Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
  • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
  • C12N15/8203—Virus mediated transformation

Definitions

• the present invention relates to plant biotechnology. More specifically the invention relates to a method for achieving enhanced protein expression in a cell of an organism.
• Molecular pharming is a technology wherein genetically engineered plants are used to produce recombinant proteins with therapeutic value, such as plant biopharmaceuticals.
• plant expression systems have the advantage of being able to produce active forms of complex proteins with post- translational modifications, such as glycosylation, which are necessary for human therapeutic proteins for correct function in vivo.
• Plant systems are, however, free of human pathogens potentially associated with mammalian cell cultures.
• McCormick et al. who used the species of tobacco plant (Nicotiana benthamiana) to manufacture a non-Hodgkin's lymphoma vaccine. Up to 1000 plants were used to manufacture the vaccine for each patient (McCormick et al., 2008. Proc. Natl. Acad. Sci. U.S.A. 105, 10131).
• Agrobacterium-mediated transient expression is a useful tool for assessing gene expression constructs in plants. It is rapid, typically giving results in 2 to 3 days after inoculation and has been demonstrated to work in whole leaf tissue from a range of plants.
• Agroinfection involves syringe or vacuum infiltration of an Agrobacterium tumefaciens suspension harboring T-DNA carrying the viral genome into plant leaves, resulting in the local transformation of the infiltrated leaf with the virus.
• Agrobacterium infects each cell in the inoculated zone and inserts its T-DNA into the plant chromosome of each cell.
• a plant promoter placed upstream of the viral cDNA induces the transcription of viral genome in the plant nucleus and viral RNA is transported to cytoplasm for viral replication.
• Plant viral vectors have emerged as an approach to achieving expression of recombinant proteins.
• plant virus- based expression systems have been developed and utilized for production of heterologous proteins in plants.
• Kleba et al., 2007 disclose the use of plant viruses as gene vectors. Plants can either be infected locally with the virus or viral RNA, or the whole plant can be transfected with Agrobacterium.
• Kleba et al. 2007 disclose a heterologous protein production system using optimized TMV vectors combined with the magnification technology. Magnification is agroinfiltration method wherein plants are fully dipped into the bacterial solution and the suspension is sucked into leaves by vacuum. Also Marillonet et al. 2005 and Kleba et al. 2004 (Current Opinion in Plant Biology, 18, 182-188, 2004) have developed virus vectors.
• the viral genome-linked protein (VPg) is a protein attached to the 5' end of RNA during RNA synthesis in a wide variety of viruses.
• the role of (VPg) has been studied in viral and host protein translation. Khan et al. 2008 (J. Biol. Chem. 283 : 1340-1349, 2008) disclose that expression from an RNA containing the 5'UTR of Tobacco etch virus (nucleotides 1-143 of TEV RNA) joined to luciferase gene and poly(A)-tail encoding sequence can be enhanced by providing VPg into the system in an in vitro system utilizing wheat germ extract. The enhancement occurs only for non-capped RNA whereas the expression of capped RNA is inhibited under these conditions. Only a modest enhancement of 1.6 fold in expression was achieved in this system.
• US2009181460 Al discloses an expression vector system for expressing heterologous proteins in plants.
• the expression system is based on using three separate gene constructs containing comovirus RNA-1, a gene construct containing comovirus RNA- 2, and a gene construct comprising a suppressor of gene silencing. Part of the RNA2 encoding sequence has been deleted from the second construct and this region is replaced with the gene encoding the protein of interest. The expression is dependent on supplying comovirus RNA-1, not on over expression of VPg.
• An object of the present invention is thus to overcome or alleviate disadvantages and problems of the prior art solutions and to provide a novel solution for achieving enhanced protein expression in an organism.
• the objects of the invention are attained by a method of achieving enhanced protein expression, which method is characterized by what is stated in the independent claims.
• the preferred embodiments of the invention are disclosed in the dependent claims.
• the present invention enables to boost up the protein expression in a cell, such as a plant cell and consequently the production of the protein of an interest.
• viral genome-linked protein (VPg) in combination with a virus genome or a fragment thereof and the virus genome comprising at least 5'untranslated region (5'UTR) has the power to enhance viral gene expression multifold compared to prior art methods.
• VPg viral genome-linked protein
• 5'UTR 5'untranslated region
• the present inventors found that when the full-length potyviral RNA serves as a transcript, there is a vast enhancement of translation in vivo. Additionally, ribosomal protein PO can be used to further enhance the protein expression.
• the present inventors show for the first time the enhancement for full-length viral RNA.
• the present inventors show that the VPg promotes translation of viral mRNAs having the 5'UTR.
• 5'UTR is an essential sequence in promotion of translation of viral RNA but it is not sufficient alone. Direct or indirect interaction of VPg with the 5'UTR is involved.
• the enhancement of translation is especially specific for viruses having VPg or for picornavirus-like viruses. Preferably, additional PVA sequences or potyviral protein(s) are required for enhanced translation.
• Ribosomal protein PO is a functional component of VPg-mediated enhancement of protein expression and can be used together with VPg to achieve maximal target protein production levels.
• the present invention relates in a first aspect to a method for achieving enhanced protein expression from a target nucleotide sequence in a cell of an organism, wherein the method comprises the steps of providing a nucleic acid construct comprising a nucleotide sequence encoding a viral genome-linked protein (VPg) or a derivative or fragment thereof; providing a nucleic acid construct comprising a nucleotide sequence encoding a virus genome or a fragment thereof having a capacity for enhanced protein expression in the presence of a VPg, wherein the virus genome or a fragment thereof comprises at least a 5' untranslated region (5'UTR) and the target nucleotide sequence operably linked to a promoter; and introducing sa id nucleic acid constructs for transient expression into a cell .
• VPg viral genome-linked protein
• the present invention also relates to a method for producing nucleotide sequences having an improved capacity of enhancing protein expression, wherein the method comprises the steps of selecting a virus genome or a fragment thereof comprising 5' untranslated region (5'UTR) and a target protein and having a capacity for enhanced protein expression in the presence of viral genome-linked protein (VPg) ; selecting a VPg or a fragment thereof capable of enhancing protein expression; producing a nucleic acid construct comprising a nucleotide sequence encoding a virus genome or a fragment thereof having a capacity for enhanced protein expression in the presence of
• VPg viral genome-linked protein
• the invention also relates to an enhancer element of protein synthesis, wherein the enhancer element comprises a nucleotide sequence encoding a virus genome or a fragment thereof having a capacity for enhanced protein synthesis in the presence of a viral genome-linked protein (VPg) ; and a nucleotide sequence encoding VPg or a fragment thereof, wherein the virus genome comprises at least a 5' untranslated region (5'UTR) and a target protein.
• VPg viral genome-linked protein
• the invention also relates to a method for heterologous production of a protein from a ta rget nucleotide sequence in a cell of an organism, wherein the method comprises the steps of providing a nucleic acid construct comprising a nucleotide sequence encoding a viral genome-linked protein (VPg) or a derivative or a fragment thereof; providing a nucleic acid construct comprising a nucleotide sequence encoding a virus genome or a fragment thereof having a capacity for enhanced protein expression in the presence of VPg; wherein the virus genome or a fragment thereof comprises at least 5'untranslated region (5'UTR) and the target protein; and introducing said nucleic acid constructs for transient expression into a cell; and producing the heterologous protein in the cell in vivo.
• the produced protein can be harvested from the cell and subsequently extracted and purified with methods known by the skilled person in the art.
• the methods and an enhancer element of the present invention comprise a nucleotide sequence encoding a ribosomal protein P0 or a derivative or a fragment thereof.
• Potyviral VPg protein is able to inhibit translation when proteins are synthesized in vitro in cell extracts.
• the role of VPg in translation is presented in vivo in cells.
• the present invention is based on the experimental studies on potyvirus PVA VPg in plants. Using a recently developed infection assay system, it is possible to quantitate the viral gene expression accurately. Agroinfiltration is used to initiate both viral and VPg gene expression simultaneously in plants.
• potyviral VPg Similarly to the in vitro system synthesis of reporter proteins from monocistronic mRNA transcripts is inhibited by the presence of potyviral VPg. Surprisingly, however, when the full-length potyviral RNA serves as a transcript, there is a vast enhancement of translation in vivo. The enhancement of translation is more pronounced with non- replicating viral transcript being average 70-fold compared to wild type replicating viral RNA with 13-fold enhancement. The present invention therefore makes it possible to boost up the production of the protein of interest which is expressed from the viral genome. As agroinfiltration is the method to initiate the VPg expression and virus infection the present technology can be used in plants which are grown under green house conditions.
• An advantageous feature of the method of the invention is that with the enhanced protein expression level it is possible to obtain target proteins in increased amounts from an organism, especially from a plant. Thus, higher expression level of the target protein reduces the amount of host organisms that are needed to satisfy the need.
• the VPg-based enhancement method of the present invention differs from the prior art methods. Compared to the method using suppressors of gene silencing, the present VPg-based enhancement method enables to enhance the production of (individual) proteins in a controlled manner. In other words, proteins which have in their mRNA a 5'UTR of a picornavirus-like virus can be enhanced. 5'UTR is an essential sequence in promotion of translation of viral RNA but it is not sufficient alone.
• VPg Direct or indirect interaction of VPg with the 5'UTR is involved.
• additional PVA sequences or potyviral protein(s) are required for enhanced translation.
• Ribosomal protein P0 can be used in the present method to achieve maximal target protein production levels. Reduced but clear enhancement of translation is achieved also with mutants deficient in the suppression function. The specificity of the enhancement supports another mechanism than general suppression of gene silencing. Examples of heterologous proteins produced in commercial scale in plants include Herceptin (Komarova et al., 2011.
• FIG. 1 is a schematic presentation of the genome and the cistrons corresponding to mature proteins in Potato virus A (PVA).
• PVA Potato virus A
• the VPg region is expanded to show details of the Lys to Ala mutations made in the nucleotide triphosphate (NTP)-binding region. Mutated residues are in bold and underlined.
• Figure 2 depicts that PVA VPg inhibits in vitro translation and has RNA binding activity. In vitro transcribed capped or non-capped flue mRNAs were translated in wheat germ extract in the presence of various amounts of wt and mutant VPgs.
• C Integrity of transcripts in the presence of wtVPg and mutant VPgs. Increasing amounts of VPgs were incubated with a constant amount of flue mRNA (2.0 pmol).
• D Coomassie-stained control gel of the recombinant proteins used in the assays.
• Figure 3 depicts that PVA VPg inhibits in vivo translation of monocistronic reporter mRNAs.
• A Expression of PVA VPg constructs in N. benthamiana initiated via Agrobacterium infiltration was verified by a western blot analysis with VPg IgGs and ECL detection.
• B Inhibition of 35S-fluc-nos (left) and 35S-rluc-nos (right) and expression in the presence of wtVPg.
• N. benthamiana leaves from nine plants were preinfiltrated with Agrobacterium culture containing 35S-VPg-nos or 35S-GUS-nos constructs (OD 600 of 0.5).
• benthamiana leaves were preinfiltrated with Agrobacterium harbouring the 35S-fluc-nos and either the wt or one of the mutant 35S-VPg-nos expression constructs at OD 6 oo values of 0.5.
• preinfiltrated regions were reinfiltrated with 35S-rluc-nos Agrobacterium at an OD 6 oo of 0.01. Accumulation of Rluc (left panel) and total protein (right panel) at 3 dpi is shown.
• two leaves in five plants were infiltrated. Four samples containing a pool of three leaf disks were collected for the analysis. The error bars indicate standard deviations.
• Figure 4 depicts that VPg supplemented in trans boosts viral gene expression. N.
• benthamiana leaves were infiltrated with a mixture of Agrobacterium containing either 35S-VPg-nos or 35S-GUS-nos construct (OD 600 of 0.5) and 35S-fluc-nos (OD 600 of 0.01) construct, together with 35S-PVA wt ::rlu nt -nos, 35S-PVA CPmut : r ⁇ ud nt -nos, or 35S-PVA ACDC :rlud nt -nos construct (OD 600 of 0.05). Each combination was infiltrated into two leaves of three plants. (A) Average Flue and (B) Rluc activities were calculated from three samples collected at 3 dpi. Each sample consisted of a pool of three leaf disks.
• the error bars indicate the standard deviations. Asterisks indicate the low viral gene expression (9995 RLUs/ ⁇ ) from 35S-PVA ACDD ': :rlud nt -nos during GUS co-expression.
• C Plant lysates were analysed by anti-Flue, -VPg, -Rluc and -CP immunoblots. Recombinant VPg (rVPg), purified PVA particles (CP), and non-infected mock plants served as positive and negative controls. Ponceau S-stained membrane shown in the lowest panel served as a loading control. The migration of molecular mass standards is indicated at left on each blot.
• Figure 5 depicts that mutations at the NTP site affect but do not abolish capacity of VPg to boost viral translation.
• N. benthamiana plants were agroinfiltrated with a mixture of Agrobacterium culture containing 35S-fluc-nos (OD 60 o of 0.01), 35S- PVA ACDD ::rlu nt -nos (OD 600 of 0.05) and either 35S-VPg-nos or 35S-GUS-nos (OD 600 of 0.5) constructs. Each combination was infiltrated to two leaves in five plants. Luciferase activities were analysed from samples (four, each containing four leaf disks) collected at 3 dpi. Error bars indicate the standard deviations.
• benthamiana plants were infiltrated with a mixture of Agrobacterium carrying 35S- fluc-nos (OD 6 oo of 0.01) and 35S-VPg-nos or 35S-GUS-nos (OD 600 of 0.5). Infiltration mixture also contained either (A) 35S-PVA ACDD ': with 5'UTR deleted or (B) 35S-PVA ACDD ': with 3'UTR deleted (OD 600 of 0.1). Each combination was infiltrated to two leaves of four plants. Samples (four, each containing five leaf disks) were collected at 3 dpi and Flue and Rluc activities were determined. (C) N.
• benthamiana plants were infiltrated with a mixture of Agrobacterium carrying 35S- fluc-nos (OD 6 oo of 0.01) and 35S-VPg-nos or 35S-GUS-nos (OD 600 of 0.5). Infiltration mixture also contained 35S-rluc-nos constructs that either contained a reference 5'UTR or the 5'UTR of PVA. Each combination was infiltrated to two leaves of four plants. Samples (five, each containing five leaf disks) were collected at 3 dpi and Flue and Rluc activities were determined. The error bars indicate the standard deviations.
• Figure 7 depicts that the amount of enhancement and inhibition of translation by VPg depends on VPg concentration.
• N. benthamiana plants were agroinfiltrated with a mixture of Agrobacterium containing 35S-fluc-nos and 35S-PVA &GDD r. rlu ⁇ -nos (OD 600 of 0.01 and 0.05) construct mixed with various OD 60 o values Agrobacterium carrying either 35S-VPg-nos or 35S-GUS-nos. Each combination was infiltrated to two leaves of four plants.
• B) Rluc activities were measured at 3 dpi from four samples, each containing four leaf disks. Error bars show the standard deviation.
• Figure 8 depicts that PVA NIa enhances viral gene expression.
• N. benthamiana plants two leaves in each of three plants
• Agrobacterium containing 35S-VPg-nos, 35S-NIa-nos or 35S-GUS-nos construct (OD 600 of 0.5) mixed with 35S- PVA ACDD ::rlu nt -nos (OD 600 of 0.05) and 35S-fluc-nos (OD 600 of 0.01) .
• Four samples consisting of four leaf disks were collected at 3 dpi.
• A Effect of NIa co-expression on Flue and Rluc accumulation.
• Lower panel shows the relative enhancement of viral gene expression in the presence of NIa and VPg when the GUS control was set to one.
• FIG. 9 depicts that PVA VPg stabilises viral RNAs. Viral RNA levels ⁇ rluc) and flue RNA levels were assessed using q-RT-PCR. Each assay consisted of three biological repiicai.es. in addition, each biological replicate consisted of three technical replicates, and their averaged values were used for downstream calculations, (A) q-PCR was carried out from co-infiltrations performed with Agrobacterium containing 35S-fluc- nos (OD 600 of 0.01) and 35S-PVA ACDD : :rlu nt -nos, 35S-PVA CPmut : :rlu nt -nos, 35S- PVA wt ::rlud nt -nos (OD 6 oo of 0.05) constructs mixed with Agrobacterium carrying either
• 35S-VPg-nos or 35S-GUS-nos (OD 600 of 0.5) constructs.
• Total RNA 1000 ng was used to synthesise cDNA. Equivalent of 11 ng of template RNA was used in q-PCR with primers annealing to flue or rluc RNA. Asterisks indicate the low viral RNA amounts (0.15 pg rluc RNA/ng of total RNA) from 35S-PVA ACDD ' : : rlu nt -nos during GUS co-expression.
• B q-PCR was carried out from co-infiltrations performed with
• Total RNA (700 ng) was used to synthesise cDNA. Equivalent of 25 ng of template RNA was used in q-PCR with primers annealing to flue or rluc RNA. Flue and rluc mRNA amounts were normalised with the amount of total RNA (ng).
• Figure 10 shows a model of PVA VPg-mediated enhancement of PVA RNA translation.
• Translation of PVA RNA benefits from the presence of VPg in a concentration- dependent manner. Simultaneously with enhanced translation the concentration of viral RNA is increasing.
• Viral RNA is protected from degradation in the presences of VPg i) because of engagement of viral RNA with ribosomes during active protein synthesis and ii) because of the interference of VPg with gene silencing.
• Figure 11 shows that the level of P0 affects viral gene expression.
• A) The effect on gene expression in PVA CPmut , PVA AGDD and FLUC due to down-regulation of P0.
• B) The effect on gene expression in PVA CPmut , PVA AGDD and FLUC due to over-expression of P0.
• Protein level of P0 was determined by western blot analysis during down- regulation (C) and over-expression (D) of P0 compared to their respective controls.
• E Over-expression of N- and C-terminally myc-tagged POs (P0 N and P0 C , respectively) and their effect on gene expression in PVA AGDD and detection by western blot analysis using anti-myc mAb.
• VPg and the viral 5 ' -UTR are involved in the PO-mediated enhancement of viral gene expression.
• FIG. 13 Multiple sequence alignment for viral 5'UTR regions with EMBOSS Needle pairwise alignment for identity percentage. The following sequences were aligned: AJ131402.1: Potato A virus RNA complete genome, isolate U 5'UTR (1-161) (SEQ ID NO:l); AF014811.2: Zucchini yellow mosaic potyvirus polyprotein gene, complete cds 5'UTR (1-143); AJ252242.1: Pea seed-borne mosaic virus complete genome 5'UTR
• M11458.1 Tobacco etch virus (highly aphid transmissible (HAT)) complete genome 5'UTR (1-143);
• X04083.1 Tobacco vein mottling virus (TVMV) RNA genome 5'UTR (1-153);
• AB027007.1 Japanese yam mosaic virus genomic RNA, complete genome 5'UTR (1-153);
• X16415.1 Plum pox virus RNA genome 5'UTR (1-149);
• HQ631374.1 Potato virus Y strain NTN isolate HN1, complete genome 5'UTR (1-188); M96425.1: Pepper mottle virus complete genome 5'UTR (1-167);
• Figure 14 Multiple sequence alignment of P0 amino acid sequences aligned with Jalview and MUSCLE 3.0 software (Robert C. Edgar, http://www.drive5.com/muscle). The following sequences were aligned: Query sequence: Partial N. benthamiana PO sequence (SEQ ID NO:28).; AF370225.1: Arabidopsis thaliana putative 60S acidic ribosomal protein PO (At3g09200) mRNA, complete cds.; BQ119027.2: EST604603 mixed potato tissues Solanum tuberosum cDNA clone STMEG62 5' end, mRNA sequence.; BF096295.1: EST360344 tomato nutrient deficient roots Lycopersicon esculentum cDNA clone CLEW11M13 5' sequence; L46848.1: Glycine max acidic ribosomal protein PO mRNA, complete cds.; CAA69256.1: Zea mays 60S acidic ribo
• EMBOSS parameters -gapopen 14; -gapextend 4; -alternatives 1; -aformat3 pair; -sproteinl; -sprotein2; Align_format: pair; Matrix: EBLOSUM62.
• AJ131402.1 Potato virus A RNA complete genome, isolate U VPg (5688-6255); AJ243766.2: Potato virus V mRNA for polyprotein, isolate DV 42 VPg (5727-6291); HQ631374.1: Potato virus Y strain NTN isolate HN1, complete genome VPg (5718- 6281); M96425.1: Pepper mottle virus complete genome VPg (5694-6258); X04083.1: Tobacco vein mottling virus (TVMV) RNA genome VPg (5610-6156); X16415.1: Plum pox virus RNA genome VPg (5720-6297); AF014811.2: Zucchini yellow mosaic potyvirus polyprotein gene, complete cds VPg (5706-6275); M11458.1: Tobacco etch virus (highly aphid transmissible (HAT)) complete genome VPg (5690- 6260); AJ252242.1: Pea seed-borne mosaic virus complete genome VPg (6029- 6554); AB
• EMBOSS Needle parameters -gapopen 10.0; -gapextend 0.5; -endopen 10.0; -endextend 0.5; - aformat3 pair; -sprotein l; -sprotein; Matrix: EBLOSUM62.
• the present invention relates to a method for achieving enhanced protein expression from a target nucleotide sequence in a cell of an organism, wherein the method comprises the steps of providing a nucleic acid construct comprising a nucleotide sequence encoding a viral genome-linked protein (VPg) or a derivative or a fragment thereof; providing a nucleic acid construct comprising a nucleotide sequence encoding a virus genome or a fragment thereof having a capacity for enhanced protein expression in the presence of VPg and wherein a virus genome or a fragment thereof comprises at least 5' untranslated region (5'UTR) and a target protein and introducing said nucleic acid constructs for transient expression into a cell.
• VPg viral genome-linked protein
• the method further comprises a host acidic ribosomal P0 protein or a derivative or a fragment thereof;
• a host acidic ribosomal P0 protein or a derivative or a fragment thereof Unless otherwise specified, the scientific and technical terms, which are used herein have the same meanings as commonly understood by a skilled person in the art to which this invention belongs. For purposes of the present invention, the following terms are defined below.
• the term "virus genome” refers to the nucleic acid which encodes the genetic information of the virus.
• the nucleic acid comprising the genome may be single- stranded or double-stranded, and in a linear, circular or segmented configuration.
• Virus genomes may contain their genetic information encoded in either DNA or RNA.
• viruses Since viruses are obligate intracellular parasites only able to replicate inside the appropriate host cells, the genome contains information encoded in a form which can be recognised and decoded by the particular type of cell parasitized. Thus, the genetic code employed by the virus must match or at least be recognised by the host organism. Similarly, the control signals which direct the expression of virus genes must be appropriate to the host.
• Single-stranded virus genomes may be: positive (+)sense, i.e. of the same polarity (nucleotide sequence) as mRNA, negative (-)sense or ambisense i.e. a mixture of the two.
• Virus genomes which consist of (+)sense RNA i.e. the same polarity as mRNA
• (+)sense vRNA is infectious when the purified viral RNA (vRNA) is applied to cells in the absence of any virus proteins. This is because (+)sense vRNA is essentially mRNA.
• the first event in a normally-infected cell is to translate the vRNA to make the virus proteins responsible for genome replication. In this case, direct introduction of RNA into cells merely circumvents the earliest stages of the replicative cycle.
• (+)sense vRNA is directly infectious when applied to susceptible host cells in the absence of any virus proteins (although it is about one million times less infectious than virus particles). There is an untranslated region (UTR) at the 5' end of the genome which does not encode any proteins and an UTR at the 3' end. These regions are functionally important in virus replication and are thus conserved in spite of the pressure to reduce genome size. Both ends of (+)stranded eukaryotic virus genomes are often modified, the 5' end by a small, covalently attached protein or a methylated nucleotide 'cap' structure and the 3' end by polyadenylation. These signals allow vRNA to be recognised by host cells and to function as mRNA.
• 5'UTR or the "five prime untranslated region” or “5' untranslated region”, also known as the leader sequence, is a particular section of messenger RNA (mRNA) and the DNA that codes for it. It starts at the + 1 position (where transcription begins) and ends one nucleotide before the start codon (usually AUG) of the coding region. It usually contains a ribosome binding site (RBS), in bacteria also known as the Shine Dalgarno sequence (AGGAGGU).
• RBS ribosome binding site
• AUGGU Shine Dalgarno sequence
• the 5'UTR may be up to a hundred or more nucleotides long. In prokaryotic mRNA the 5' UTR is normally short. Some viruses and cellular genes have unusual long structured 5'UTRs which may have roles in gene expression.
• 5'UTR Binding sites for proteins, that may affect the mRNA's stability or translation, for example iron responsive elements, which occur in the 5'UTRs of a small number of eukaryotic mRNAs that regulate gene expression in response to iron. 5'UTR may also contain sequences that promote the initiation of translation .
• a nucleotide sequence of 5'UTR of Potato virus A is presented in SEQ ID NO : l .
• the 5'UTR is at least 35% identical to SEQ ID NO : l, preferably the identity is 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% to SEQ ID NO : 1.
• RNA virus refers to a loosely defined group of positive-sense single-stranded RNA viruses that are major pathogens of animals, plants and insects.
• Viruses from the six divergent families (the Picornaviridae, Caliciviridae, Comoviridae, Sequiviridae, Dicistroviridae and Potyviridae) that comprise the picornavirus-like virus superfamily have the following features in common : a genome with a protein attached to the 5 ' end and no overlapping open reading frames, all the RNAs are translated into a polyprotein before processing, and a conserved RNA-dependent RNA polymerase (RdRp) protein .
• RhdRp conserved RNA-dependent RNA polymerase
• Potyviruses belong to genus Potyvirus, family Potyviridae. They have a positive sense ssRNA genome of about 9.7 kb. Virions are flexuous filaments, 680-900 nm long and 11- 13 nm wide. Several sites within potyviral genome have been shown to tolerate a gene-sized foreign insert. Proteins which have been expressed from PVA Nib/CP cloning site include e.g. GFP, GUS, Rluc and human S-COMT (Kelloniemi et al ., 2006.
• PVA icDNA has three cloning sites, namely PI, Pl/HCpro and Nib/CP.
• PVA with three inserts (gfp/Rluc/uidA) has a genome ca . 38 % longer as compa red to wt PVA. Flexuous rod-shaped PVA particles grow in relation to the growing genome size. Even with three inserts the virus is able to cause systemic infection (Kelloniemi et al ., 2008. Virus Res. 135, 282-291) .
• Potato virus A is a member of the genus Potyvirus within the picornavirus-like viruses.
• the PVA genome is expressed as a single polyprotein that is subsequently cleaved by three virus-encoded proteinases to yield up to ten mature proteins ( Figure 1) .
• One of the mature virus-encoded proteins "viral genome-linked protein” or "VPg”, is covalently linked to the 5' terminus of viral genomic RNA via a Tyr residue.
• the VPg is a multifunctional protein that has a role in virus replication (translation and RNA synthesis) .
• VPg plays a role in potyviral movement and functions as an avirulence determinant in certain resistant hosts in which the potyvirus fails to achieve systemic infection.
• An amino acid and a nucleotide sequence of Potato virus A is a member of the genus Potyvirus within the picornavirus-like viruses.
• the PVA genome is expressed as a single polyprotein that is subsequently cleave
• VPg are presented in SEQ ID NO : 2 and SEQ ID NO : 3, respectively.
• Potyvirus VPg is typically 25 kDa, while the size of the VPg in some other viruses can be 8- 12 kDa .
• the length of viral VPg is typically from 22 to 200 amino acids.
• a common structure of VPgs comprises a lysine-rich region (Fig . 15) .
• Potyviral VPgs have been shown to interact with eukaryotic translation initiation factor 4E (eIF4E) or its isoform iso4E (eIFiso4E) (reviewed by Robaglia and Caranta, 2006). After initial investigations, these interactions have received considerable attention.
• eIF4E eukaryotic translation initiation factor 4E
• eIFiso4E isoform iso4E
• the first suggestion is that this interaction is involved in translation by either promoting translation of viral mRNAs or inhibiting translation of cellular mRNAs.
• the eIF4E might be recruited to translate viral mRNAs via its interaction with VPg that is located at the 5'end of viral RNA.
• VPg-eIF4E interaction may be involved in sequestering eIF4E from cellular mRNAs resulting in translation inhibition. Both hypotheses are supported by the finding that the eIF4E-VPg interaction increases the affinity of eIF4E to eIF4G, a key component in the assembly of the translation preinitiation complex.
• PVY Potato virus Y
• TMV Tobacco etch virus
• TuMV Turnip mosaic virus
• TuMV stimulates the translation of uncapped IRES-containing mRNAs (Khan et al., 2008). Therefore, VPg appears to have a dual role in translation.
• the interaction with eIF4E may also serve other functions.
• VPg interacts with polyA binding protein (PABP) that binds to the 3'terminal polyA tail of potyviruses.
• PABP polyA binding protein
• Positioning of VPg to the 3'UTR via eIF4E-VPg interaction might function in RNA replication to facilitate initiation of minus strand synthesis. In picornaviruses, circularization of the genome is required for RNA replication.
• the eIF4E interaction may also be involved in viral movement. Intracellular trafficking may be achieved via interaction of the eIF4E-VPg complex with eIF4G that targets the complex to microtubules.
• Another study showed that eIF4E is involved in cell-to-cell trafficking.
• VPg-Pro (NIa) co-localises with eIF4E in the nucleus, whereas in the presence of 6K2 the co-localisation takes place in 6K2-VPg-Pro-induced vesicles. This supports the suggestion that the interaction may have several functions in the cell.
• potyviral 5'UTRs can function as internal ribosome entry sites (IRESs) (Gallie, 2001) recruitment of the translation preinitiation complex into the 5'end of the potyviral mRNAs may be unnecessary. This is supported by the finding that eIF4G binds directly to the TEV 5'UTR (Ray et al., 2006), and that eIF4E is not required for translation initiation from the TEV 5'UTR (Gallie, 2001).
• PVA VPg The native structure of PVA VPg is a partially disordered molten globule-like form (Rantalainen et al., 2008).
• the N-terminal part of PVA VPg is predominantly unstructured .
• Amino acids 38-44 of PVA VPg are responsible for nucleotide triphosphate (NTP)-binding and their deletion debilitates uridylylation of VPg (Puustinen and Makinen, 2004) .
• NTP nucleotide triphosphate
• this region conta ins the C-terminal part of the bipartite nuclear localization signal (NLS) of PVA (Rajamaki and Valkonen, 2010) .
• Region B (amino acids 41-50) of the N LS of PVA pa rtly overlaps with the NTP- binding site.
• the NTP-binding region has been suggested to be part of a natively unstructured stretch of amino acids (Rantalainen et al ., 2008), which may allow this region to be multifunctional and structurally flexible, and thereby facilitate its participation in various interactions.
• a proteolytic fragment containing the corresponding NTP-binding site of PVY VPg was pulled out with eIF(iso)4E, indicating that the NTP-binding region may exert its effect on PVA infection at the level of the eIF4E/(iso)4E interaction (Cotton et al ., 2006, Grzela et al ., 2006) .
• Numerous studies are actively focused on deciphering the exact molecula r mechanism underlying the effect of eIF4E on virus multiplication .
• VPg enhances translation of viral RNAs but inhibits reporter mRNA translation in planta. VPg enhances the viral gene expression.
• the identity percentage between PVA VPg (SEQ ID NO : 2) and VPg of the present invention is at least 42%.
• the identity is at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%.
• the similarity percentage between PVA VPg (SEQ ID NO : 2) and VPg is at least 58%.
• the similarity is at least 58%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%.
• target protein refers to the product of a gene inserted into the viral icDNA.
• One or more genes or inserts or nucleotide sequences encoding a target protein or ta rget proteins can be inserted into the vector in question .
• the "ribosomal protein P0" or “P0” relates to a lateral protruding structure found on the large ribosome subunit, being referred to as the ribosomal stalk (Gonzalo and Reboud, 2003) .
• the eukaryotic ribosomal stalk commonly consists of the 60S acidic ribosoma l proteins P0 (SEQ ID NO : 4 and SEQ ID NO : 5), PI and P2 and P3 that is plant specific.
• P-proteins Collectively these proteins are referred to as P-proteins.
• P0 binds to the ribosome via a 28S rRNA interaction and the remaining P-proteins anchor to the ribosome to form the stalk via P0.
• P-proteins exist in the cytoplasm in a free pool outside the ribosome, and P-proteins appear to shuttle between the ribosome and the cytoplasmic free pool .
• Recent studies in yeast have implied that the stalk may assemble outside of the ribosome and thereby join the ribosome as a ready-made stalk structure (Francisco-Velilla and Remacha, 2010) .
• P-proteins have also been implied to affect translation of specific mRNAs. When PI and P2 were depleted from yeast, a change in the protein expression pattern was observed . This suggested that the P-proteins affect to the differential translation of specific mRNAs. P-proteins derive their name from phosphoproteins as they are frequently phosphorylated . Anaerobic conditions were reported to alter both stalk composition and P-protein phosphorylation in maize, and its role in selective translation as a response to anaerobic stress was considered . Also, the P-protein phosphorylation status was proposed to be tightly regulated during seed germination, possibly taking part in regulating specific protein synthesis.
• IRES-driven translation is a common strategy amongst viruses, and a fraction of cellular mRNAs also possesses IRES-elements that mediate their translation . Functioning in selective IRES translation appears therefore as one possible mean by which P-proteins could regulate translational patterns a nd be involved in virus translation.
• the identity percentage of P0 of the present invention or a derivative or a fragment thereof is at least 45% identical with SEQ ID NO : 28.
• the identity is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical with SEQ ID NO : 28.
• the similarity percentage of the P0 of the present invention or a derivative or a fragment thereof is at least 60%, preferably at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% similar with SEQ ID NO : 28.
• a "fragment" of VPg comprises an amino acid sequence wherein one or more amino acid residues are deleted from the VPg amino acid sequence while the fragment essentially retains its capacity to enhance protein expression of viral mRNA having 5'UTR.
• a "derivative" of VPg as used in this application comprises a VPg-derived polypeptide that has been chemically modified e.g . via conjugation to another chemical moiety, phosphorylation, and glycosylation or other natural or artificial chemical modification while essentially retaining its capacity to enhance protein expression of RNA, preferably viral RNA.
• polypeptide include in addition to the full-length or native amino acid sequences also derivatives, variants (wherein a substituted amino acid has simila r structural or chemical properties as the wild-type amino acid), fragments, and muteins of the polypeptide, examples of which are described below.
• An “enhancer element” of protein synthesis comprises a nucleotide sequence encoding a virus genome or a fragment thereof having a capacity for enhanced protein synthesis in the presence of a viral genome-linked protein (VPg) ; and a nucleotide sequence encoding VPg or a fragment thereof, wherein the virus genome comprises at least a 5' untranslated region (5'UTR) and a target protein.
• VPg viral genome-linked protein
• the present invention relates to a method for achieving enhanced protein expression from a target nucleotide sequence in a cell of an organism, wherein the method comprises the steps of providing a nucleic acid construct comprising a nucleotide sequence encoding a viral genome-linked protein (VPg) or a derivative or fragment thereof; providing a nucleic acid construct comprising a nucleotide sequence encoding a virus genome or a fragment thereof having a capacity for enhanced protein expression in the presence of VPg, wherein a virus genome or a fragment thereof comprises at least 5'untranslated region (5'UTR) and a target nucleotide sequence operably linked to a promoter; and introducing said nucleic acid constructs for transient expression into a cell .
• VPg viral genome-linked protein
• the method further comprises a step of providing a nucleic acid construct comprising a nucleotide sequence encoding ribosomal protein PO or a derivative or a fragment thereof.
• PO may originate from a host cell or from the cell used for ta rget protein expression.
• PO or a derivative or a fragment thereof may be from any organism.
• the virus genome and VPg or derivatives or fragments thereof originate from a picornavirus-like virus, preferably a potyvirus. Enhancement of translation is especially specific for such mRNA molecules, which have 5'UTR of a picornavirus-like virus, such as a potyvirus.
• a superg roup of picornavirus-like plant viruses include the como-, nepo- and potyviruses.
• Picornavirus-like animal viruses include Picornaviridae and Caliciviridae.
• the virus belongs to Potyviridae family. More preferably the virus genome and VPg originate from a potyvirus.
• Potyviruses include for example Potato virus Y (PVY), Potato virus A (PVA), Tobacco vein mottling virus (TVMV), Beet mosaic virus (BtMV), Clover yellow vein virus (CIYVV) or Plum pox virus (PPV).
• PVY Potato virus Y
• PVA Potato virus A
• TVMV Tobacco vein mottling virus
• BtMV Beet mosaic virus
• CIYVV Clover yellow vein virus
• PSV Plum pox virus
• the method of the present invention can be in vitro or in vivo method.
• the method is an in vivo method.
• Enhanced protein expression from a target nucleotide sequence is achieved in a cell of an organism.
• the organism can be a eukaryote, such as an animal, preferably a non-human animal or a plant or a fungus, such as yeast.
• the enhanced expression can be detected by any method known to a skilled person in the art.
• Agroinfiltration may be used to initiate both viral and VPg expression simultaneously in cells.
• synthesis of reporter proteins from monocistronic mRNA transcripts is inhibited by the presence of potyviral VPg.
• An aspect of the invention is that when the full-length potyviral RNA serves as a transcript there is a vast enhancement of translation in vivo.
• plants can be infiltrated with Agrobacterium culture that contains two types of bacteria : one which is carrying the gene of interest inserted into the viral genome and one which is carrying the construct for VPg expression. Furthermore, a construct for expressing PO is introduced into a cell.
• An Agrobacterium-mediated infection assay may be used for quantitating the viral gene expression (Eskelin, K. et al. 2010).
• the infection assay uses Renilla luciferase (Rluc) as a reporter for virus activity and firefly luciferase (Flue) as an internal control was used to address the early stages in PVA infection.
• the reporter can be any suitable detectable protein, such as a marker gene.
• the present invention also relates to a method for producing nucleotide sequences having an improved capacity of enhancing protein expression, wherein the method comprises the steps of selecting a virus genome or a fragment thereof comprising 5'UTR and a target protein and having a capacity for enhanced protein expression in the presence of VPg; selecting a VPg or a fragment thereof; producing a nucleic acid construct comprising a nucleotide sequence encoding a virus genome or fragments thereof having a capacity for enhanced protein expression in the presence of VPg, wherein the virus genome comprises 5'UTR and a target protein; producing a nucleic acid construct comprising a nucleotide sequence encoding VPg or a fragment thereof.
• the method further comprises a step of selecting a ribosomal protein P0 or a derivative or a fragment thereof capable of enhancing protein expression in the presence of VPg.
• the virus genome and VPg or derivatives or fragments thereof can originate from a picornavirus-like virus.
• the virus is a potyvirus.
• VPg and P0 can be produced as a fusion.
• the present invention is also related to an enhancer element of protein synthesis, wherein the enhancer element comprises a nucleotide sequence encoding a virus genome or a fragment thereof comprising a 5'untranslated region (5'UTR) and a target protein and having a capacity for enhanced protein synthesis in the presence of VPg; and a nucleotide sequence encoding VPg or a derivative or a fragment thereof capable of enhancing protein synthesis.
• the enhancer element further comprises a nucleotide sequence encoding ribosomal protein PO or a derivative or a fragment thereof.
• the invention also relates to a plasmid comprising said enhancer element.
• the invention also relates to an expression vector comprising said enhancer element according to the present invention.
• the invention also relates to a host cell comprising said enhancer element.
• the invention also relates to use of said enhancer element to enhance protein expression in a cell in vivo.
• the present invention also provides vectors and host cells comprising the polynucleotides according to the present invention.
• vector refers to a vector in which a nucleic acid is under the control of, and operably linked to, an appropriate promoter or other regulatory elements for transcription in a host cell.
• the vector may be a bifunctional expression vector which functions in multiple hosts.
• the vector may contain its own promoter of other regulatory elements and/or an appropriate promoter or other regulatory elements for expression in the host cell. Skilled persons in the art are familiar with the expression vectors which can be used in the present invention for the expression of nucleic acid constructs.
• Nucleic acid constructs comprising for example VPgs or derivatives or fragments thereof may be prepared by any appropriate method used in the art.
• mutant VPgs in the in vivo translation inhibition experiments are prepared as follows: VPgs are cloned from the recombinant protein expression constructs by PCR using appropriate oligonucleotides as PCR primers. The primers with suitable introduced restriction sites are used to insert the products into a vector containing the appropriate promoter and terminator, from which the promoter-target gene fragment is cloned into the final vector using appropriate restriction sites.
• the method further relates to a method for heterologous production of a protein from a target nucleotide sequence in a cell of an organism, wherein the method comprises the steps of providing a nucleic acid construct comprising a nucleotide sequence encoding viral genome-linked protein (VPg) or a derivative or a fragment thereof; providing a nucleic acid construct comprising a nucleotide sequence encoding a virus genome or a fragment thereof having a capacity for enhanced protein expression in the presence of VPg, wherein a virus genome or a derivative or fragment thereof comprises at least 5'untranslated region (5'UTR) and a target protein; introducing sa id nucleic acid constructs comprising VPg and/or a virus genome into a cell ; and producing the heterologous protein in the cell in vivo.
• VPg viral genome-linked protein
• the method further comprises a step of providing a nucleic acid construct comprising a nucleotide sequence encoding ribosomal protein PO or a derivative or a fragment thereof.
• heterologous is used broadly to indicate that the target gene or sequence of nucleotides in question have been introduced into to the cells in question using genetic engineering .
• a heterologous gene may replace an endogenous equivalent gene i .e. one which normally performs the same or similar function, or the inserted sequence may be additional to the endogenous gene or other sequence.
• Nucleic acid heterologous to a cell may be a non-naturally occurring in cells of that type, va riety or species.
• the heterologous nucleic acid may comprise a coding sequence of, or derived from, a particular type of cell, placed within the context of a cell of different type or species or variety.
• nucleic acid sequence to be placed within a cell in which it or a homologue is found naturally, but wherein the nucleic acid sequence is linked and/or adjacent to nucleic acid which does not occur naturally within the cell, or cells of that type or species or va riety, such as operably linked to one or more regulatory sequences, such as promoter sequence, for control of expression .
• the heterologous protein produced in the present invention may be any protein .
• the viral genome-linked protein (VPg) of Potato virus A translation of viral RNAs inhibits reporter mRNA translation in planta.
• VPg is co-expressed with PVA genome, replication-deficient PVA genome or movement-deficient pvA CPmut RNA, viral gene expression from PVA AGDD ,PVA CPmut , and PVA wt RNA is enhanced.
• the enhancing effect of VPg on viral translation is mediated through the 5' untranslated region (UTR).
• UTR 5' untranslated region
• the 5'UTR alone is not sufficient to enhance monocistronic reportermRNA expression, suggesting that either some additional viral sequence or protein is required for enhanced expression.
• PVA VPg inhibits reporter gene expression from monocistronic flue and rluc mRNAs. Inhibition of reporter gene expression also occurs in the presence of VPg in vitro.
• virus genome As shown in the Examples below virus genome, VPg and PO have a specific synergistic influence on the protein synthesis of viral RNA molecules having 5'untranslated region.
• the presence of a virus genome, VPg, 5'UTR and/or PO enable the enhancement of translation of desired viral RNA molecules or fragments thereof.
• virus vectors such as potyviruses can be used in the present invention.
• An example of a virus vector is potato virus A (PVA) vector.
• VPg from different origins can be used as well as various host organisms.
• An example of an organism is N. benthamiana.
• the applications of the present invention include heterologous protein production in plants i.e. molecular farming. Examples of such proteins include therapeutic proteins (e.g.
• heterologous proteins and nucleic acids encoding them are not limited to the ones listed above, but can be of any origin.
• the method according to the present invention is also suitable for determining variations in genome expression.
• Lys residues (K) were substituted with Ala residues (A) within PVA VPg, as shown in Fig. 1.
• the (His) 6 /pQE30 (Qiagen) construct was used to express wild-type VPg (wtVPg) Merits et al. 1998) (SEQ ID N0: 6), and used as a template for the production of the VPg-NTPl (SEQ ID NO: 7), VPg-NTP2 (SEQ ID NO: 8), VPg-NTP3 (SEQ ID NO :9) and VPg-A38-44 (SEQ ID NO : 10) mutants. Mutations were introduced using standard PCR reactions with Phusion DNA polymerase (Finnzymes).
• Oligonucleotide 5 ' -ACAAAGGGCAAAACACATGGAATGGGGAAA-3 ' was used as the forward primer (the underlined adenine indicates the mutation in the Ncol restriction site, which was used to screen mutated plasmids from the wild-type plasmids) and the following oligonucleotides were used as reverse primers: 5 ' - GCTTCCAAAATAGTGCTCTAACGTGGAGTC-3 ' (SEQ ID NO : 12), for VPg-A38-44; 5 ' - TG CTCCTTTCTTTGTGTACG CG CTTCC A AA- 3 ' (SEQ ID NO: 13), for VPg-ntpl ; 5 ' - TGCTCCGGCCTTTGTGTACGCGCTTCCAAA-3 ' (SEQ ID NO: 14), for VPg-ntp2; and 5 ' - TG CTCCG G CTG CTGT
• VPgs were cloned from the recombinant protein expression constructs by PCR using the oligonucleotides 5'-AATAGGTACCATGACAGGATCGCATCA-3' (SEQ ID NO: 16), as a forward primer, and 5 '- AATATCTAG ATTACTCG AATTCAACCG ACTC- 3 ' (SEQ ID NO : 17), as a reverse primer. These primers introduced Kpnl and Xbal restriction sites, respectively, at the ends of the PCR products.
• Viral 35S-PVA: :rlucInt-nos constructs used in the Agrobacterium-mediated infection experiments have been described previously (Eskelin et al 2010). Bacterial expression of the rluc gene was prevented by insertion of the first intron from ribulose-1,5- bisphosphate carboxylase/oxygenase (Rubisco, RBC-1I). The constructs were based on full-length infectious cDNA copies of PVA-B11 (Oruetxebarria et al. 2001) tagged with the rluc-gene (35S-PVA: :rluc) (Gabrenaite-Verkhovskaya et al. 2008).
• PVA GDD 3VTR :rlucInt-nos.
• monocistronic 35S-PVA 5VTR-rluc-nos cassette was cloned into pRD400.
• Nicotiana benthamiana plants were grown in a greenhouse under a regimen of 16 h light at 24°C and 8 h dark at 18°C. The plants were watered daily, and fertilizer was applied once a week (the N : P: K ratio was 6 : 1 : 5; Substral Vital Active, the Scotts Company, USA).
• Agrobacterium tumefaciens strain C58C1, containing the helper plasmid pGV2260 for w ' r-gene expression was transformed with the binary vector constructs described above. Transformation and cultivation of agrobacteria as well as the infiltration procedure has been described previously (8). Before infiltration, Agrobacterium cultures were mixed with each other at the required ratios to form the final infiltration culture. In the in vivo translation inhibition assays the VPg expressing agrobacteria and the infiltration control (35S-GUS-nos or 35S-fluc-nos, depending on the experiment) were infiltrated at an OD 6 oo of 0.5 unless otherwise noted.
• the reporter protein constructs (35S-fluc-nos and/or 35S-rluc-nos) were infiltrated at an OD 6 oo of 0.01 or 0.005.
• 35S-PVA::rlu nt -nos constructs were infiltrated at an OD 6 oo of 0.05 unless otherwise stated.
• Each Agrobacterium mixture was infiltrated to two leaves of at least three plants.
• a single sample usually consisted of 2-5 leaf disks pooled from various infiltrated leaves. The disks were immediately frozen in liquid nitrogen and stored at -80°C. Each experiment was performed at least twice, each of which was done with minimum of three replicates.
• VPg and P0 Co-expression of VPg and P0 with PVA AGDD and their effect on viral gene expression was carried out essentially as reported for VPg here.
• P0 was cloned from a cDNA library of Arabidopsis thaliana and inserted into the binary vector pMDC32 (Curtis and Grossniklaus, 2003), and transiently over-expressed in plants using agro- transformation.
• Luciferase activity determination All samples in a particular experiment were analysed concurrently, using the same set of reagents. Activity measurements were carried out as described previously (8) using a Dual Luciferase Assay Kit (Promega) and a Luminoscan TL Plus device (Thermo Labsystems) . Mean values for Rluc and Flue activities were calculated from parallel samples, and were used in calculating the Rluc/Fluc activity relation or to normalize Rluc with the formula Rluc*Fluc/Fluc aV erage- Standard deviations of mean values were determined. Error bars normally represent the standard deviation within four (luciferase activity) or three (qPCR) replicate samples in a particular experiment. SDS-PAGE and western blotting
• Equal volumes of plant lysates used for dual luciferase measurements were pooled from each treatment and heated in the presence of SDS-PAGE loading buffer containing ⁇ -mercaptoethanol. Proteins were separated on 12% SDS polyacrylamide gels. After the run, proteins were blotted by electrophoretic transfer to PVDF membranes (Immobilon-P, Millipore).
• Recombinant proteins were produced in E. coli M 15 cells. Expression and purification of proteins were performed according to the manufacturer's protocol, under denaturing conditions, using Ni 2+ -NTA agarose. Prior to further analysis, proteins were renatured by overnight dialysis against MQ or diethylpyrocarbonate-treated H 2 0. Protein concentration was measured with a BioPhotometer (Eppendorf).
• Linear control DNA encoding Flue was used to synthesize capped and non-capped flue mRNA using the RiboMax Kit (Promega). Template DNA was removed using RQ1 RNase-Free DNase (Promega), and RNA was purified with Qiagen RNeasy columns. RNA concentration was determined with a BioPhotometer (Eppendorf), and integrity of the mRNA was verified by agarose gel electrophoresis.
• mRNAs were translated in wheat germ extract (Promega) in the presence of recombinant wt or mutant VPgs.
• VPgs (20, 40, or 60 pmol) were added to in vitro translation reactions together with 0.5 pmol of flue mRNA in a total volume of 12 ⁇ . Reactions were carried out at room temperature in the absence of ribonuclease inhibitors. Flue activity was measured with a luminometer (Luminoskan TL Plus) using the Luciferase Assay System (Promega).
• PVA 5'UTR was cloned in front of the rluc gene in pGEM-T (Promega).
• PVA-Rluc RNA was synthesized as above.
• PVA-rluc and flue mRNAs were mixed in a 1 : 1 molar ratio (20 pmol of each) and used for in vitro translation as above. Reactions were supplemented with 60 pmol of PVA VPg or BSA. Flue and Rluc activities were measured after 60 min using a Dual Luciferase Assay system (Promega).
• Primers used to anneal to rluc gene were 5 '- A ATCTG GTAATG GTTCTTATAG GTTAC-3 ' (SEQ ID NO : 25) (underlined nucleotide represents the exon-exon junction) and 5 '-CAACATG GTTTCCACG AAG A- 3 ' (SEQ ID NO : 26).
• q-PCR reaction was performed in 15 ⁇ mixtures containing LightCycler 480 SYBR Green I Master (Roche), 0.5 ⁇ each primer and 5 ⁇ of diluted cDNA.
• cDNAs and master mixes were pipeted on 384-well plates using CAS1200- BIOROBOT (Corbett). Three technical replicates were done for each cDNA.
• the PGR reaction was performed according to the following parameters: 1) Pre-incubation : 95°C 10 rnin, ramp rate .8°C/s, 2) Amplification : 45 cycles a) 95°C 10 s, ramp rate 4.8°C/s, b) 60°C 10 s, ramp rate 2.5°C/s, c) 72°C 15 s, ramp rate 4,8°C/s (single data collection), 3) Melting : a) 95°C 5 s, ramp rate 4.8°C/s f b) 65°C 60 s, ramp rate 2.5°C/s, and c) 97°C 0 s (continuous data co!iection); 4) Cooling : 40°C 10 seconds, ramp rate 2°C/s.
• RNA in each test template was measured by its crossing point (C p ) and determined from the standard curve.
• Standard curve was generated from 10-fold serial dilutions (0.5 pg-5 ng) of Flue- and Rluc encoding plasmids.
• C p _values were plotted against DNA amount to construct the standard curve. Quantification of flue and PVA RNA (rluc) in each test sample was back calculated and expressed as pg/ng total RNA.
• a gateway-compatible cDNA of A. thaliana 60S acidic ribosomal protein P0 (RPP0C) (NM_111960) was generated by PCR from an A. thaliana cDNA library.
• the P0 cDNA was recombined into pDONRZTM / Zeo (Invitrogen) and further into pMDC32 (Curtis and Grossniklaus, 2003), pGWB14, 15 and 42 (Nakagawa et al., 2007) to generate P0 and myc- or YFP-fusions using standard gateway cloning.
• gateway compatible cDNA fragments were generated from a N.
• Agrobacteria carrying PVA, flue and, possible co-expressed proteins were infiltrated four days after hairpin-infiltrations a nd gene expression analysed three days later. Gene-silencing was verified by reverse transcription (RT)- PCR.
• RT reverse transcription
• total RNA was extracted from plant leaves four days after infiltration of Agrobacteria carrying hairpin constructs. 1 ⁇ g of total RNA was treated with DNAse I (Promega) followed by RT with M-MLV RT enzyme (Finnzymes) using an oligo (dT) primer. The same primers that were used for generation of the hairpin sequences used for silencing were also used here in PCR with Dynazyme II DNA polymerase (Finnzymes) .
• the potyviral NIa/VPg region has been reported to inhibit cell-free protein synthesis (Cotton et al ., 2006, Grzela et al ., 2006, Khan et al ., 2008) . Accordingly, a proteolytic fragment containing the NTP-binding site of PVY VPg was pulled out with eIF(iso)4E, indicating that the NTP-binding region may be involved in the 4E/(iso)4E interaction.
• lysines in the NTP-binding site of VPg was assessed using PVA VPg-NTPl (K44A), VPg-NTP2 (K41A, K42A), VPg-NTP3 (K41A, K42A, K44A), and VPg-A38-44 mutants presented in Fig. 1.
• Capped flue mRNA was incubated with increasing concentrations of wt or mutant VPgs in in vitro translation reactions, and aliquots drawn at specific times were assayed for Flue accumulation .
• the wtVPg, NTP1-3, and ⁇ 38-44 proteins were also tested for in vitro translation inhibition activity in the presence of non-capped flue mRNA.
• the translation of non- capped flue mRNAs resulted in four times lower levels of Flue activity when compared to capped flue mRNAs, indicating that the capping reaction was successful.
• the translation reaction supplemented with 60 pmol of wtVPg yielded ⁇ 19% of the Flue activity measured in the control reaction lacking VPg at 60 min. Again, we found that wtVPg had the strongest inhibitory effect on translation of reporter mRNA.
• TuMV VPg has been shown to enhance translation of mRNAs having the TEV 5'UTR (Khan et al., 2008).
• the 5'UTR of PVA was cloned in front of the rluc gene and synthesised transcripts were used for in vitro translation in the presence of an equimolar amount of capped flue mRNA having non-viral 5'UTR.
• the addition of 60 pmol of VPg into the translation reactions resulted in a specific increase in Rluc accumulation in translations where the mRNA contained the PVA 5'UTR.
• Flue accumulation By contrast, there was no effect on Flue accumulation.
• the increase in Rluc accumulation was modest for both uncapped mRNAs (1.4-fold) and capped mRNAs (1.7-fold).
• potyviral NIa also has deoxyribonuclease and ribonuclease activities (Anindya et al., 2005, Cotton et al., 2006), we decided to test whether variation in translation inhibition might be caused by differences in nuclease activity of the VPg mutants.
• a clear mobility shift resulting from non-specific binding between VPg and the added mRNA was evident, whereas no RNA degradation was observed (Fig. 2C).
• RNA degradation was detected under these conditions.
• the quantity and purity of protein were checked by Coomassie staining (Fig. 2D). From these experiments we concluded that PVA VPg has no ribonuclease activity. It has been suggested that the NTP- binding site could be a sign of RNAse activity as many plant ribonucleases have phosphate binding sites but no other homology to known ribonucleases.
• the RNase assay showed that the wtVPg, as well as the mutant VPgs, bound flue RNA and retarded its migration in the gel.
• we assayed RNA binding activities of the wtVPg and mutant VPgs by incubating 10, 40 and 60 pmol of VPg with RNA. Higher VPg concentrations resulted in more retarded movement of the RNA, whereas the addition of 10 pmol of VPg only slightly retarded the migration of RNA.
• wtVPg The effect of wtVPg on reporter protein expression was studied.
• the wtVPg and GUS expression constructs were preinfiltrated next to each other in the same plant leaves. On the next day, the same regions were infiltrated with a mixture of agrobacteria containing the 35S-fluc-nos and 35S-rluc-nos constructs.
• Rluc activity arising from monocistronic reporter mRNAs is referred to as monocistronic Rluc (mRluc) to separate it from Rluc activity originating from viral genome.
• mRluc monocistronic Rluc
• Luciferase measurements at 3 dpi showed that reporter protein expression was lower in the VPg expressing plant versus the GUS control (Fig. 3B).
• the level of inhibition varied from plant to plant, which may be related to developmental status of the leaf or occurrence of the VPg expressing construct and reporter protein constructs in the same cell. Flue expression was inhibited to 39-89% of control, the average being 60 ⁇ 17% (Fig. 3B, left panel). Rluc expression was inhibited to 37-76% of control; the average was 58 ⁇ 12% (Fig. 3B, right panel). Pair wise comparison of VPg and GUS treatments showed that the inhibition was statistically significant (t-test, one-tail, P F
• VPg expression constructs or the 35S-fluc-nos construct were preinfiltrated into the plants at an OD 6 oo of 0.5.
• the 35S-rluc-nos construct was infiltrated to the preinfiltrated region at an OD 6 oo of 0.01. Rluc expression and total protein amount was measured at 3 dpi.
• all VPgs appeared to inhibit Rluc expression to some extent (Fig. 3C, left panel), whereas total protein amounts were comparable (Fig.
• mutant VPgs were as efficient as wtVPg in inhibiting Rluc expression; in fact, the NTP1 mutant appeared to have the strongest inhibitory effect on translation.
• the NTP1 mutant inhibited Rluc expression by 55%, whereas in the presence of the other VPgs Rluc expression reached ⁇ 75% of that of the control plants.
• VPg enhances viral gene expression in vivo
• GUS-nos with expression constructs for PVA WT 35S-PVA wt : :rlu nt -nos
• a movement-deficient mutant 35S- PVA ⁇ r. rlu ⁇ -nos
• the Agrobacterium mix also contained the 35S-fluc-nos construct as an internal control.
• VPg co-expression inhibited Flue expression (Fig. 4A).
• VPg co-expression increased accumulation of a specific CP form of approximately 47 kDa. Only this form of CP was detected in the pvA CPmut sample co-expressed with VPg. The sensitivity of the assay was not high enough to be able to detect any CP-specific bands in PVA AGDD samples. The effect of NTP mutations on viral translation was tested next.
• 35S-GUS-nos construct was coinfiltrated into N. benthamiana leaves together with 35S-PVA GDD ::rlu nt -nos and 35S-fluc-nos. Luciferase activities were assayed at 3 dpi. All NTP mutants inhibited expression of the internal control Flue, and expression was inhibited to the same extent (35-43%) irrespective of the VPg used (Fig. 5A). This result was in agreement with results obtained in coexpression of NTP mutants with reporter proteins (see Fig. 3B). In contrast, mutants had different effects on viral translation (Fig. 5A). In this experiment the boost in translation was even higher than in the experiment presented in Fig. 4. WtVPg produced the largest boost in translation (92-fold).
• NTP1 promoted translation 79-fold, NTP2 13-fold and NTP3 31-fold.
• the corresponding Fluc-normalised Rluc values were 186-, 161-, 26-, and 31-fold.
• Western blot analysis verified that all VPgs were expressed in similar quantities (Fig. 5B).
• VPg co-expression stimulated Rluc accumulation 26-fold when the 5'UTR was present but the 3'UTR was deleted (Fig. 6B). From these experiments we conclude that the stimulatory effect of VPg on viral translation requires the viral 5'UTR but not the 3'UTR. Finally, the PVA 5VTR-rluc cassette was cloned between the 35S promoter and nos- terminator and transferred into a binary vector. This was done to determine whether the 5'UTR was the only requirement for enhanced viral gene expression in the presence of VPg in vivo.
• the effect of VPg was also assayed in the presence of 35S- rluc-nos that had a multicloning site as the 5'UTR.
• VPg co-expression inhibited Flue expression to 50-80% of that in GUS co-expression.
• co-expression of PVA VPg together with the 35S-PVA5VTR- rluc-nos did not improve Rluc accumulation (Fig. 6C).
• PVA 5'UTR was not alone responsible for enhanced translation in the context of the viral genome.
• the PVA 5'UTR alone did not enhance translation, as the mRluc activities were ⁇ 70% of that of the reference 35S-rluc-nos construct.
• PVA NIa enhances viral gene expression in vivo
• NIa The proteolytic processing site between PVA VPg and Pro domain in NIa protein is slowly processed (Merits et al., 2002) and full-length NIa is abundant in infected cells (Haren et al., 2010). We asked whether NIa has the capacity to boost viral gene expression and simultaneously inhibit cellular translation as well.
• 35S-NIa-nos or 35S-GUS-nos constructs were agroinfiltrated together with PVA AGDD and 35S-fluc-nos internal control into N. benthamiana leaves and assayed at 3 dpi. Flue accumulation was not much reduced in the presence of NIa, whereas VPg alone reduced Flue expression to 41% (Fig. 8A). The reduction in Flue amount was visible in western blot analysis (Fig. 8C). An eight-fold increase in Rluc amount was observed when co-expressed with NIa (Fig. 8B), while the boost mediated by VPg was still 8- times higher than that.
• RNA amounts in the presence of GUS co-expression was similar to our earlier results (Eskelin et al ., 2010) .
• VPg co-expression increased viral RNA levels.
• viral RNA levels were 10-times higher, whereas in PVA wt the increase was 5-fold when compa red to corresponding GUS controls (Fig . 9A, left panel) .
• Measured Rluc/Fluc activates correlated with the rluc/fluc mRNA ratios (Ta ble 1) .
• RNA levels resulted in an approximate 100- fold increase in the measured ratio of enzymatic activities (Table 1), which is also in line with our previous results (Eskelin et al ., 2010) .
• Table 1 Comparison of rluc/fluc mRNA ratios to Fluc/Rluc enzymatic ratios from assay on the effect of VPg on flue mRNA and viral gene expression among PVA WT , pvA CPmut and PVA AGDD . Samples were homogenised in liquid nitrogen in 1.5 ml tubes. Pestle used for g rinding the sample was dipped in Passive lysis buffer and used to measure Rluc and Flue activities.
• Rluc mRNA amounts were normalised with flue mRNA amounts and are shown in the left part of the table. Equally, enzymatic activities of Rluc were normalised with Flue activities a nd are shown in right. First columns show the absolute value. In addition relative mRNA and enzymatic levels, where the GUS control was set to one is shown for each treatment. rluc/fluc mRNA Rluc/Fluc enzyme
• RNA levels and Rluc/Fluc enzymatic ratios were also analysed from sa mples in which pvA AGDD wgs co-expressed with 35S-fluc-nos and either with wtVPg, NTP mutants, NIa or GUS.
• pvA AGDD wgs co-expressed with 35S-fluc-nos and either with wtVPg, NTP mutants, NIa or GUS.
• rluc mRNA levels monitoring the accumulation of viral RNA decreased g radually when more lysines were mutated to alanines in the NTP mutants of VPg (Fig . 9B, right panel) .
• NIa co-expression increased viral RNA accumulation only ⁇ 2.5-fold as compared to GUS control, which may result from the low a mount of NIa expression (See Fig . 8) . Again, the Rluc/Fluc ratios of enzymatic activities a nd RNA amounts correlated nicely (Table 2) .
• VPg does not boost translation of reporter mRNA having PVA 5'UTR as the only virus-derived sequence
• the 5'UTR was cloned in front of the rluc gene and synthesised transcripts were used for translation in the presence of an equimolar amount of flue mRNA in vitro.
• Flue accumulation originating from mRNAs that lacked the PVA 5'UTR was modest for uncapped mRNAs ( 1.4-fold) and for capped mRNAs ( 1.7- fold) .
• the PVA5VTR-rluc cassette was cloned under the 35S promoter and transferred into a binary vector. This was done to determine whether the 5'UTR was the only requirement for enhanced viral gene expression in the presence of VPg in vivo.
• Agrobacterium harbouring the 35S-PVA5VTR-rluc, and either 35S-VPg or 35S- GUS, were mixed and infiltrated into N. benthamiana.
• the effect of VPg was also assayed in the presence of 35S-rluc that had a multicloning site as the 5'UTR.
• VPg co-expression inhibited Flue expression to 50-80% of that in GUS co-expression.
• P0 was down-regulated through transient hairpin (hpPO) expression in N. benthamiana in conjunction with a recently described infection assay for PVA (Eskelin et al., 2010).
• the infection assay is based on quantitating virally expressed Renilla luciferase (RLUC), and viral gene expression is initiated by Agrobacterium-mediated transformation of PVA infectious cDNAs into plants. Firefly luciferase (flue) is co- infiltrated and functions as an internal control to report for Agrobacterium-mediated and virus-independent gene expression. Two different viral constructs were used; pvA CPmut and PVA AGDD .
• PVA CPmut replicates but is incapable of cell-to-cell movement.
• PVA AGDD is replication-deficient and hence, functions to report for non-replication associated viral gene expression.
• transcripts of PVA AGDD have so far shown a specific response to both the viral coat protein (Hafren et al., 2010) and VPg (Eskelin et al., 2011) when compared to flue.
• Down-regulation of P0 via hpPO expression caused elevated gene expression in PVA CPmut , whereas that in PVA AGDD was hardly affected and control FLUC reduced.
• the presented figure represents an average derived from five independent experiments.
• As control to hpPO expression a hairpin containing a GFP-fragment (hpGFP) was expressed similarly.
• PO or GUS control was over-expressed and the responses in gene expression were evaluated using the same infection assay as above.
• the viral 5 ' -UTR, VPg, and PO are linked in enhancing viral gene exression
• PVA A5UTR showed an attenuated response to PO over-expression, whereas the relative increase in gene expression was even higher in PVA A3UTR , compared to PVA AGDD . This suggests that the 5 ' -UTR is involved and supports the response to PO over-expression.
• 5'UTR of PVA RNA is 36-58% identical to the analogous region in the other potyviral RNAs. Some areas of the 5'UTRs are more conserved than others which can be seen from the alignment (made with the Jalview program) of the 5'UTRs ( Figure 13). The most conserved regions are between nucleotides 1-40, 62-65, 70-80, 114-160, and 180-203. Ribosomal protein PO
• Identity refers to an exact match between two nucleotides or amino acids. Similarity refers to a resemblance between two residues that is greater than one would expect at random. The similarity is feasible only for amino acid sequence alignments. The highest identity (82.9%) and similarity (87.1%) was found between the query and soya bean P0 sequence (L46848.1 : Glycine max acidic ribosomal protein P0 mRNA).
• P0 proteins of maize (CAA69256.1 : Zea mays 60S acidic ribosomal protein P0; identity 74.3% and similarity 84.3%) and rice (BAC66723.1 : Oryza sativa Japonica Group 60S acidic ribosomal protein P0; identity 72.9% and similarity 84.3.%) are also very similar to N. benthamiana protein. Potato and tomato sequences were aligned only on a ten amino acids long stretch. The identity / similarity percentages of yeast homologs from Saccharomyces cerevisiae and Pichia pastoris were 47.1 / 61.4 % and 45.7 / 62.9 %, respectively. Human sequence is more similar to N.
• the identity percentage between PVA VPg and potato virus V, potato virus Y, pepper mottle virus, tobacco vein mottle virus, plum pox virus, zucchini yellow mosaic potyvirus, tobacco etch virus, pea seed-borne mosaic virus and japanase yam mosaic virus on amino acid level was 45.8%, 50.5%, 48.4%, 51.6%, 52.3%, 50.3%, 45.5%, 42.7% and 47.9%, respectively.
• the similarity percentage between PVA VPg and potato virus V, potato virus Y, pepper mottle virus, tobacco vein mottle virus, plum pox virus, zucchini yellow mosaic potyvirus, tobacco etch virus, pea seed-borne mosaic virus and japanase yam mosaic virus on amino acid level was 68.4%, 68.4%, 68.9%, 71.9%, 67.9%, 67.0%, 62.3%, 58.9% and 65.6%, respectively.
• the so called NTP-binding region which extends in PVA VPg from amino acid 38 to 44 is conserved among all potyviral VPg sequences.
• This region also shares a limited amino acid homology between poliovirus and potyvirus VPgs (Puustinen and Makinen, 2004. J . Biol . Chem. 279 : 38103-38110) .
• the identity percentage of VPg gene was between 53.1 - 57.2%, the least homologous being Japanese yam mosaic virus and the most homologous being potato virus Y.
• VPg The role of VPg in in vivo translation has not been reported previously. In this study, we studied the effect of PVA VPg on monocistronic reporter mRNA and viral gene expression in vivo. Since the capacity of PVA VPg to inhibit translation in vitro has not been clarified ea rlier, this analysis was performed first. Some differences amongst potyviral VPgs in their capacity to inhibit translation are apparent. TuMV VPg inhibits translation of capped mRNAs having TEV 5'UTR but promotes translation if mRNAs are uncapped (Khan et al ., 2008) . We also observed a reproducible 1.4- to 1.8-fold translational boost for mRNAs with PVA 5'UTR in vitro.
• RNA-binding capacity of VPg decreased as more Lys to Ala mutations was made in the NTP-binding region (Rantalainen et al ., 2011) . These results support the possibility that differences in the capacity of the wt and mutant VPgs to inhibit in vitro translation may result from their different affinities to RNA.
• nt wt and NTP-binding site mutant VPgs were incubated in the in vitro translation mixes with capped or uncapped mRNAs. All VPgs inhibited tra nslation in vitro, but a gradual decrease in inhibition with increasing number of mutations at the NTP-binding site was detected (Fig . 2B) .
• RNAse assay showed that neither the wt nor the mutant VPgs possess RNase activity. Thus, the inhibition of translation in vitro was not caused by RNase activity of the PVA VPg . However, the RNase assay indicated that all VPgs had an affinity towards the RNA. This was not surprising, since PVA VPg binds RNA unspecifically in vitro (Merits et al ., 1998) .
• Our recent computer- based modeling of PVA VPg structure showed that the NTP-binding site is located on one side of the VPg molecule, which has a positively charged surface. Mutation of the Lys residues to Ala at the NTP-binding site gradually reduces the positive charge of the surface in the model.
• RNA-binding capacity of VPg decreases, even though it is not totally abolished.
• the NTP-binding site is followed by positively charged amino acids that possibly enable the mutant VPgs to still bind RNA to some extent. Also, the N-terminus of the protein is positively charged.
• the Lys residues of the NTP-binding region form part of the nuclear localization region B of PVA VPg and they are needed for gene silencing suppression activity of PVA VPg (Rajamaki and Valkonen, 2010a). Therefore observed boost in virus-derived Rluc production could result, in part, from gene silencing suppression activity of PVA VPg.
• Substitution of two or three amino acids at NLS region B totally abolishes auxiliary function of the VPg in gene silencing suppression (Rajamaki and Valkonen, 2010a). For instance, a K42AK44A mutation, identical to our NTP2 mutant, did not result in suppression of gene silencing.
• mutant VPgs confirmed that the translational enhancement function of wtVPg may partially be related to its gene silencing suppression activity. Interestingly, if gene silencing suppression is behind the effect, the suppressor activity of VPg appears to act specifically on viral RNA.
• Ribosomal protein PO is a functional component of VPg-mediated enhancement of protein expression and can be used together with VPg to achieve maximal target protein production levels. This suggests that PO acts at a more general level apart from translation promoting mRNA stability and that VPg can hijack PO to act specifically on the viral mRNA, promoting both its stability and translation.
• Tyrosine 66 of Pepper vein banding virus genome-linked protein is uridylylated by RNA-dependent RNA polymerase. Virology 336:154-162.
• Potyviral VPg enhances viral RNA translation but inhibits reporter mRNA translation in planta.
• Cylindrical inclusion protein of Potato virus A is associated with a subpopulation of particles isolated from infected plants. J. Gen. Virol.89:829-838.
• HSp70 and its cochaperone CPIP promote potyvirus infection in Nicotiana benthamiana by regulating viral coat protein functions. Plant Cell 22:523-535.
• Turnip mosaic virus VPg interacts with Arabidopsis thaliana eIF(iso)4E and inhibits in vitro translation. Biochimie 90:1427-1434.
• Oruetxebarria I., D. Guo, A. Merits, K. Makinen, S. Saarma, and J. P.
• Potato virus A genome-linked protein VPg is an intrinsically disordered molten globule-like protein with a hydrophobic core. Virology 377:280-288.
• Tobacco etch virus mRNA preferentially binds wheat germ eukaryotic initiation factor (elF) 4G rather than eIFiso4G. J. Biol. Chem.281:35826-35834.
• elF wheat germ eukaryotic initiation factor

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