Expression of a Sheep Pox Virus Gene in Plant Systems under the Control of Plant Viral Regulatory Elements and with SubCellular Targeting

The aim of this work was to investigate the feasibility of producing the sheep pox virus surface antigen that is an ortholog of vaccinia viral A27L in wheat germ cell free system and then in transgenic tobacco plants. Insertion into the A27L-mRNA 5'-untranslated region (5' UTR) of various translational enhancers (TEs) from plant viruses, as well as an artificial TE, allowed to increase the level of A27L protein synthesis in a wheat germ cell-free system. When the 5' UTR of potato virus Y genomic RNA was used as a TE, synthesis of an additional polypeptide that was approximately 2 kDa larger than the primary product of translation was observed in both plant and mammalian cell-free systems. The DNA constructs used for stable transformation of tobacco plants, besides 5' TEs, contained the transit-peptide-coding sequences for targeted accumulation of synthesized A27L protein. The amount of recombinant protein in the leaves of transgenic plants varied from 0.01 to 0.03 % of the total soluble protein.

Sheep pox virus (SPPV) is the causative agent of a highly dangerous disease in small ruminants and can cause substantial economic losses as a consequence.Attenuated SPPV strains are used as vaccines for prophylactic control of the disease 1 .The "NISKHI" strain is used in the Republic of Kazakhstan and Central Asia.The engineering of transgenic plants that produce viral proteins offers an alternative strategy for vaccine development 2,3 .
The SPPV 17.5 kDa protein is the ortholog of the A27L protein of vaccinia virus (VACV), which is localized in the lipoprotein membrane of the intracellular mature virion (IMV).Antibodies produced against the A27L protein of VACV were able to neutralize IMVs in vitro.Mice immunization with recombinant A27L protein synthesized in Escherichia coli or injection of anti-A27L monoclonal antibodies provides full protection against a lethal dose of VACV 4,5 .
We investigated the feasibility of producing the SPPV surface antigen in wheat germ cell free system and then in transgenic tobacco plants.We used the 5' untranslated region (5' UTR) of alfalfa mosaic virus (AMV) genomic (g)RNA4, 5' UTRs from gRNAs of potato virus Y (PVY) and tobacco etch virus (TEV), an artificial 5' UTR ARC1 in five repeats (5×ARC1), and the 3' UTR from a gRNA of tobacco mosaic virus (TMV) as translational enhancers (TEs) to increase the expression of the transgene.The ability of these UTRs to increase the expression of mRNAs has been shown earlier [6][7][8][9][10] .
Heterologous proteins synthesized in plant cells may undergo degradation by proteases in the cytoplasm.In addition to using TEs, specific intracellular localization with the help of signal peptides can provide greater accumulation of recombinant protein 11 .We used transit peptides from the small RuBisCO subunit of tomato (Lycopersicon esculentum) 12 and calreticulin of tobacco (Nicotiana plumbaginifolia) 13 that help to transport synthesized protein into the chloroplast and endoplasmic reticulum, respectively.From the endoplasmic reticulum, the synthesized recombinant protein can be further translocated into the apoplast 14,15,16 .

Western blot analysis
Electrophoresis of proteins in a 15 % polyacrylamide gel in the presence of 0.1 % of sodium dodecyl sulfate (SDS-PAGE) was performed according to standard techniques.After electrophoresis, proteins from in vitro translation reactions were transferred from the gel to a PVDF membrane that was treated with anti-oligo-histidine antibodies conjugated with horseradish peroxidase (5 Prime), and then recombinant proteins containing oligo His were detected by treatment with 3,3' diaminobenzidine (Sigma Aldrich).For detection of recombinant proteins in transgenic plants, polyclonal rabbit antibodies specific to the A27L protein were used as primary antibodies.Goat anti-rabbit antibodies conjugated with alkaline phosphatase (Sigma Aldrich) were used as secondary antibodies.Alkaline phosphatase activity was detected by the Fast Red TR/ Naphthol AS MX substrate (Sigma Aldrich).
To estimate the level of A27L protein synthesis, densitometric analysis of blots was performed using the ImageJ software (NIH, USA).On each electrophoretic track, an equal rectangle was scanned covering the zones from 15 to 30 kDa and areas under the peaks were calculated as percentages and arbitrary units.

Transformation of plants and analysis of transgene expression
A. tumefaciens EHA105 cells were mixed with plasmids and electroporated at 1.8 kV, 25 µF, 200 Ω.Leaf discs of Nicotiana tabacum L. cv.Samsun NN were transformed by co cultivation with electroporated agrobacterial cells.DNA from tobacco leaves was isolated according to Dilworth 18 .RNA was isolated by Trizol reagent (Sigma Aldrich) and treated with DNase I (Thermo Scientific).A27L mRNA was detected by reverse transcription (RT) and PCR as follows: first strand cDNA was synthesized by M MuLV Reverse Transcriptase (Thermo Scientific) with the reverse primer for the A27L gene.PCR analysis after RT was performed with both direct and reverse primers for the A27L gene.Two equal leaf discs were cut out of each plant and homogenized in 150 µl of CellLytic™ P Cell Lysis Reagent for plant cell lysis (Sigma Aldrich) with addition of the protease inhibition cocktail (Santa Cruz).The debris was removed by centrifugation in a 1.5 ml tube for 5 min at 14000 g, and protein concentration was measured by the Bradford procedure.Total soluble protein (TSP) from each transgenic plant in 50 µg amounts was mixed with sample buffer and after heating for 3 min at 70 °C was analyzed by electrophoresis in a 15 % SDS-PAG.

In vitro synthesis of A27L protein
To test the ability of A27L mRNA to correctly function in plant systems under the control of different TEs we first constructed a set of plasmids for transcription in vitro.All constructs contained the 3' TMV and differed only in the structure of the 5' UTR (Fig. 1).In this work, we used five different leaders: "pl" (polylinker derived), 5' UTRs of the plant viruses AMV, TEV and PVY, and the artificial 5¹ UTR "5×ARC1", which contained five copies of a synthetic oligonucleotide (10 nt long) complementary to the region 1115-1124 of the plant 18S rRNA 7 .
The structure of the mRNAs containing the A27L ORF under the control of different 5' UTRs is schematically shown in Fig. 2.
Plasmids were linearized with EcoRI before in vitro synthesis of uncapped full-length mRNAs.After transcription, the mRNAs were isolated and checked for integrity by electrophoresis in 1 % agarose.Equal amounts of mRNAs were translated in the wheat germ cell free system for 1 h at 26 °C, and then the recombinant A27L protein was analyzed by SDS-PAGE and subsequent western blotting (Fig. 3).Anti-His tag antibodies readily detected a polypeptide that corresponded to recombinant A27L.Recombinant A27L protein synthesized in vitro seem to have larger size because of the high voltage applied during electrophoresis, but correspond in size with positive control.The efficiency of mRNA translation was evaluated by densitometry of developed blots using the program ImageJ 1.46q.
The amount of protein synthesized from the mRNA 'pl-A27L' was taken as one arbitrary comparative unit because the polylinker derived leader "pl" does not enhance translation 19 .The presence of the PVY, TEV or "5×ARC1" 5¹ UTRs increased translation efficiency more than 2 fold with respect to the control leader "pl", while the AMV 5' UTR gave almost no enhancement.Notably, the PVY 5' UTR caused synthesis not only of A27L protein of expected size (20.3 kDa), but also of an additional polypeptide of larger size, which was apparently a variant of A27L extended by several amino acids at the N terminus.
Interestingly, when the uidA mRNA was translated under the control of the PVY 5' UTR in the rabbit reticulocyte cell free system in the presence of [ 35 S] methionine, we also observed the formation of two protein products: (i) a main one with a molecular mass of about 68 kDa that corresponded to β glucuronidase (GUS) and (ii) an additional one (GUS*) with a molecular mass approximately 2 kDa larger (Fig. 4a).The additional polypeptide of larger size was not observed when two copies of the artificial translational enhancer ARC1 (2×ARC1) was used as the 5' UTR.
The amount of additional polypeptide was about 20-30 % of the primary one as evaluated by densitometry of the images presented in Figs. 3  and 4 using the ImageJ 1.46q software.
Synthesis of the additional larger polypeptide in the wheat germ (Fig. 3) and rabbit reticulocyte (Fig. 4a) cell-free systems programmed by mRNAs with the PVY 5' UTR suggests that this 5' TE contains an additional upstream initiation site that can be recognized by the translation apparatus of both plants and animals.The universality of this initiation site is emphasized by the fact that it functions with various ORFs.Because the 5' UTR PVY does not contain upstream AUG codons (Fig. 2), it is obvious that initiation of the larger protein synthesis begins with a non AUG codon.To verify that the aberrant peptide is the larger one, we generated additional construct with PVY 5' UTR containing in frame stop codon before initiating AUG codon (Fig. 5).
Inserted stop codon led to the termination of the synthesis of the larger peptide in wheat germ cell free system (Fig. 4b).The average molecular weight of an amino acid is 110 Da.Assuming the aberrant peptide is about 2 kDa larger, initiating codon should be located some 17-18 triplets upstream of the ORF's start AUG codon.
A similar effect was observed earlier when mRNA with the TMV 5' UTR (Ω sequence) was translated in plant systems in vitro and in vivo.It was established that several upstream AUU codons in the Ω sequence could be used for translation initiation, leading to the synthesis of undesired by product proteins extended at the N terminus 20,21 .Because the PVY 5' UTR induces the synthesis of undesired by product proteins we did not use this 5' TE to increase transgene expression in vivo.Nevertheless, the PVY 5' UTR can provide a significant increase in the mRNA translation rate 7,22 .Non canonical start codons in this 5' TE deserve additional examination to identify and prevent undesired polypeptide synthesis by inserting stop codons, as was suggested earlier 22 .Moreover, the reproducibility and universality of translation initiation at non AUG codons in the PVY 5' UTR allow us to use this phenomenon as a model system to study its molecular mechanism.

Synthesis and accumulation of A27L protein in transgenic tobacco plants
Binary vectors carrying the A27L gene were constructed and transfected into A. tumefaciens EHA105 cells, which were then used for co cultivation with leaf discs to transform tobacco cells.As shown in Fig. 6, the expression cassettes contained the following functional segments: the constitutive CaMV 35S promoter, various 5' UTRs (5×ARC1, AMV 5' UTR, TEV 5' UTR), the A27L gene without or with N terminal signal peptides (from the tomato RuBisCO small subunit or tobacco calreticulin), and the polyadenylation signal from the nopaline synthase gene (253 bp).The TMV 3' UTR (3' TMV, 246 bp) that was used in constructs for in vitro translation was intentionally excluded from agrobacterial DNA constructs between the A27L gene and NOS terminator, because its presence would result in the mRNA having too long a 3' UTR.It was shown recently that 3' UTRs longer than 300 nucleotides cause mRNA instability in plant cell nuclei 23 .
After transformation, tobacco plants were regenerated from leaf segments on regeneration medium containing 50 mg kanamycin/ l.The DNA of most regenerated plants directed the synthesis of the expected PCR product, which confirmed insertion of the A27L gene into their genomes.In many plants, the A27L-mRNA was detected by RT of total RNA and PCR amplification using specific primers (Fig. 7).
Accumulation of the recombinant A27L protein was determined in samples of total soluble protein (TSP) extracted from the leaves of transgenic plants that were positive in RT PCR analysis.After SDS PAGE, the recombinant protein was identified by immunoblotting using A27L specific polyclonal antibodies.In some transgenic lines, a polypeptide of expected size was detected as shown in Fig. 8.It can be seen that the viral and artificial translation enhancers promoted synthesis of approximately equal amounts of A27L protein (compare lanes 1, 2 and 5).Additionally to transgenic plants used for western blot of the proteins in Fig. 8, we also obtained transgenic plants with subcellular localization to apoplast and chloroplast that did not show the presence of the A27L protein.
According to our estimates, the amount of SPPV A27L in the leaves of transgenic tobacco plants varied from 0.01 to 0.03 % of the total soluble protein.Approximately the same level of VACV A27L protein production was achieved previously in plants transformed through the nuclear genome 24 .

CONCLUSIONS
In this work, we compared several 5'-UTRs of plant viruses and an artificial translational enhancer (ARC1) by their ability to stimulate translation of a downstream ORF.Unlike the leader of the naturally capped AMV RNA4, 5' UTRs of the naturally uncapped gRNAs of TEV and PVY, as well as the artificial 5' UTR ARC1, increased the translation of uncapped mRNAs in a wheat germ cell-free system.The PVY 5' UTR contains a non AUG start site (approximately 17-18 triplets upstream of the ORF's AUG codon) that is functional in both wheat germ and rabbit reticulocyte cell free systems.
We obtained transgenic tobacco plants expressing the SPPV-A27L gene for the envelope protein of the intracellular mature virion under the control of AMV, TEV, and ARC1 5' TEs, and with targeting of the protein product to chloroplasts and the apoplast.Enhancers of mRNA translation and targeting of recombinant protein into chloroplasts and the apoplast allowed A27L accumulation in plant cells to a level sufficient for detection by western blot analysis, however, the amount of A27L originally synthesized in the cytoplasm was apparently the same.

Fig. 3 .Fig. 4 .Fig. 5 .
Fig. 3.Western analysis of A27L protein synthesizedin a wheat germ cell-free system through translation of mRNAs with different 5'-UTRs.Detection with anti-His-tag antibodies, C+, positive control (50 ng of A27L expressed in E. coli); C-, negative control (cell free system without A27L mRNA); pl, PVY, ARC1, AMV and TEV, translation products of mRNAs with the respective 5' UTRs; M, marker proteins with known molecular masses (indicated at the left).Numbers beneath the lanes indicate arbitrary units of respective mRNA translation efficiency; an additional polypeptide is marked with an asterisk