucleotide that separates the coding regions of p12 and CP. The nucleotide changes giving rise to the five-amino-acid covariation in the CP have not been included, since they are shown in Table 3.


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DISCUSSION
 
To gain insights into the factors that influence molecular variability of PFBV, the sequences of the 3'-terminal half of the genomic RNA of 19 PFBV isolates have been compared. The nucleotide changes detected among the sequences derived from a given isolate indicate that PFBV, like many other RNA viral pathogens, propagates as a population of similar but not identical molecular variants termed quasispecies (15). The degree of nucleotide variability within PFBV isolates (0.16%, on average) fell within the range of those estimated for other RNA viruses (2, 4, 17, 25, 51) and revealed high sequence homogeneity in each propagating population. Considerable sequence conservation was also observed between isolates despite their very diverse origins, in good agreement with other viral plant pathogens (2, 8, 18, 35, 50) and suggesting that the PFBV genome is under strong selective constraints. These strong selective constraints have been evaluated by comparing the rates of synonymous and nonsynonymous substitutions across codon sites. Overall, we have shown that negative selection is a major evolutionary force shaping nucleotide diversity of the three coding regions analyzed. Several specific amino acid sites have been identified as targets of this type of selection, although in no instance did the prediction pass the more stringent significance level imposed by Bonferroni's method for multiple tests of the same null hypothesis. This lack of statistical power is clearly a consequence of the reduced sample size. Sequences from more isolates, rather than clones from the same isolate, would be desirable to solve this statistical problem. By contrast, not a single case of positive selection has been detected after comparing the sequences from different isolates. However, a test build on different theoretical grounds has detected acceleration in the rate of nonsynonymous substitution in the branch leading to the DSMZ isolates. As discussed below in more detail, we provide unquestionable experimental evidence of host-driven positive selection when PFBV isolates adapt to the C. quinoa.

Besides the selective constraints on coding regions, the need to maintain a certain conformation seems to restrict sequence heterogeneity in the 3' UTR. This observation is consistent with the critical role of this genomic region, which, as reported for numerous RNA viruses, might contain cis elements essential for viral replication. Indeed, studies with several members of the Tombusviridae have revealed that the 3'-proximal stem-loop comprises the minimal promoter for RdRp recognition and is needed for efficient in vivo replication of the viral RNAs (7, 9, 16, 53, 55). In addition, a sequence located about 50 nt upstream of the 3' terminus and part of the large internal loop of a hairpin has been shown to act, through a base-pairing interaction with the last 3' nucleotides, as a replication silencer in Tomato bushy stunt virus and in Turnip crinkle virus (43, 60, 61), and similar interactions are predicted for other tombusviruses and carmoviruses, including PFBV (62). Remarkably, the internal loop containing the putative PFBV replication repressor, which is asymmetrical in contrast with most carmoviruses (60, 62), becomes symmetrical in the sequence variant harboring the A insertion at position 3831 (Fig. 1), suggesting that asymmetry in this loop is not an absolute requirement for viral viability.

The considerable sequence conservation found in p7 and p12 resembles that reported for the corresponding proteins of another carmovirus, CarMV, although the numbers of amino acid changes affecting the p7 and p12 homologs were higher and lower, respectively, than those found for the PFBV proteins (8). The N-terminal region of PFBV p12 concentrated an important part of the variability observed among isolates, suggesting a noticeable sequence flexibility for this region of the molecule, which, moreover, corresponds to an extension exclusive to this protein (the homologous proteins of most carmoviruses have estimated sizes of 8 or 9 kDa in contrast with the 12 kDa of PFBV p12). On the other hand, the detection of conservative amino acid changes in the putative leucine zipper of PFBV p12, its presumed enlargement in one of the isolates, and the strict preservation of the key L residues argue in favor of the functional relevance of the motif, a question which is currently under investigation.

Concerning the CP, the higher conservation of the S domain than of the R and P domains is in agreement with the general layout from sequence comparisons among CPs of carmoviruses and other members of the family Tombusviridae (5, 10, 11). Phylogenetic analysis based on the CP sequences displayed a clear segregation of isolates DSMZ1 and DSMZ2 which have been transferred many times in the local host C. quinoa. A first evidence of natural selection driving adaptation to C. quinoa came from the observed acceleration in the rate of nonsynonymous substitutions occurring in the DSMZ lineage. This finding was later experimentally confirmed by the analysis of the molecular evolution of a single PFBV variant subjected to serial transfers in C. quinoa. The evolution experiments clearly showed that the main differences between the two groups of isolates are attributable to host adaptation and that clustering of the DSMZ isolates in the CP-based phylogeny (Fig. 4) is likely the consequence of convergent evolution rather than common ancestry. Recent reports have shown that serial passages of Hibiscus chlorotic ringspot virus (HCRSV), another member of the genus Carmovirus, in C. quinoa also results in alteration of the CP amino acid composition of the progeny (32). Similarly to what we have found for PFBV, the changes detected in the HCRSV CP after successive transfers in C. quinoa corresponded to a covariation of eight site-specific amino acids which was consistent and reproducible in independent serial passage experiments. Paralleling those results, the finding of the same five amino acid substitutions in the CP of the progeny of a single PFBV-SP18 variant subjected to serial transfers into C. quinoa and of isolates largely propagated in this experimental host, such as DSMZ1 and DSMZ2, indicates that these substitutions are not likely to be random events but must be due to host-associated positive selection. Moreover, this selection must act on molecular variants that appeared de novo, since genetic variability in the initial inoculum was null as it was produced by in vitro transcription from clone pSP18-IC.

The precise virus-host interaction acting as the driving selective force leading to the covariation equilibrium observed is currently unknown. However, it is interesting to note that the first host-specific mutations were detected in the protruding domain of the CP. They may have been selected because they enhanced the specificity or the strength of interactions with some host-specific factor(s). These initial mutations could have led to the fixation of the other three amino acid substitutions to readjust the stability of the viral particle. The concomitant fixation of abundant nucleotide substitutions is remarkable and suggests a concerted shaping of different regions of the viral RNA to improve fitness. The necessity of maintaining middle-range RNA-RNA interactions in the viral genome or RNA-protein (such as the CP and/or other viral proteins or host factors) interactions may account for the covariations observed in the coding and noncoding regions of PFBV.

The rapid alteration of the genetic structure of the populations of PFBV and HCRSV after consecutive transfers in an experimental host contrasts with that reported for other plant viral pathogens, such as Tobacco mosaic virus, Cucumber mosaic virus, or Cowpea chlorotic mottle virus, which showed high genetic stability after serial passages in different hosts with no mutation or only one mutation becoming fixed (48, 49). In line with these results, no evidence for host-driven selection was obtained upon periodical transfer of Wheat streak mosaic virus in distinct cereal host species (24), and no nucleotide changes were detected in the 3' UTR of Cherry leaf roll virus after more than 40 passages in C. quinoa (44). The unlike behavior of these viruses suggest more-specific interactions among carmoviruses and their hosts than those established by members of other taxonomic groups and/or an intrinsic capability of carmoviral populations to evolve rapidly. The distinctive features of the proteins involved in replication in the different viral pathogens (PFBV and HCRSV belong to supergroup II, whereas TMV, CMV, CCMV, CLRV, and WSMV are members of the other two supergroups in the RdRp-based classification of positive-strand RNA viruses [28]) could lead to higher mutation rates in carmoviruses, thus facilitating a quicker optimization of the viral populations after challenge with new environments, although at this stage, this proposal is merely speculative.

In the case of HCRSV, the adaptation to C. quinoa led to avirulence in the original host kenaf (32), which has been recently linked with the reduced ability of the CP with the C. quinoa-induced mutations to suppress posttranscriptional gene silencing (37). Whether the PFBV variants selected through passages onto C. quinoa have lost any fitness in the original host, Pelargonium zonale, remains to be explored.


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ACKNOWLEDGMENTS
 
We are grateful to R. Wölk for providing PFBV isolates from Germany and Kenya, to V. Mokrá for the Czech isolate, and to M. Borja for the Spanish isolates and for valuable comments in the course of this work.

This research was supported by grants AGL2000-0942 and AGL2003-04249 (to C.H.) and BMC2003-00066 and BFU2005-23720-E/BMC (to S.F.E.) from the Spanish MEC-FEDER, by grant Grupos 03/064 from the Generalitat Valenciana (to C.H. and S.F.E.), and by the EMBO Young Investigator Program (to S.F.E.). P.R. was the recipient of predoctoral fellowships from the Generalitat Valenciana and from CSIC (I3P program).


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FOOTNOTES
 
* Corresponding author. Mailing address: Instituto de Biología Molecular y Celular de Plantas (CSIC-UPV), Campus Universidad Politécnica de Valencia, Avenida de los Naranjos s/n, 46022 Valencia, Spain. Phone: 34 963 877 869. Fax: 34 963 877 859. E-mail: cahernan{at}ibmcp.upv.es. Back


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Journal of Virology, August 2006, p. 8124-8132, Vol. 80, No. 16
0022-538X/06/$08.00+0     doi:10.1128/JVI.00603-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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