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1 Sezione di Genetica e Genomica Vegetale, ENEA CR Casaccia, Via Anguillarese 301, 00060 Rome, Italy
2 Proteomics and Mass Spectrometry Laboratory, ISPAAM, National Research Council, 80147 Naples, Italy
Correspondence
Selene Baschieri
selene.baschieri{at}casaccia.enea.it
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Supplementary methods are available in JGV Online.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). A promising application of engineered plant viruses is the development of epitope vaccines by the generation of chimeric virus particles (CVPs) that display peptides of interest on their surface as fusions to the coat protein (CP) (Scholthof et al., 1996). The potexvirus Potato virus X (PVX) has been shown to be an ideal, highly ordered, multivalent scaffold to be used to this purpose (Brennan et al., 1999; Marusic et al., 2001). PVX could also be used as a carrier of whole proteins, although this approach presents some disadvantages in vaccine applications due to the unpredictability of the number of chimeric CP units per virion, precluding an exact determination of the vaccine dose (Santa Cruz et al., 1996).
PVX filamentous particles consist of a single-stranded, positive-sense RNA molecule embedded in a capsid made of approximately 1300 units of the same protein. Although no atomic-resolution data are available defining the organization of the virus particle and CP folding, it is assumed that PVX has a helical structure, with the CP N terminus exposed on the viral surface (Baratova et al., 1992a, b; Parker et al., 2002). For this reason, the construction of PVX CVPs requires the fusion of exogenous sequences at the 5' terminus of the cp gene. The genetic manipulation of PVX has been greatly improved by the development of expression vectors, such as pPVX201, that contain the cDNA encoding the complete viral genome (Baulcombe et al., 1995; Chapman et al., 1992a).
In this work, we have isolated a spontaneous PVX mutant characterized by a deletion at the 5' terminus of the cp gene. Two pPVX201-derived vectors encoding the cp mutant gene have been constructed and were used to fuse, to the 5' end of the cp gene, sequences encoding a panel of peptides varying in both length and amino acid composition. From the observation of the infectious phenotypes induced in planta by the different constructs, we concluded that cell-to-cell and phloem movement of PVX CVPs is critically affected by the occurrence of tryptophan (Trp), as well as the isoelectric point (pI) of the peptide.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) encoding the peptide KSS (amino acid sequence CKSSGKLISLC) (Scala et al., 1999) as an N-terminal fusion with the CP was performed as described previously (Marusic et al., 2001), adopting a strategy that removes the duplicated cp subgenomic promoter. To construct the viral expression vector pPVXCC, the 5'-deleted portion of the mutant cp gene was substituted to the wild-type cp in pPVX201. Briefly, an RT-PCR fragment was obtained by using total RNA extracted from the systemically infected leaves of pPVX201-KSS-inoculated plants as a template and primers PVXBack (5'-CTGGGGAATCAATCACAGTGTTG-3') and PVXXho (5'-GACGTAGTTATGGTGGTGGTAG-3'). The PCR product was digested with the restriction enzymes NheI and XhoI and ligated into similarly digested pPVX201. To construct the viral expression vector pPVXSma, in which sequences recognized by the restriction enzyme SmaI are inserted immediately downstream of the start codon of the mutant cp gene, sense (5'-TCGACATGCCCGGGACTCCTGCCACAGCTTCAGG-3') and antisense (5'-CCTGAAGCTGTGGCAGGAGTCCCGGGCATG-3') primers were designed and annealed in vitro to generate a fragment with NheI-compatible 5' and StuI-compatible 3' ends. This fragment was ligated into NheI–StuI-digested pPVXCC.
To insert sequences encoding the heterologous peptides as 5'-end fusions with the cp gene in pPVXCC, primer pairs were designed (following the PVX cp codon usage) and annealed in vitro to obtain fragments with NheI-compatible 5' and StuI-compatible 3' ends to be ligated into the NheI–StuI-digested vector. All of the sequences encoding the peptides were preceded by an ATG codon and followed by the cp gene portion that is lost by StuI digestion.
To insert the sequences encoding the heterologous peptides as fusions with the cp gene into pPVXSma, oligonucleotide pairs were designed to obtain NheI-compatible 5'- and SmaI-compatible 3'-end DNA fragments that were ligated into the NheI–SmaI-digested vector. As reported previously, sequences encoding the peptides were preceded by an ATG codon.
The biochemical features of the N-terminal CP peptides encoded by pPVXCC- and pPVXSma-derived constructs were calculated by using the ProtParam tool (http://www.expasy.org/tools/protparam.html).
Plant infection.
Nicotiana benthamiana plants were inoculated with the different constructs or with pPVX201 (as control) as described previously (Marusic et al., 2001). To verify the genomic stability and infectivity of virus particles that cause systemic infection, repeated cycles of reinfection were performed using leaf sap, prepared by homogenizing infected tissue in 1x PBS and centrifuging the sample for 3 min at 20 000 g and 4 °C. The supernatant was used directly to infect the plants. Briefly, sap obtained from symptomatic systemic leaves of the plants inoculated with the construct of interest (#1) was used to inoculate a second group of plants (#2). Afterwards, a third group of plants (#3) was infected by using the sap of the symptomatic systemic leaves from #2 plants. The presence of the expected cp gene was assessed by RT-PCR on total RNA extracted from systemic leaves of plants #1, #2 and #3, as described below.
RNA extraction and RT-PCR.
Ten to twelve days post-infection (p.i.), expression of the chimeric cp genes was verified by RT-PCR. Briefly, total RNA from systemically infected leaves was extracted by using an RNeasy plant mini kit (Qiagen) and RT-PCRs were performed by using a GeneAmp RNA PCR Kit (Perkin Elmer). cDNA was synthesized by using oligo d(T)16 and PCR was carried out with PVXBack and PVXNew (5'-CAGTCTAGCTCTGCTGATGCCGTTG-3') primers. PCR fragments were purified and verified by sequencing.
Western blot analysis and ELISA.
Protein extracts were obtained from inoculated and systemic leaves as described previously (Marusic et al., 2001). Aliquots (5 µg for extracts of symptomatic leaves or 20 µg for extracts of asymptomatic leaves) were separated on a 12.5 % (w/v) SDS-PAGE gel before transferring onto a PVDF membrane (Millipore). Immunodetection was performed as described previously (Donini et al., 2005).
Alternatively, the presence of PVX CP was revealed by ELISA using an Agdia Inc. kit (catalogue no. SRP10 000/0500) following the manufacturer's instructions.
Site-directed mutagenesis.
The mutagenesis of pPVXSma-NYESO and pPVXSma-NYESOSh constructs was performed by using a QuikChange Multi site-directed mutagenesis kit following the manufacturer's instructions (Stratagene). The same oligonucleotide (5'-Pho-AGCCTACTAATGGGGATTACACAATGT-3') allowed the TGG(Trp)
GGG(glycine, Gly) codon substitution in both constructs.
Electron microscopy.
Small pieces of the inoculated leaves, including necrotic ringspots, were fixed in 2.5 % (v/v) glutaraldehyde solution in 0.08 M phosphate buffer (pH 7.0) and then transferred into 1 % (w/v) osmium tetroxide. After dehydration in ethanol solutions, the samples were embedded in araldite resin. Ultrathin sections prepared by using an Ultracut-E ultramicrotome (Reichert–Jung) were collected on copper grids and stained with 1 % (w/v) uranyl acetate solution. Sections were then analysed by using a transmission electron microscope EM208 (Philips).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) was modified by inserting at the 5' end of the cp gene the sequence encoding a peptide of interest (KSS) for vaccine formulations. The resulting construct, pPVX201-KSS, was used to inoculate N. benthamiana plants with the aim of obtaining CVPs displaying the KSS peptide on their surface. Ten days p.i., the systemic leaves showing veinal chlorosis (a typical symptom of PVX infection) were sampled. To verify the presence of the expected modified viral genome within the leaves, total RNA was extracted, reverse-transcribed and amplified in the region including the 5' end of the cp gene. Surprisingly, we found a rearrangement of the cp gene, causing the loss of nt 4–67 from the 5' end. Analysis of the deduced amino acid sequence revealed that residues 2–22 at the N terminus were lost and substituted by a cysteine (Cys) residue in the correct reading frame.
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). To verify the ability of pPVXCC to produce infectious virus particles, the vector was used to inoculate N. benthamiana plants. Ten days p.i., systemic leaves of the plants displayed the same symptoms induced by pPVX201 (Fig. 1c, e) and contained the expected viral RNA, as assessed by RT-PCR and sequence analysis (not shown). Moreover, after repeated infection cycles with sap from systemic leaves of pPVXCC-inoculated plants, the sequence of the viral genome did not change, demonstrating viral genome stability.
To facilitate the insertion of sequences encoding heterologous peptides into pPVXCC, the vector was engineered by adding at the 5' end of the deleted cp gene a unique restriction site (SmaI) (Fig. 1f). This modification produced the substitution of Cys (CP position 2) and alanine (Ala) (CP position 3) with proline (Pro) and Gly, respectively (Table 1). The resulting pPVXSma vector induced symptoms on systemic leaves that were identical to those of the controls (Fig. 1g). The stability of the genome of these virus particles was verified as described previously (data not shown).
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). The panel of peptides included epitopes selected from melanoma-associated (MART, MARTSh, NYESO, NYESOSh) (Kirkin et al., 1998) or human immunodeficiency virus type 1 (Z13, Z13Cys, 2F5L, 2F5LCys, FZ, FZSh, FZShCys, SL9, P18, NefB, NefC) (Hurwitz et al., 2005; Shiver & Emini, 2004) proteins. Other peptides, associated with coeliac disease (EB1, EB2, EB3) (Shan et al., 2002) or endowed with antimicrobial activity (PK, SK) (Donini et al., 2005; Polonelli et al., 2003), were also included. To elucidate a possible role of the Cys residue in position 2 of pPVXCC CP on viral stability, some of the peptide-encoding sequences were designed both with and without this amino acid in this position.
Symptoms and molecular analysis of plants inoculated with the chimeric CP-encoding constructs
Ten to twelve days p.i., systemic leaves were examined to evaluate the presence of virus symptoms. Subsequently, plant tissues were analysed for the presence of the expected viral RNAs and CPs by sequencing and Western blotting. The constructs were divided easily into two main groups: those able to induce systemic infection (pPVXSma-SL9, pPVXSma-EB1, pPVXSma-EB2, pPVXSma-EB3, pPVXSma-MARTSh, pPVXSma-KS, pPVXSma-SK, pPVXSma-NefB, pPVXSma-NefC) and those that were not. Viral RNA was present in systemic leaves of plants inoculated with constructs able to induce systemic infection (group I; Fig. 2a). Although major sequence rearrangements did not occur, occasional point mutations affected the heterologous nucleotide sequence selectively, resulting in amino acid substitutions in the displayed peptide. This phenomenon was mostly evident during reinfections, but in one case (pPVXSma-EB1), the substitution occurred during the infection cycle produced by plasmid inoculation (Table 2). In view of the possible application of the systemically moving and genetically stable CVPs to biopharmaceutical research, some of them were produced on a large scale in plants and, after purification, the nature of CP–peptide fusion proteins was ascertained by extensive mass mapping experiments (see supplementary material, available in JGV Online).
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) and a group unable to induce symptoms (pPVXCC-Z13, pPVXCC-Z13Cys, pPVXCC-2F5L, pPVXCC-2F5LCys, pPVXCC-FZ, pPVXCC-FZSh, pPVXCC-FZShCys, pPVXSma-NYESOSh, pPVXSma-NYESO) (group III; Fig. 2c). ELISA analysis of the leaves inoculated with constructs belonging to group III revealed that, although local spreading was not detectable, these constructs were able to direct the synthesis of CP (data not shown).
Effect of amino acid composition of the heterologous peptide on genetic stability and on viral cell-to-cell movement
With the goal of defining general rules for the rational design of chimeric CPs in PVX, attributes correlating with the in vivo performance of each group of chimeric CPs were evaluated. We considered as heterologous peptide every non-consensus N-terminal sequence extending over the threonine (Thr) residue in position 24 of the CP encoded by pPVX201 (Table 1). This allowed a comparative analysis of pPVXCC-, pPVXSma- and pPVX201-derived constructs. The first trait to be analysed was peptide length, as the capability of generating assembled virus particles able to infect plants systemically could have been related primarily to the extent of peptide steric hindrance. Peptides were thus ordered by length and considered in terms of the in vivo behaviour of the CP–peptide fusion-encoding construct. As shown in Fig. 3 (upper panel), this criterion did not consistently explain the overall performance of our constructs in planta, as group I, II and III constructs extended randomly over the different peptide lengths (10–24 aa).
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). Although definite amino acid residues have not been identified as strictly necessary for CVP systemic movement, we observed that Trp occurred only and always in peptides encoded by non-permissive group III constructs (Table 1). The only exception was the permissive construct pPVX201-2F5, which, however, showed a significantly low Trp content (3.7 %) when compared with the lowest value (6.2 %) of group III-encoded peptides. To demonstrate that Trp may hinder virus movement, we designed a Trp to Gly substitution in the peptides encoded by pPVXSma-NYESO (Trp content 6.2 %) and pPVXSma-NYESOSh (Trp content 10 %) group III constructs by site-directed mutagenesis. The new chimeric constructs, named pPVXSma-NYESOM and pPVXSma-NYESOShM, were evaluated in their ability to induce local and/or systemic symptoms. By removing Trp, both mutated constructs were able to induce local and systemic lesions, acquiring the characteristics of group I constructs (Fig. 4).
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, lower panel). In contrast, peptides encoded by group II and group III constructs had a pI value outside this range. The only exceptions were the non-permissive group III constructs pPVXSma-NYESO and pPVXSma-NYESOSh, encoding peptides characterized by a high Trp content (Table 1).
To prove that PVX systemic movement (i.e. access to the phloem) is somehow affected by the pI of the displayed peptide, the pI values of the peptides encoded by two group II constructs (pPVXSma-p18 and pPVXSma-MART) and by one group III construct (pPVXSma-Z13) were modified. Modification to a permissive pI value was achieved by cloning, into pPVXSma, fragments designed to append one or two suitable amino acid residues to the ends of the original peptides, thus preserving their immunological properties. The sequence of the peptide P18 (pI 12.00) was modified by adding to each end an aspartic acid residue (Asp), generating the peptide P18DD (pI 5.96); the sequence of the peptide MART (pI 3.80) was modified by adding to each end a lysine residue (Lys), generating the peptide MARTKK (pI 6.14); finally, the sequence of the peptide Z13 (pI 3.80) was modified by adding to the C terminus a Lys residue, generating the peptide Z13K (pI 5.84) (Table 1). The inoculated and systemic leaves of the plants inoculated with the new constructs (pPVXSma-P18DD, pPVXSma-MARTKK and pPVXSma-Z13K) were examined carefully for the presence of symptoms and harvested for molecular analysis. Whilst no effect was observed for the construct originally belonging to group III, modification in group II constructs generated functional CVPs able to move systemically, despite the increase in peptide length (Figs 3 and 4).
Electron microscopy analysis of the leaves inoculated with pPVXSma-p18 and pPVXSma-p18DD
To understand how the pI of the peptides fused N-terminally to the CP could affect systemic spreading, leaves of the plants inoculated with the vector pPVXSma and with the constructs pPVXSma-P18 and pPVXSma-P18DD were analysed by electron microscopy. Analysis was focused on the lesions of inoculated leaves. The results revealed that pPVXSma-P18DD was able to induce not only the laminated inclusion components (LICs) (Shalla & Shepard, 1972) typical of PVX, but also large, cytoplasmic, fibrous aggregates of assembled virus particles (Lesemann, 1988) (Fig. 5b). Conversely, these virus-particle aggregates were not identified in the local lesions induced by pPVXSma-P18, which were characterized only by the presence of LICs in the cytoplasm of the infected cells (Fig. 5c, d).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). This could be an important feature when chimeric PVX is used as carrier of vaccine peptides, an application that requires repeated administration (Borisova et al., 1999; Tangri et al., 2005). pPVXCC and its derivative pPVXSma vectors, harbouring the mutated cp gene, were able to induce plant infections indistinguishable from those induced by the wild-type pPVX201 vector, with the formation of virus particles carrying genetically stable genomes. These data, together with a recent study aimed at defining PVX structure (Shanmugam et al., 2005), indicate that the formation of functional virus particles is not affected significantly by the removal of CP N-terminal amino acids. However, our observations also demonstrated that the fusion of heterologous peptides of varying length and composition to the truncated N terminus does not always result in systemic infection. This could be expected, as CP is essential for PVX movement (Baulcombe et al., 1995; Chapman et al., 1992a, b) through both plasmodesmata and phloem (Fedorkin et al., 2001; Santa Cruz et al., 1998). By the visual inspection of inoculated plants, the chimeric constructs were classified into three main groups. In the case of groups I (symptoms on both inoculated and systemic leaves) and II (symptoms only on inoculated leaves), the infection phenotype matched perfectly with the molecular analysis performed. On the contrary, group III constructs, despite being unable to induce detectable symptoms, were able to direct the synthesis of the CP in the inoculated leaves, although at low levels. This result demonstrated that the performance of these constructs could not simply be ascribed to transcriptional problems, but rather to a functional failure of the chimeric CP that prevents the transport of the viral genome from initially infected cells to the neighbours.
Because group III peptides limited viral local movement, whereas group II peptides were able to hinder CP function selectively in phloem transport, our data indicate that the CP exerts distinct functions in cell-to-cell and systemic movement. To shed light on how peptide attributes affect each function selectively, we tried to identify common physicochemical parameters that characterized each group of peptides unambiguously. Ranking peptides according to their length resulted in a random distribution of the three groups, indicating that this parameter was unrelated to ability (group I) or not (group II and group III) to generate viral forms moving systemically. Peptide groupings were further analysed in terms of amino acid composition. This approach revealed that, although systemic movement was not related to a specific amino acid composition, the presence of Trp residues was peculiar to group III peptides. One exception to this rule was pPVX201-2F5 (group I), yet encoding the peptide with the lowest Trp content. Trp residues are often found associated with the end of hydrophobic regions in transmembrane proteins and are supposed to act as ‘floats' at the membrane boundary, by fixing the protein to the lipid bilayer (Lee, 2003). On this basis, it could be hypothesized that the inability of group III constructs to promote cell-to-cell spreading might be ascribed to peptide-mediated membrane anchoring of chimeric CP, inhibiting cell-to-cell transfer. This hypothesis was supported by the fact that group III peptides were mainly putative transmembrane or membrane-proximal peptides (Chen et al., 1997; Salzwedel et al., 1999) and was finally confirmed by site-directed mutagenesis experiments showing that Trp to Gly substitution completely restored the ability of the CP to induce systemic infection.
Another correlation found to be highly significant was between the pI range of group I peptides and systemic movement. The robustness of this correlation was tested by modifying the pI value of peptides encoded by group II and group III constructs (i.e. MART, P18 and Z13). The results of these experiments showed that, whilst pI refinement of group II peptides resulted in the generation of viral forms able to move systemically (hence acting as group I peptides), pI adjustment of group III peptides had no effect. These data support the idea that the influence of pI on systemic infection may be hindered by a particularly high Trp content, interfering with earlier stages of viral movement. Thus, we can conclude that the effect of pI is selective on systemic movement, whilst Trp affects cell-to-cell movement independently of the pI value.
Despite the fact that all constructs belonging to group I conformed to the Trp and pI ‘rules’, some of them were subjected to point mutations in the sequences encoding the heterologous peptides. These mutations occurred mainly during reinfection cycles and were mainly biased towards the increase of Ser/Thr content, supporting findings that identify in these residues the phosphorylation sites involved in virus unpackaging (Baratova et al., 2004; Kozlovsky et al., 2003).
Electron microscopy studies, aimed to define differences between group II and pI-remodelled group I constructs, showed that whilst LICs were present in both samples, formation of virus-particle aggregates in infected cells was evident only after pI value adjustment. These results indicate that a non-permissive pI value (i.e. a charged N terminus) could interfere with the correct assembly of chimeric CP subunits to form complete virions. It is indeed a common belief that the introduction of repulsive charges in the N-terminal domain through phosphorylation is associated with PVX disassembly (Atabekov et al., 2001; Kozlovsky et al., 2003).
The major drawback to the production of CVPs harbouring peptides of interest in plants has been ascribed primarily to the steric interference of the foreign peptide with the correct assembly of virus particles (Scholthof et al., 1996), but peptide pI/charge has also been shown to affect viral fitness. In the case of rod-shaped Tobacco mosaic virus, this parameter affects cell-to-cell movement and this phenomenon has been ascribed to a lethal effect of the CP–peptide fusion on host cells (Bendahmane et al., 1999). Conversely, in both icosahedral Cowpea mosaic virus and filamentous Zucchini yellow mosaic virus, the effect is on systemic movement and is explained in terms of interference in the interaction between the CP and undefined host components (Kimalov et al., 2004; Porta et al., 2003). From these data, it is evident that common physicochemical features of a displayed peptide can influence the fitness of diverse viral genera (i.e. Tobamovirus, Comovirus and Potyvirus), but that the effect is different depending on the different role played by virus particles and coat proteins in the virus life cycle. This is further confirmed by our work that has extended the knowledge base of the effects of peptide display to the filamentous potexvirus PVX. Our findings, by providing new insights into the prediction of peptide sequences compatible with the production of infectious CVPs for candidate vaccines, reinforce the notion that PVX moves from cell to cell and through the phloem in two structurally different forms (i.e. cell-to-cell movement as a ribonucleoproteic complex and systemic movement as assembled virus particles) (Lough et al., 1998, 2000).
From the current and prior studies, we can conclude that information derived from peptide-display technology applied to viruses of different phylogenetic origins has important implications both to produce candidate vaccines and to deepen knowledge of virus biology.
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ACKNOWLEDGEMENTS |
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Received 4 April 2006;
accepted 26 June 2006.
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