Since the integration of foreign sequences in the PVX genome, or the modification of the CP, may cause genomic rearrangements 1819, we have verified the integrity of the chimeric transcript. RT-PCR carried out with PVX and epitope specific primers (Fig. 1, Table 1) resulted in a single amplification product of the expected size in cDNA samples derived from plants infected with PVX6HisE2¹2ACP or PVX6HisE2²2ACP plasmids (Fig. 2). No amplification products were observed in samples when the RT step was omitted (data not shown).

Cellular extracts from plants inoculated with plasmid DNAs were used to infect new plants (second order infections) and those obtained from second order infected plants to inoculate other plants (third order infections). Only bands of the expected sizes were visualized in RT-PCR experiments carried out on samples derived from plants of second and third order infections. Figure 2 shows the results of RT-PCR experiments carried out on mRNAs isolated from systemic leaves derived from third order infections. These results demonstrated that the epitope sequences are correctly retained during PVX replication. Direct sequencing of the RT-PCR products obtained from the third-order samples, with PVX specific primers, showed the absence of point mutations (data not shown), leading to the conclusion that epitope coding sequences were replicated with high fidelity during viral infections.

Total soluble proteins, extracted from symptomatic leaves 15 dpi with PVX6His2ACP, PVX6HisE2¹2ACP, PVX6HisE2²2ACP constructs or from mock-infected plants, were analyzed by Western blotting with a polyclonal anti-CP antibody. The conformation of the 2A within the P ribosome site may cause the cleavage of the nascent chain with the subsequent release of the N terminal product from the ribosome. Some ribosomes however will continue to produce the downstream protein as a discrete entity 2021. Processing of the 2A site in synthetic polyproteins can be incomplete and the proportion of cleaved versus uncleaved polyprotein may vary depending on the sequence context upstream the 2A site 20.

Translation of chimeric transcripts is therefore expected to produce: i) a full length uncleaved polyprotein (Fig. 3), ii) the N-terminal cleaved portion of the polyprotein and iii) the CP cleaved protein corresponding to the C-terminal portion of the polyprotein (Fig. 3). A single broad band of about 31–34 kDa corresponding to the cleaved CP and to the uncleaved 6His2ACP polyproteins, respectively, were visualized in PVX6His2ACP samples (Fig. 4 A3 and B3). Accordingly, in Western blotting of N. benthamiana infected with a similar PVX2A vector, Santa Cruz et al. 22, detected the cleaved CP protein at 31 kDa.

In PVX6HisE2¹2ACP-infected samples, two bands of about 31 and 42 kDa were visualized (Fig. 4 A1). These bands likely correspond to the cleaved CP protein and to the uncleaved 6HisE2¹2ACP protein, respectively (Fig. 3A–C). In PVX6HisE2²2ACP-infected samples, the 37 and 31 kDa bands (Fig. 4 B1) correspond to the uncleaved 6HisE2²2ACP polyprotein and to the cleaved CP protein, respectively (Fig. 3D–F). No bands were visualized in the sample of mock infected plants (Fig. 4 A2, B2, C2 and D2).

Duplicate blots were also probed with an anti-6His antibody. Only a single band of 42 kDa and 37 kDa were visualized in PVX6HisE2¹2ACP and PVX6HisE2²2ACP-infected samples (Fig. 4 C1 and D1). While a signal of about 34 kDa was visualized in PVX6His2ACP infected plants (Fig. 4 C3 and D3), no signal was detected in uninfected plants (Fig. 4 C2 and D2). Bands of about 11 and 6 kDa, corresponding to the predicted molecular weights for 6HisE2¹2A and 6HisE2²2A were never observed. These results demonstrate that the PVX6His-recombinant vectors are able to drive the expression of an uncleaved 6His-epitope-2ACP polyprotein.

Recombinant virions were partially purified by centrifugation of cellular extracts from PVX6His2ACP and PVX6HisE2¹2ACP infected plants after incubation on ice for 2 h in presence of 8% PEG-8000 as reported by AbouHaidar et al. 23. The purification yielded about 150 and 120 μg of virus particles per gram of leaves infected with PVX6His2ACP and PVX6HisE2¹2ACP, respectively. An aliquot of 250 ng of purified protein for each construct was fractionated by SDS-PAGE and stained with Comassie Brilliant Blue. Two bands of 42 and 31 kDa were visualized over a very weak background (Fig. 5A). The 42 kDa:31 kDa relative amount, quantified with GeneSnap software (Syngene), was 1:2. Western blotting with anti-6His (Fig. 5B upper) and anti-CP (Fig. 5B lower) antibodies confirmed that these bands correspond to the uncleaved 6HisE2¹2ACP polyprotein and to cleaved CP protein.

To verify that the epitopes produced in planta are immunogenic, rabbits were immunized with 50 μg of purified PVX6HisE2¹2ACP virions (group A) or with purified PVX6His2ACP (group C). The presence of anti-E2 antibodies in rabbit sera was tested by Western analyses of purified E2 proteins. Western blotting tests were performed with serial dilutions of sera from rabbit immunized with PVX6HisE2¹2ACP (Fig. 6A–D) and PVX6His2ACP (Fig. 6E–H). The E2 specific band was detected with a PVX6HisE2¹2ACP sera dilution up to 1/100 (Fig. 6A–D) but not with PVX6His2ACP at any dilution (Fig. 6E–H). To verify the immunogenic response of rabbits inoculated with PVX6His2ACP proteins (Fig. 5C), western blotting analysis were carried out using their sera against proteins purified from mock-inoculated control plants (Fig. 5 C1) and from plants infected with PVX6His2ACP (Fig. 5 C2). As shown in Figure 5D, a 55 kDa band, corresponding to the E2 protein, was visualized in blots probed with rabbit sera but not in duplicate blots probed with pre-immune sera or sera bled from B and C group rabbits (data not shown). The A group rabbit sera recognized both the 42 kDa and 31 kDa proteins in total protein extracts derived from PVX6HisE2¹2ACP-infected plants (Fig. 5D). These results suggest that the PVX6HisE2¹2ACP antibody is able to induce anti-E2 antibodies in immunized rabbits.

To express peptides fused to PVX coat protein (CP), an in-frame sequence encoding 16 amino acids of the 2A oligopeptide (NFDLLKLAGDVESNPG) of the Foot-and Mouth Disease Virus (FMDV) 32, was cloned between the 3' end of a sequence encoding for a 6 Histidine tag (6His) and the fourth codon of the coat protein coding sequence (CDS) (Fig. 1). Transcription of the chimeric messenger was driven by a duplicated CP promoter of PVX 33. The double-stranded 2A oligolinker was prepared by mixing 500 pmol of the 2A upper (5'-TCGACAATTTTGATTTATTAAAGTTAGCAGGTGATGTTGAATACAATCCAGGTCCAG-3') and 2A lower (5'-CTGGACCTGGATTTGATTCAACATCACCTGCTAACTTTAATAAATCAAAATTGTCGA-3') oligos. The 6His-tag double stranded oligolinker was prepared by mixing 500 pmol of 6His upper (5'-TCGAGATGCACCACCACCACCACCACG-3') and 6His lower (5'-TCGACGTGGTGGTGGTGGTGGTGCATC-3') oligos. The oligonucleotide mixtures were heated at 95°C for 10 min and then cooled at room temperature to allow annealing.

The PVX201 vector (kindly provided by David Baulcombe, The Sainsbury Laboratory, Norwich, UK, 34) which contains the PVX cDNA under the transcriptional control of the Cauliflower 35S promoter and the nopaline synthase terminator 33 was digested with NheI (New England Biolabs) and then blunt-ended by Klenow polymerase (New England Biolabs). The linearized vector was then ligated to the 2A double stranded oligolinker to produce the PVX2ACP vector.

The PVX2ACP vector was then linearized with SalI and ligated to the 6His-tag oligolinker to produce the PVX6His2ACP vector. This vector contains a unique SalI site to be used for cloning coding sequences for the gene of interest that is fused in frame to the 3' terminus of the 6His-tag and to the 5' end of the 2A sequence.

The E2¹ (region between amino acids 790 and 860) and E2² (region between amino acids 854 and 894) sequences of the E2 protein were amplified by PCR using specific primers (Table 1) that contained a SalI site to allow subsequent cloning into PVX6His2ACP.

The amplification fragments were cloned into a PCR4 TOPO vector using the TOPO TA cloning for sequencing kit (Invitrogen) and sequenced to verify the absence of point mutations. The E2¹ and E2² fragments were excised from the respective PCR4 vectors using SalI and separated on a 1% agarose gel. The bands were then purified from the gel using the GFX™ PCR DNA and Gel Band Purification kit (Amersham Biosciences).

The PVX6His2ACP vector, linearized with SalI and dephosphorylated using calf intestinal alkaline phosphatase (New England Biolabs), was ligated to either the E2¹ or E2² fragments to produce PVX6HisE2¹2ACP and PVX6HisE2²2ACP, respectively.

Nicotiana benthamiana plants were grown at 22°C with a day-light cycle of 16 h of light and 8 h of dark. Two healthy leaves per plant were infected using 20 μg of either PVX6His2ACP, PVX6HisE2¹2ACP or PVX6HisE2²2ACP plasmid as described by Marusic et al. 16.

Total RNAs were isolated from symptomatic leaves two weeks after the infection using the GenElute Mammalian Total RNA miniprep kit (Sigma) according to the manufacturer's instructions. Total RNA was purified from residual genomic DNA using the DNA-free kit (Ambion). First strand cDNA was synthesized from 1 μg of total RNA in a volume of 50 μl according to Sambrook and Russell 35. RT-PCR reactions were performed in a total volume of 50 μl containing 1 μl of first-strand cDNA, 1 μM of each primer, 1× PCR buffer, 1.5 mM MgCl₂, 0.2 μM dNTPs and 2 U of recombinant Taq DNA polymerase (Invitrogen). The DNA was denatured at 94°C for 1 min, and then subjected to 25 cycles of 94°C for 1 min, Tm (specific for each primer pair, Table 1) for 1 min and 72°C for 1 min, plus a final extension at 72°C for 10 min. Table 1 gives the sequence of primers used for RT-PCR experiments (Fig. 2). Sequencing was performed using an ABI Prism 377 (Applied Biosystems) and the ABI Prism Big Dye Terminator v1.1 Ready Reaction Cycle Sequencing kit (Applied Biosystems). Primers used for sequencing reactions were PVX FOR and PVX REW (Table 1).

N. benthamiana leaves were ground to a fine powder in liquid nitrogen. Approximately 0.5 g of leaf powder was resuspended in 0.7 ml of extraction buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM PMSF, 0.2% Triton X-100, pH 8). The homogenates were centrifuged at 15,000 × g at 4°C for 20 min and the supernatants were quantified using the Bradford protein assay kit (Bio-Rad) using Bovine Serum Albumin (BSA) as reference standard.

Equal amounts of total soluble proteins have been loaded on 12% SDS-polyacrylamide gels according to Sambrook and Russell 35. Gels were either stained with Coomassie Brilliant Blue (Bio-Rad) according to Sambrook and Russell 35 or transferred to a Hybond C⁺ membrane (Amersham) using a semidry blotting apparatus (Bio-Rad) at 20 V for 12 h at 4°C. The membranes were blocked with 10% non-fat dry milk (Bio-Rad) in T-TBS for 1 h at room temperature. A 1: 10,000 rabbit polyclonal anti-CP antibody (1:10,000; kindly provided by CNR-IVV) and an anti-rabbit IgG peroxidase conjugated (1:5,000; Sigma) were used as primary and secondary antibodies, respectively. A mouse monoclonal anti-6His antibody (1:3,000; BD Biosciences) and an anti-mouse peroxidase conjugate (1:10,000; Sigma) were used for Western blotting. Hybridized blots were exposed with Kodak AR films (Kodak) after the chemiluminescent reaction carried out using the ECL kit (Pierce).

Virus particles were purified from plant leaves infected with PVX6His2ACP or PVX6HisE2¹2ACP according to a modified method of AbouHaidar et al. 23. Briefly, powdered infected leaves (5 g) were suspended in 20 ml Tris-borate buffer containing 0.2% β-mercaptoethanol. The suspension was filtered through four layers of cheesecloth and n-butanol was added to a final concentration of 6%. The mixture was kept on ice for 2 h with constant stirring and then centrifuged for 10 min at 15,000 × g. PEG 8,000 was added to a final concentration of 8% in the presence of 2% NaCl on ice for 2 h with constant stirring, decanted for another 2 h, and then centrifuged at 15,000 × g for 10 min. The pellet was resuspended in Tris-borate buffer, pH 7.5 at 4°C overnight. The virus solution was then centrifuged three times at 7,500 × g for 5 min each. After precipitation, particles were directly dialyzed against 1× PBS. The concentration of virus was calculated using the PVX extinction coefficient (2.97) at 260 nm as reported by Uhde et al. 18.

Three months old rabbits (New Zealand Breed) were divided in groups A, B and C of four rabbits each and injected by subcutaneous route using 50 μg of purified proteins containing Freund's adjuvant. Immunizations were repeated after 21 and 45 days without adjuvants. The A group was immunized with PVX6HisE2¹2ACP, the B group with PBS only and C group with PVX6His2ACP purified virions. Fifteen days after the last immunization, rabbits were bled and serum was recovered. Preimmune sera (Fig 6A and 6E) and sera dilutions (1/10, 1/100, 1/1000) from A and C groups (Fig. 6B–D and 6F–H) were then tested for antibody reactivity against E2 following standard procedure. E2 protein was specifically recognized only by sera from A group rabbit dilutions up to 1/100 (Fig. 6B–D). Baculovirus produced E2 protein, kindly provided by the Italian Reference Centre for Classical Swine Fever (Istituto Zooprofilattico dell'Umbria e delle Marche), was resuspended in sample buffer and loaded in 12% SDS-PAGE and blotted to Hybond C⁺ membrane (Amersham).