WO2003104449A2 - Improvements in or relating to protein production - Google Patents

Abstract

Improved method for protein production in plant cells comprising introducing into plant cells nucleic acid components that encode for RNA dependent RNA polymerase proteins of a cucumovirus and further introducing into the said plant cells a second isolated nucleic acid that encodes a recombinant cucumoviral RNA 3 molecule in the minus-sense orientation comprising at least an RNA sequence that codes for a minus-sense RNA sequence of a target protein, vectors, host cells and uses thereof.

Description

Improvements In or Relating to Protein Production

Background

The present invention relates to a method for producing heterologous or exogenous proteins in plant cell material such as transformed plant cells in culture or in plant tissue derived from transformed plant cells. In particular, the method relates to a method for producing pharmaceutical proteins in plant material, the genetic material required therefore, such as DNA and RNA, vectors, host cells, methods of introduction of genetic material into plant cells, and uses thereof.

In the past, pharmaceutical proteins have been produced using a variety of transformed cell systems, including cell culture systems such as those derived from bacteria, yeast, fungi, insect or mammalian cell lines (Kudo T. 1994, In: Y. Muroo a and T. Amanaka (Eds.) Recombinant microbes for industrial and agricultural applications, pp. 291-299, Marcel Dekker, New York; Harashima S., Bioproc. Technol. 1994, 19: 137-158; Archer D.B. 1994, In: Y. Murooka and T. Amanaka (Eds.) Recombinant microbes for industrial and agricultural applications, pp. 373- 393, Marcel Dekker, New York; Goosen M.F.A. 1993, In:

M.F.A. Goosen, A. Baugulis and P. Faulkner (Eds.) Insect cell culture engineering, pp. 1-16, Marcel Dekker, New York; Hesse F. & Wagner R. , Trends in Biotechnol. 2000, 18(4): 173-180).

Bacteria are prokaryotic organisms and are not suited for the expression of eukaryotic genes. Proteins produced in prokaryotic backgrounds may not be post-translationally modified in a similar manner to that of eukaryotic proteins produced in eukaryotic systems, e.g. they may not be glycosylated with sugars at particular amino acid residues, such as aspartic acid (N) residues (N-linked glycosylation) . Furthermore, folding of bacterially- produced eukaryotic proteins may be inappropriate due to, for example, the inability of the bacterium to form disulfide bridges. Moreover, bacterially-produced recombinant proteins frequently aggregate and accumulate as insoluble inclusion bodies.

Eukaryotic cell systems are better suited for the production of pharmaceutical proteins, such as human proteins, since such cell systems may effect post- translational modification of the produced proteins in a more efficient manner to that of prokaryotic cell systems. However, the yield of recombinant protein using certain eukaryotic cell systems, such as yeast expression systems is generally low. Furthermore, yeasts possess poly annose glycans and contaminants in purified protein preparations derived from yeast cell cultures that may be antigenic to humans (Harashima S., Bioproc. Technol . 1994, 19: 137-158; Jenkins N. et al . , Nat. Biotechnol. 1996, 14: 975-981).

Fungi have rigid cell walls and appear to be suitable mostly for the production of extra-cellular proteins that are secreted, such as industrial enzymes (Archer D.B. 1994, In: Y. Murooka and T. Amanaka (Eds.) Recombinant microbes for industrial and agricultural applications, pp. 373-393, Marcel Dekker, New York) .

The maintenance of insect and mammalian cells in culture requires complex and expensive media comprising blood- derived proteins. Furthermore, there is a considerable risk of contamination with infectious agents such as viruses and prions. The yield of recombinant proteins in animal cell systems is often low, thus making commercial production using animal cells appear relatively unattractive. It is thought that the main reason for relatively low yields is that the target proteins are expressed from one or only a few transgene copies that are successfully integrated into the chromosomal DNA.

Using recombinant replicating viruses as vehicles for protein production can circumvent low protein yields in certain eukaryotic protein expression systems. Although in principle protein yields should increase when using recombinant replicating viruses, the majority of described systems have been found to be unreliable.

Viruses with RNA genomes replicate in the cytoplasm of infected cells. Recombinant viral RNA genomes typically undergo deletions upon replication and as a consequence, inserted functional genes of interest may be rapidly lost, or are rendered dysfunctional (Covey S.N. and Hull R. 1992, In: Genetic engineering with plant viruses, T.M.A. Wilson and J.W. Davies (Eds.) pp. 217-249, CRC Press, Boca Raton, Fla; Hohn T. and Goldbach R.W., 1994, In: Encyclopedia of Virology, R.G. Webster and A. Granoff (Eds.), pp. 1536-1543; Sleatt D.E. and Wilson T.M.A. , In: Genetic engineering with plant viruses, T.M.A. Wilson and J.W. Davies (Eds.) pp. 55-113, CRC Press, Boca Raton, Fla) .

Baculoviruses have been used in insect cells to produce biopharmaceuticals (Goosen M.F.A. 1993, In: M.F.A. Goosen, A. Baugulis and P. Faulkner (Eds.) Insect cell culture engineering, pp. 1-16, Marcel Dekker, New York; Luckow V.A. and Summers M.D. , Bio/technology 6: 47-55, 1988). These viruses have DNA genomes and replicate in the nuclei of infected cells. The recombinant viruses however are rapidly overgrown by defective interfering particles, which out-compete the recombinant viruses and thereby severely suppress the generation of potentially high yields of recombinant protein.

Relatively recently plants have been considered as systems to produce biopharmaceuticals such as oral/edible vaccines, referred to as ^Λmolecular pharming' . (Daniel H. et al . , Trends in Plant Sci. 6(5): 219-226, 2001).

A number of experiments have demonstrated that plant glycoproteins are poorly immunogenic (Chargelegue D. et al . , Transgenic Res. (: 187-194, 2000). Such reported observations are consistent with the fact that animals, including humans, that eat plants or plant parts as part of their diet, are exposed to high levels of intake of plant oligosacharides and glycoproteins. However, for some well-known plant allergens it has been shown that the xylosyl and fucosyl residues are the key epitopes responsible for the observed allergenicity in hypersensitive individuals (Van Ree R. et al . , J. Biol. Chem. 275: 11451-11458, 2000).

An advantage of using plant cell cultures as expression systems over mammalian or insect cell cultures is that the maintenance of plant cell cultures is relatively simple requiring simple synthetic media comprising sugars, nutrient salts and synthetic plant growth regulators. Using plants per se in the field or under cover as expression systems may be disadvantageous since they can be subject to different abiotic and biotic stresses and the like.

Certain researchers have turned to the production of proteins in transgenic plant cell cultures. USP 5824856 teaches that certain nucleic acid elements of the brome mosaic virus (the model virus of the cucumovirus group) , along with an exogenous nucleic acid sequence of choice may be inserted into the genome of the brome mosaic virus, and have been used to produce exogenous or heterologous proteins in plants. The components that are taught as having been expressed include cDNA molecules of the RNA dependent RNA polymerase or replicase (hereinafter polymerase) genes from brome mosaic virus, and a cDNA of a recombinant brome mosaic virus genomic

RNA in which the coat protein gene is partially or wholly replaced with an exogenous gene.

However, certain features that are associated with the invention of USP 5824856 render the described system problematic for the reliable production of large amounts of exogenous proteins of interest and/or the use of it in, for example, dicotyledonous ( dicot' ) plant cell systems. Firstly, BMV has a narrow host range and only infects a number of cereal grains belonging to the Graminae (ie monocotyledonous ( ^Λmonocot' plants) . Protoplasts of many dicot species can be infected with BMV, but the virus is not capable of cell-to-cell movement or of systemic movement in dicot plants. Moreover, the amount of virus particles present in infected dicot cells is relatively low (DeJong W. and Ahlquist P. 1991, Semin. Virol. 2: 97) and generally far lower than the amount of virus present in infected natural host plants. In practice, the amount of recombinant protein produced in this system is relatively low since the replication and transcription rate of BMV in dicot cells is relatively low. Secondly, in the process of the invention of USP 5824856 it is suggested that cDNA encoding a BMV RNA 3 in the viral plus-sense orientation and carrying the nucleic acid of interest is expressed and is then acted upon by the viral polymerase components, giving rise to a minus-sense BMV RNA 3. This RNA molecule in turn is then acted upon by the viral polymerase components giving rise to the production of progeny plus-sense BMV RNA 3 and the subgenomic BMV RNA 4 from which the nucleic acid of interest is translated. Hence the recombinant protein is produced after at least one cycle of replication and at least one cycle of transcription (that is, a two cycle process) .

In cells infected with plus strand RNA viruses, it is known that more than 95% of the virus-specific RNA molecules are of plus-sense polarity whereas less than 5% of the molecules are of minus-sense polarity (Ball L.A. 1994, Proc. Natl. Acad. Sci. USA 91: 12443-12447).

Thirdly, the amounts of viral RNA molecules in infected cells are controlled by two different cytoplasmic enzymatic activities. The viral polymerase is responsible for the production of viral RNA molecules and a host inducible enzyme complex is responsible for the degradation of viral RNA molecules. This latter complex is thought to be responsible for an intracellular process referred to as RNA silencing (Ratcliff F.G. et al . 1999, The Plant Cell 11: 1207-1215; Ding S.W. 2000, Curr. Opin. Biotechnol. 11: 152-156). Some viruses, such as cucumoviruses carry genes which are involved in suppression of RNA silencing, thereby allowing the viral RNA molecules to accumulate at very high levels. BMV does not encode a viral RNA silencing suppressor. The absence of such a gene in USP 5824856 is thought by the present inventors to be the reason for the limited replication rate of BMV RNA molecules in dicot plant cells. As a consequence, it is considered that BMV, as a model system used in representing the Bromovirxdae has certain flaws and failings as indicated above, and as such may not represent an adequate model for the cucumovirus family.

The present inventors have found that minus-sense viral RNA molecules are much more efficient templates for replication and transcription than plus sense viral RNA molecules. By expressing a minus-sense RNA 3 molecule in plant cells in which the movement protein gene and/or the coat protein gene is partially or wholly replaced with an exogenous gene, the recombinant protein is produced after only a single cycle of replication or transcription. This single cycle mechanism is thought to represent a more efficient protein production system than the ^xtwo cycle' process as taught in USP 5824856.

Thus, there exists a need for an alternative protein production method in plant cells over those of the prior art.

The basis for the present invention, which does not appear to have been realised in the prior art is to use a virus, preferably a cucumovirus such as cucumber mosaic virus (CMV) , which naturally infects dicot plant species, and which carries a gene ( a suppressor' ) involved in the suppression of RNA silencing.

The present invention relates to the production of transgenic plant cells comprising i) a nucleic acid derived from a Cucumovirus that is expressed in the form of a recombinant viral minus-sense RNA 3 molecule ("carrier RNA") harbouring at least a minus-sense nucleic acid sequence corresponding to at least a target protein; ii) compatible cucumovirus viral polymerase proteins, which can replicate and transcribe the said carrier RNA molecule; and iii) a plant virus-derived gene which encodes a viral suppressor protein that acts in the suppression of host cell RNA silencing. Transgenic plant cells harbouring such introduced elements are designed to accumulate high levels of the carrier mRNA in the plus- sense orientation, and hence accumulate large amounts of target protein.

Detailed description

According to the present invention there is provided a method of producing at least a heterologous or exogenous target protein in a plant cell that comprises:

1) introducing into said plant cell a first isolated nucleic acid that comprises nucleic acid components that code for RNA dependent RNA polymerase proteins of a cucumovirus wherein each of said RNA dependent RNA polymerase nucleic acid components is operably linked to an exogenous promoter that drives expression in a plant cell; and

2) introducing into the said plant cell a second isolated nucleic acid that comprises nucleic acid operably linked to an exogenous promoter that drives expression in a plant cell wherein said nucleic acid encodes a recombinant cucumoviral RNA 3 molecule in the minus-sense orientation said minus-sense RNA 3 molecule comprising at least an RNA sequence that codes for a minus-sense RNA sequence of a target protein.

In an embodiment of the present invention the first introduced nucleic acid of step 1) may further comprise a nucleic acid component in the plus-sense orientation that encodes a compatible viral RNA silencing suppressor protein, such as CMV P2b, operably linked to an exogenous promoter that drives expression in a plant cell.

In a further embodiment of the invention, the second isolated nucleic acid introduced into the plant cell may further include a nucleic acid component which encodes for the compatible viral RNA silencing suppressor protein in the minus-sense configuration. In this embodiment, the first nucleic acid does not contain a nucleic acid sequence encoding a suppressor protein in any orientation. The second nucleic acid component comprises the nucleic acid encoding for RNA 3 in which i) the movement protein (in the case of CMV, the P3 protein) is replaced wholly or in part by the target nucleic acid encoding the one or more target protein sequence and ii) the coat protein gene is replaced wholly or in part by nucleic acid encoding a compatible viral RNA silencing suppressor protein in the minus sense orientation.

Naturally, the man skilled in the art will appreciate that in one variant of this embodiment of the invention, the movement protein may be replaced wholly or in part by the nucleic acid encoding for the suppressor protein and the coat protein may be replaced wholly or in part by nucleic acid encoding the target sequence, in appropriate orientation. Thus, as a further embodiment of the present invention there is provided a method of producing at least a heterologous or exogenous target protein in a plant cell that comprises:

1) introducing into said plant cell a first isolated nucleic acid that comprises nucleic acid components that code for RNA dependent RNA polymerase proteins of a cucumovirus wherein each of said RNA dependent RNA polymerase nucleic acid components is operably linked to an exogenous promoter that drives expression in a plant cell; and

2) introducing into the said plant cell a second isolated nucleic acid that comprises nucleic acid operably linked to an exogenous promoter that drives expression in a plant cell wherein said nucleic acid encodes i) a recombinant cucumoviral RNA 3 molecule in the minus-sense orientation said minus-sense RNA 3 molecule comprising at least an RNA sequence that codes for a minus-sense RNA sequence of a target protein and ii) a compatible viral RNA silencing suppressor protein wherein the nucleic acid encoding the said viral RNA silencing suppressor protein is in the minus-sense orientation.

The heterologous or exogenous target protein is contemplated to be any protein of interest that may be produced by the method of the invention. Types of target proteins that are contemplated for production in a method of the present invention include pharmaceutical proteins for use in mammals, including man, such as insulin, preproinsulin, proinsulin, glucagon, interferons such as a-interferon, a-interferon, a-interferon, blood-clotting factors selected from Factor VII, VIII, IX, X, XI, and XII, fertility hormones including luteinising hormone, follicle stimulating hormone growth factors including epidermal growth factor, platelet-derived growth factor, granulocyte colony stimulating factor and the like, prolactin, oxytocin, thyroid stimulating hormone, adrenocorticotropic hormone, calcitonin, parathyroid hormone, somatostatin, erythropoietin (EPO) , enzymes such as a-glucocerebrosidase, haemoglobin, serum albumin, collagen and the like. Furthermore, the method of the invention can be used for the production of specific monoclonal antibodies or active fragments thereof and of industrial enzymes.

All proteins mentioned hereinabove are of the human type. Other proteins that are contemplated for production in the present invention include proteins for use in veterinary care and may correspond to animal homologues of the human proteins mentioned.

One or more target proteins can be generated using the process of the invention from RNA dependent RNA polymerase proteins and RNA 3 molecules (carrier RNA molecules) derived from one RNA virus or from functionally related viruses (belonging to one virus family) . Suitable RNA viruses are those that have split genomes or those which express their genes using sub- genomic mRNA molecules, e.g. viruses which belong to the order of Nidovirales, or more specifically of the family Nodaviridae, Tombusviridae , Flaviviridae, Togaviridae, Bromoviridae or Closteroviridae (ICTV, 1998) . Viruses with segmented RNA genomes are preferred as vehicles for the expression of recombinant proteins in methods according to the invention. Preferably, the virus of choice is a cucumovirus, such as, tomato aspermy virus (TAV) or cucumber mosaic virus (CMV) of subgroup 1 or subgroup 2. Most preferably, the cucumovirus is cucumber mosaic virus and is a CMV subgroup 1 virus.

Cucumoviruses with a tripartite plus-sense RNA genome appear suited as vehicles. The RNA dependent RNA polymerase is encoded by the large two RNA molecules, RNA 1 and RNA 2 respectively encoding the subunits Pi and P2. RNA 2 encodes a second protein from a subgenomic mRNA, denoted P2b. This protein is a suppressor of RNA silencing (Brigneti et al . 1998, EMBO J. 17: 6739-6746). RNA 3 is bi-cistronic encoding the viral movement protein, P3 and the coat protein (CP) . The CP cistron is expressed from a subgenomic mRNA molecule, denoted RNA 4. The intercistronic region on RNA 3 serves as a subgenomic promoter for the production of RNA 4 by the cucumoviral polymerase.

All cucumoviral polymerase activities required for replication of the cucumoviral genomic RNA' s are comprised by the PI and P2 proteins (Hayes R.J. and Buck K.W. 1993, In: Molecular Virology, a practical approach, A.J. Davison and R.M. Elliott (Eds.), IRL Press at Oxford University Press) .

A compatible viral RNA silencing suppressor protein is one which is capable of suppressing RNA silencing, such that the target sequence is expressed at detectably high levels relative to wild type background levels, if any. Such compatible viral suppressor proteins may include suppressor proteins that are known in the art such as the P2b suppressor protein of CMV, the p25 protein of potato virus X, the AC2 protein of African cassava mosaic virus (ACMV) , the PI protein of rice yellow mottle virus, the 19K protein of tomato bushy stunt virus, and potyviral HC-Pro proteins (Li & Ding, Curr. Opin. in Biotech. 12:150-154); and the NS3 silencing suppressor of rice hoja blanca virus (RHBV) , the NS_S silencing suppressor of tomato spotted wilt virus (TSWV) (Bucher et al (2003) Journal of Virology 77: 1329-1336). Further compatible viral suppressor proteins may also include vertebrate suppressor proteins as disclosed in European Patent application no. 02079257.8.

The carrier RNA ( = RNA 3 component) may represent the minus-sense cucumoviral RNA 3 sequence, which may comprise one or more target protein nucleic acid sequences in minus-sense orientation, such that when the cucumoviral polymerase acts on it, plus-sense RNA 3 and RNA 4 are produced from which one or more target proteins may be translated. Such target nucleic acid minus-sense sequences may be located in tandem on the said second isolated nucleic acid or may be located adjacent to one another. The target nucleic acid minus-sense sequences can also be separated by nucleotide spacers, such as the cucumovirus inter-cistronic region (or subgenomic promoter sequences) on the second isolated nucleic acid. The target nucleic acid minus-sense sequences preferably replace all or part of the coat protein cistron or movement protein cistron found on the carrier RNA as alluded to hereinabove. Preferably it is the movement protein component of the RNA 3 component that is wholly or in part replaced by the target sequence (s) of interest.

Alternatively, the coat protein cistron plus the intercistronic subgenomic promoter region located on RNA 3 may be deleted and the movement protein cistron may be wholly or in part replaced with an exogenous gene. Essential for recognition and subsequent replication of the carrier RNA by the viral polymerase, are the 5' and 3' untranslated terminal sequences. It is therefore important that the RNA 3 molecules (= carrier RNA) have similar terminal sequences as the viral RNA molecules.

In order to avoid the presence of redundant and non-viral sequences at the 5'- and 3' -termini of the carrier RNA, which may act to inhibit recognition and subsequent replication by the viral polymerase, the cDNA encoding the carrier RNA may be fused to the transcription initiation site of the promoter. Redundant 3' -terminal sequences may be removed by using RNA sequences which are capable of auto-catalytical, self-cleavage, such as ribozymes. The DNA encoding a ribozyme may be located downstream of the DNA corresponding with the viral carrier RNA. Examples of ribozymes include ribonuclease P, Tetrahymena L-19 intervening sequence, hammerhead ribozymes, Hepati tis delta virus RNA, Neurospora mitochondrial VS RNA and the like (Symons, R.H. Ann. Rev. Biochem. 61: 641, 1992) . The Hepatitis del ta antigenomic ribozyme is a self-cleaving RNA sequence which cleaves without the need of a specific consensus sequence upstream of the cleavage site and for this reason are suitable for removing downstream redundant sequences

(Ball, L.A. Proc. Natl. Acad. Sci. USA 91: 12443-12447, 1994) .

An exogenous promoter is one that denotes a promoter that is introduced in front of a nucleic acid sequence of interest and is operably associated therewith. Thus an exogenous promoter is one that has been placed in front of a selected nucleic acid component as herein defined and does not consist of the natural or native promoter usually associated with the nucleic acid component of interest as found in wild type circumstances. Thus a promoter may be native to a plant cell of interest but may not be operably associated with the nucleic acid of interest in front in wild-type plan cells. Typically, an exogenous promoter is one that is transferred to a host cell or host plant from a source other than the host cell or host plant.

The cDNA' s encoding the polymerase proteins, the RNA silencing suppressor and the carrier RNA molecules contain at least one type of promoter that is operable in a plant cell, for example, an inducible or a constitutive promoter operatively linked to a first and/or second nucleic acid sequence or nucleic acid sequence component as herein defined and as provided by the present invention. As discussed, this enables control of expression of the gene. The invention also provides plants transformed with said first or second nucleic acid sequence or construct and methods including introduction of such a first or second nucleic acid sequence or construct into a plant cell and/or induction of expression of said first or second nucleic acid sequence or construct within a plant cell, e.g. by application of a suitable stimulus, such as an effective exogenous inducer .

The term "inducible" as applied to a promoter is well understood by those skilled in the art. In essence, expression under the control of an inducible promoter is "switched on" or increased in response to an applied stimulus (which may be generated within a cell or provided exogenously) . The nature of the stimulus varies between promoters. Some inducible promoters cause little or undetectable levels of expression (or no expression) in the absence of the appropriate stimulus. Other inducible promoters cause detectable constitutive expression in the absence of the stimulus. Whatever the level of expression is in the absence of the stimulus, expression from any inducible promoter is increased in the presence of the correct stimulus. The preferable situation is where the level of expression increases upon application of the relevant stimulus by an amount effective to alter a phenotypic characteristic. Thus an inducible (or "switchable") promoter may be used which causes a basic level of expression in the absence of the stimulus which level is too low to bring about a desired phenotype (and may in fact be zero) . Upon application of the stimulus, expression is increased (or switched on) to a level, which brings about the desired phenotype. One example of an inducible promoter is the ethanol inducible gene switch disclosed in Caddick et al (1998) Nature Biotechnology 16: 177-180. A number of inducible promoters are known in the art .

Chemical-regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator. Depending upon the objective, the promoter may be a chemical-inducible promoter, where application of the chemical induces gene expression, or a chemical-repressible promoter, where application of the chemical represses gene expression. Chemical-inducible promoters are known in the art and include, but are not limited to, the maize In2-2 promoter, which is activated by benzenesulfona ide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre-emergent herbicides, and the tobacco PR-la promoter, which is activated by salicylic acid. Other chemical-regulated promoters of interest include steroid- responsive promoters (see, for example, the glucocorticoid-inducible promoter in Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88: 10421-10425 and McNellis et al. (1998) Plant J. 14 (2) : 247-257 ) and tetracycline- inducible and tetracycline-repressible promoters (see, for example, Gatz et al. (1991) Mol. Gen. Genet. 227:229- 237, and U.S. Patent Nos. 5,814,618 and 5,789,156), herein incorporated by reference.

Where enhanced expression in particular tissues is desired, tissue-specific promoters can be utilized. Tissue-specific promoters include those described by Yamamoto et al. (1997) Plant J, 12 (2)255-265; Kawamata et al. (1997) Plant Cell Physiol. 38 (7) : 792-803; Hansen et al. (1997) Mol. Gen Genet. 254 (3) : 337-343; Russell et al. (1997) Transgenic Res. 6(2) : 157-168 ; Rinehart et al.

(1996) Plant Physiol. 112(3) 1331-1341; Van Camp et al. (1996) Plant Physiol. 112(2) 525-535; Canevascini et al.

(1996) Plant Physiol. 112(2) 513-524; Yamamoto et al.

(1994) Plant Cell Physiol. 35 (5) : 773-778; Lam (1994)

Results Probl. Cell Differ. 20:181-196; Orozco et al.

(1993) Plant Mol Biol. 23 (6) : 1129-1138 ; Matsuoka et al (1993) Proc Natl. Acad. Sci. USA 90 (20) : 9586-9590; and

Guevara-Garcia et al. (1993) Plant J. 4 (3) : 495-505.

So-called constitutive promoters may also be used in the methods of the present invention. Constitutive promoters include, for example, CaMV 35S promoter (Odell et al. (1985) Nature 313:810-812); rice actin (McElroy et al. (1990) Plant Cell 2:163-111); ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12:619-632 and Christensen et al . (1992) Plant Mol . Biol . 18:675-689) ; pEMU (Last et al . (1991) Theor. Appl . Genet . 81:581-588); MAS (Velten et al . (1984) EMBO J. 3:2723-2730) ; ALS promoter (U.S. Application Serial No. 08/409,297), and the like. Other constitutive promoters include those in U.S. Patent Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; and 5,608,142.

Naturally, the man skilled in the art will appreciate that terminator DNA sequences will be present in constructs used in the invention. A terminator is contemplated as a DNA sequence at the end of a transcriptional unit which signals termination of transcription. These elements are 3' -non-translated sequences containing polyadenylation signals, which act to cause the addition of polyadenylate sequences to the 3' end of primary transcripts. For expression in plant cells the nopaline synthase transcriptional terminator (A. Depicker et al . , 1982, J. of Mol. & Applied Gen. 1:561-573) sequence serves as a transcriptional termination signal.

Those skilled in the art are well able to construct vectors and design protocols for recombinant nucleic acid sequence or gene expression. Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. For further details see, for example, Molecular Cloning: a Laboratory Manual : 2nd edition, Sambrook et al , 1989, Cold Spring Harbor Laboratory Press. Many known techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Current Protocols in Molecular Biology, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992. The disclosures of Sambrook et al . and Ausubel et al. are incorporated herein by reference. Specific procedures and vectors previously used with wide success upon plants are described by Bevan (Nucl. Acids Res. 12, 8711-8721 (1984)) and Guerineau and Mullineaux (1993) (Plant transformation and expression vectors. In: Plant Molecular Biology Labfax (Croy-RRD ed.) Oxford, BIOS Scientific Publishers, pp 121-148).

Naturally, the skilled addressee will appreciate that each nucleic acid sequence will be under regulatory control of its own exogenous promoter and terminator. When two or more target proteins are destined to be produced from a single carrier RNA it is preferable if they are able to be readily separated, for example by binding to different protein-specific antibodies

(monoclonal or polyclonal) in the harvesting phase of the plant cell culture system. Preferably, one target protein of interest is produced from the RNA 4 nucleic acid in the method of the invention.

Selectable genetic markers may facilitate the selection of transgenic plants and these may consist of chimaeric genes that confer selectable phenotypes such as resistance to antibiotics such as kanamycin, neomycin, hygromycin, puramycin, phosphinotricin, chlorsulfuron, methotrexate, gentamycin, spectinomycin, imidazolinones and glyphosate. When introducing selected nucleic acid sequences according to the present invention into a cell, certain considerations must be taken into account, well known to those skilled in the art. The nucleic acid to be inserted should be assembled within a construct, which contains effective regulatory elements, which will drive transcription. There must be available a method of transporting the construct into the cell. Once the construct is within the cell membrane, integration into the endogenous chromosomal material either will or will not occur. Finally, as far as plants are concerned the target cell type must be such that cells can be regenerated into whole plants.

Plants transformed with DNA segments containing sequences of interest as provided herein may be produced by standard techniques, which are already known for the genetic manipulation of plants. DNA can be transformed into plant cells using any suitable technology, such as a disarmed Ti-plasmid vector carried by Agrobacterium exploiting its natural gene transfer ability (EP-A- 270355, EP-A-0116718, NAR 12(22) 8711 -87215 1984), particle or micro projectile bombardment (US 5100792, EP- A-444882, EP-A-434616) microinjection (WO 92/09696, WO 94/00583, EP 331083, EP 175966, Green et al . (1987) Plant Tissue and Cell Cul ture, Academic Press) , electroporation (EP 290395, WO 8706614) other forms of direct DNA uptake (DE 4005152, WO 9012096, US 4684611), liposome mediated DNA uptake (e.g. Freeman et al . Plant Cell Physiol . 29: 1353 (1984)), or the vortexing method (e.g. Kindle, PNAS U. S . A. 87: 1228 (1990d) Physical methods for the transformation of plant cells are reviewed in Oard, 1991, Biotech . Adv. 9: 1-11. Thus once a nucleic acid sequence or gene has been identified, it may be reintroduced into plant cells using techniques well known to those skilled in the art to produce transgenic plants of the appropriate phenotype.

Agrobacterium transformation is widely used by those skilled in the art to transform dicotyledonous species. Production of stable, fertile transgenic plants in almost all economically relevant monocot plants is also now routine: (Toriyama, et al. (1988) Bio/Technology 6, 1072- 1074; Zhang, et al . (1988) Plant Cell Rep. 7, 379-384; Zhang, et al. (1988) Theor. Appl . Genet 76, 835-840; Shima oto, et al. (1989) Nature 338, 274-276; Datta, et al. (1990) Bio/Technology 8, 736-740; Christou, et al . (1991) Bio/Technology 9, 957-962; Peng, et al . (1991) International Rice Research Institute, Manila, Philippines 563-574; Cao, et al. (1992) Plant Cell Rep. 11, 585-591; Li, et al . (1993) Plant Cell Rep. 12, 250- 255; Rathore, et al. (1993) Plant Molecular Biology 21, 871-884; Fromm, et al. (1990) Bio/Technology 8, 833-839; Gordon-Kamm, et al. (1990) Plant Cell 2, 603-618; D'Halluin, et al. (1992) Plant Cell 4, 1495-1505; Walters, et al. (1992) Plant Molecular Biology 18, 189- 200; Koziel, et al . (1993) Biotechnology 11, 194-200; Vasil, I. K. (1994) Plant Molecular Biology 25, 925-937; Weeks, et al. (1993) Plant Physiology 102, 1077-1084; Somers, et al. (1992) Bio/Technology 10, 1589-1594; W092/14828) . In particular, Agrobacterium mediated transformation is now a highly efficient alternative transformation method in monocots (Hiei et al . (1994) The Plant Journal 6, 271-282) .

The generation of fertile transgenic plants has been achieved in the cereals rice, maize, wheat, oat, and barley (reviewed in Shimamoto, K. (1994) Current Opinion in Biotechnology 5, 158-162.; Vasil, et al . (1992) Bio/Technology 10, 667-674; Vain et al., 1995, Biotechnology Advances 13 (4): 653-671; Vasil, 1996, Nature Biotechnology 14 page 702) . Wan and Lemaux (1994) Plant Physiol . 104: 37-48 describe techniques for generation of large numbers of independently transformed fertile barley plants.

Micro projectile bombardment, electroporation and direct DNA uptake are preferred where Agrobacterium is inefficient or ineffective. Alternatively, a combination of different techniques may be employed to enhance the efficiency of the transformation process, e.g. bombardment with Agrobacterium coated micro particles

(EP-A-486234) or micro projectile bombardment to induce wounding followed by co-cultivation with Agrobacterium (EP-A-486233) .

Following transformation, a plant may be regenerated, e.g. from single cells, callus tissue or leaf discs, as is standard in the art. Almost any plant can be entirely regenerated from cells, tissues and organs of the plant. Available techniques are reviewed in Vasil et al., Cell Culture and Somatic Cell Genetics of Plants, Vol . I, II and III, Laboratory Procedures and Their Applications , Academic Press, 1984, and Weiss Bach and Weiss Bach, Methods for Plant Molecular Biology, Academic Press, 1989.

The particular choice of a transformation technology will be determined by its efficiency to transform certain plant species as well as the experience and preference of the person practising the invention with a particular methodology of choice. It will be apparent to the skilled person that the particular choice of a transformation system to introduce nucleic acid into plant cells is not essential to or a limitation of the invention, nor is the choice of technique for plant regeneration.

The invention further encompasses a host cell transformed with vectors or constructs as set forth above, especially a plant or a microbial cell. Thus, a host cell, such as a plant cell, including nucleotide sequences of the invention as herein indicated is provided. Within the cell, the nucleotide sequence may be incorporated within the chromosome.

Also according to the invention there is provided a plant cell having incorporated into its genome at least a nucleotide sequence, particularly heterologous nucleotide sequences, as provided by the present invention under operative control of regulatory sequences for control of expression as herein described. The coding sequence may be operably linked to one or more regulatory sequences which may be heterologous or foreign to the nucleic acid sequences employed in the invention, such as not naturally associated with the nucleic acid sequence (s) for its (their) expression. The nucleotide sequence according to the invention may be placed under the control of an externally inducible promoter to place expression under the control of the user. A further aspect of the present invention provides a method of making such a plant cell involving introduction of nucleic acid sequence (s) contemplated for use in the invention or a suitable vector including the sequence (s) contemplated for use in the invention into a plant cell and causing or allowing recombination between the vector and the plant cell genome to introduce the said sequences into the genome. The invention extends to plant cells containing a nucleotide sequence according to the invention as a result of introduction of the nucleotide sequence into an ancestor cell.

The term "heterologous" may be used to indicate that the gene/sequence of nucleotides in question have been introduced into said cells of the plant or an ancestor thereof, using genetic engineering, ie by human intervention. A transgenic plant cell, i.e. transgenic for the nucleotide sequence in question, may be provided. The transgene may be on an extra-genomic vector or incorporated, preferably stably, into the genome. A heterologous gene may replace an endogenous equivalent gene, ie one that normally performs the same or a similar function, or the inserted sequence may be additional to the endogenous gene or other sequence. An advantage of introduction of a heterologous gene is the ability to place expression of a sequence under the control of a promoter of choice, in order to be able to influence expression according to preference. Furthermore, mutants, variants and derivatives of the wild-type gene, e.g. with higher activity than wild type, may be used in place of the endogenous gene. Nucleotide sequences heterologous, or exogenous or foreign, to a plant cell may be non- naturally occurring in cells of that type, variety or species. Thus, a nucleotide sequence may include a coding sequence of or derived from a particular type of plant cell or species or variety of plant, placed within the context of a plant cell of a different type or species or variety of plant. A further possibility is for a nucleotide sequence to be placed within a cell in which it or a homologue is found naturally, but wherein the nucleotide 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 variety of plant, such as operably linked to one or more regulatory sequences, such as a promoter sequence, for control of expression. A sequence within a plant or other host cell may be identifiably heterologous, exogenous or foreign.

Plants which include a plant cell according to the invention are also provided, along with any part or propagule thereof, seed, selfed or hybrid progeny and descendants. Particularly provided are transgenic crop plants, which have been engineered to carry genes identified as stated above. Examples of suitable plants include tobacco and other Nicotiana species, carrot, vegetable and oilseed Brassica' s as provided for above, melons, Capsicums, grape vines, lettuce, strawberry, sugar beet, wheat, barley, maize, rice, soybeans, alfalfa, peas, sorghum, sunflower, tomato, and potato.

In addition to a plant, the present invention provides any clone of such a plant, seed, selfed or hybrid progeny and descendants, and any part of any of these, such as cuttings, seed. The invention provides any plant propagule, that is any part which may be used in reproduction or propagation, sexual or asexual, including cuttings, seed and so on. Also encompassed by the invention is a plant which is a sexually or asexually propagated off-spring, clone or descendant of such a plant, or any part or propagule of said plant, offspring, clone or descendant.

The present invention also encompasses the polypeptide expression product of a nucleic acid molecule according to the invention as disclosed herein or obtainable in accordance with the information and suggestions herein. Also provided are methods of making such an expression product by expression from a nucleotide sequence encoding therefore under suitable conditions in suitable host cells e.g. E. coli . Those skilled in the art are well able to construct vectors and design protocols and systems for expression and recovery of products of recombinant gene expression.

Preferred polymerase polypeptides and the P2b silencing suppressor protein encoded by CMV RNA' s 1 and 2 are as described in Rizzo T.M. and Palukaitis P. 1988, J. Gen. Virol. 69: 1777-1787; Rizzo T.M. and Palukaitis P. 1989, J. Gen. Virol. 70: 1-11. The carrier RNA is derived from CMV RNA 3 as described in Owen J. et al . 1990, J. Gen. Virol. 71: 2243-2249.

A polypeptide according to the present invention may be an allele, variant, fragment, derivative, mutant or homologue of the (a) polypeptides as mentioned herein. The allele, variant, fragment, derivative, mutant or homologue may have substantially the same function of the polypeptides alluded to above and as shown herein or may be a functional mutant thereof.

"Homology" in relation to an amino acid sequence of the invention may be used to refer to identity or similarity, preferably identity. As noted already above, high level of amino acid identity may be limited to functionally significant domains or regions, e.g. any of the domains identified herein. In particular, homologues of the particular CMV-derived polypeptide sequences provided herein, are provided by the present invention, as are mutants, variants, fragments and derivatives of such homologues. Such homologues are readily obtainable by use of the disclosures made herein. Naturally, the skilled addressee will appreciate that homologues of the target protein sequences per se, other than those homologues that due to the degeneracy of the genetic code give rise to amino acid sequences that are true copies (i.e. 100% identical) of the mammalian proteins of interest, and especially of human proteins of interest, are encompassed within the present invention. Thus the present invention also extends to polypeptides which include amino acid sequences with CMV function as defined herein and as obtainable using sequence information as provided herein. The CMV homologues may at the amino acid level have homology, that is identity, with the amino acid sequences described in Rizzo T.M. and Palukaitis P. 1988, J. Gen. Virol. 69: 1777-1787; Rizzo T.M. and Palukaitis P. 1989, J. Gen. Virol. 70: 1-11; Owen J. et al . 1990, J. Gen. Virol. 71: 2243-2249, preferably at least about 50%, or at least 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80% homology, or at least about 85 %, or at least about 88% homology, or at least about 90% homology and most preferably at least about 95% or greater homology provided that such proteins have a polymerase activity and an RNA silencing suppressor activity that fits within the context of the present invention.

In certain embodiments, an allele, variant, derivative, mutant derivative, mutant or homologue of the specific sequence may show little overall homology, say about 20%, or about 25%, or about 30%, or about 35%, or about 40% or about 45%, with the specific sequence. However, in functionally significant domains or regions, the amino acid homology may be much higher. Putative functionally significant domains or regions can be identified using processes of bioinformatics, including comparison of the sequences of homologues.

Functionally significant domains or regions of different polypeptides may be combined for expression from encoding nucleic acid as a fusion protein. For example, particularly advantageous or desirable properties of different homologues may be combined in a hybrid protein, such that the resultant expression product, with CMV protein function, may include fragments of various parent proteins, if appropriate.

Similarity of amino acid sequences may be as defined and determined by the TBLASTN program, of Altschul et al . (1990) J. Mol . Biol . 215: 403-10, which is in standard use in the art. In particular, TBLASTN 2.0 may be used with Matrix BLOSUM62 and GAP penalties: existence: 11, extension: 1. Another standard program that may be used is BestFit, which is part of the Wisconsin Package, Version 8, September 1994, (Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA, Wisconsin 53711) . BestFit makes an optimal alignment of the best segment of similarity between two sequences. Optimal alignments are found by inserting gaps to maximize the number of matches using the local homology algorithm of Smith and Waterman (Adv. Appl . Math . (1981) 2: 482-489). Other algorithms include GAP, which uses the Needleman and Wunsch algorithm to align two complete sequences that maximizes the number of matches and minimizes the number of gaps. As with any algorithm, generally the default parameters are used, which for GAP are a gap creation penalty = 12 and gap extension penalty = 4. Alternatively, a gap creation penalty of 3 and gap extension penalty of 0.1 may be used. The algorithm FASTA (which uses the method of Pearson and Lipman (1988) PNAS USA 85: 2444-2448) is a further alternative.

Use of either of the terms "homology" and "homologous" herein does not imply any necessary evolutionary relationship between compared sequences, in keeping for example with standard use of terms such as "homologous recombination" which merely requires that two nucleotide sequences are sufficiently similar to reco bine under the appropriate conditions. Further discussion of polypeptides according to the present invention, which may be encoded by nucleic acid according to the present invention, is found below.

According to the present invention there is provided transgenic plant cells producing recombinant viral RNA molecules or carrier RNA molecules that harbour the gene(s) encoding the target protei (s); polymerase proteins of the matching RNA virus and a viral RNA silencing suppressor protein. In the transgenic plant cells, the carrier RNA molecules are capable of replication and transcription, by the polymerase proteins. The viral RNA silencing suppressor protein further increases the amounts of carrier RNA and derivative transcripts in plant cells and thereby further increases the yields of target protein (s). The skilled addressee will appreciate that a plant cell or plant cells comprised in plants can be transgenic for the polymerase components and movement protein as hereinbefore described and other nucleic acid components may be introduced transiently using Agrobacterium tumefaciens as a vector. Such vectors can comprise one or more RNA 3 molecules including one or more target sequences in minus-sense orientation and one or more RNA silencing suppressor sequences also in minus-sense orientation. It is thought that at least one coat protein component of RNA 3 should remain intact since this appears to be necessary for the systemic spread or movement of the recombinant RNA 3 molecules throughout the plant.

The viral recombinant RNA molecules or carrier RNA molecules, which are replicated and/or transcribed by the viral polymerase, are either transformed into the cells expressing the viral polymerase proteins or introduced into the polymerase-expressing cells by sexual crossing of plants. Thus one parent plant line may be transgenic for the polymerase and silencing suppressor protein and the other parent plant line may be transgenic for at least an RNA 3 molecule or carrier molecule in minus- sense orientation comprising at least one target sequence of interest (also in minus orientation) as hereinbefore described. In an alternative embodiment, a first parent line can be transgenic for the polymerase components as hereinbefore described and a second parent line can be transgenic for an RNA 3 molecule including the target sequence in minus-sense orientation and an RNA silencing suppressor sequence also in minus-sense orientation. In this alternative embodiment, the coat protein component and the movement protein component of the RNA 3 molecule may be wholly or partly replaced by the said target sequence and a suppressor sequence as hereinbefore described. By having parent plant lines carrying different components as outlined above, the protein expression system of the present invention is rendered more versatile compared to those systems expressing the polymerase gene(s) and the genes encoding the target proteins from the same replicating recombinant viral RNA molecule or from the same DNA construct used for expression of all the above mentioned elements in transgenic cells. As such, in this system of the invention wherein plant parent lines carry different genetic components as outlined above, an almost inexhaustible supply of initial plant material may be crossed and the resulting progeny plants containing all genes of interest may be used as starting materials for providing plant cells for culture in bioreactors. In such a system, there is no absolute requirement to breed crossed plant lines in which the crossed plants of interest breed true for introduced and desired characteristics since all that is required is cellular material from the crossed plants in which the relevant genes of interest are expressed giving rise to target protein production, can be fed into a reactor and grown using conventional plant cell culturing technology.

Examples

Example 1 : Isolation of CMV particles and RNA therein

A CMV subgroup 1 isolate was collected from lily and maintained in N. benthamiana by mechanical passaging. Virus particles are purified from systemically infected N. benthamiana plants following the procedure of Francki et al [(1979) CMI/AAB Descr. Of plant viruses 213]. Approximately 100 μg of virus in a volume of 250 μl is extracted with phenol, then with a mixture of phenol & chloroform and finally with chloroform. RNA is precipitated with ethanol and collected by centrifugation. The pellet is dissolved in 20 μl of water.

Example 2 : Molecular cloning of the CMV Pi and P2 polymerase genes and the P2b RNA silencing suppressor gene

The sequences of the Pi, P2 and P2b genes are isolated using RNA-based PCR. Oligonucleotides Plf (Seq id no.l) : GGGCGGCCGCAATTCCTATGGCGACGTCC and Plr (Seq. Id. no.2) : GGGCGGCCGCCTAGGCACGAGCAACAC are designed, which are homologous or complementary to the RNA sequences on RNA 1 flanking the translational start codons of the PI protein. Oligonucleotides P2f (Seq Id no.3) GGGCGGCCGCTTTCTCATGGCTTTCCCC and P2r (Seq Id no.4): GGGCGGCCGCTCAGGCTCGGGTAACTCC are designed, which are homologous or complementary to the RNA sequences on RNA 2 flanking the translational start codons of the P2 protein. Oligonucleotides P2bf (Seq Id no.5): GGGCATGCGAAAGAAATATGGAATTG and P2br (seq Id no.6): GGGCATGCTCAGAAGGCTCCTTCCGC are designed, which are homologous or complementary to the RNA sequences on RNA 2 flanking the translational start codons of the P2b protein. The Plf, Plr, P2f and P2r oligonucleotides contain a an Notl recognition site, which is absent in CMV RNA 1- and 2-derived cDNA sequences to enable further cloning of the amplified PI and P2 cDNA molecules. Purified CMV RNA is subjected to RNA-based PCR using oligonucleotides Plf and Plr yielding the PI cDNA. Purified CMV RNA is subjected to RNA-based PCR using oligonucleotides P2f and P2r yielding the P2 cDNA. Purified CMV RNA is subjected to RNA-based PCR using oligonucleotides P2f and P2r yielding the P2 cDNA. The resulting PCR fragments are digested with Notl that cleaves within the oligonucleotide sequences and cloned into pSK+ (Stratagene) , yielding the recombinant plasmids pSK-Pl and pSK-P2 respectively. Purified CMV RNA is subjected to RNA-based PCR using oligonucleotides P2bf and P2br yielding the P2b cDNA. The resulting PCR fragment is digested with Sphl that cleaves within the oligonucleotide sequences and cloned into pUC19 (Stratagene), yielding the recombinant plasmid pSK-P2b.

Example 3 : Molecular cloning of CMV RNA3

The sequence of the CMV RNA3 is isolated using RNA-based PCR. Two oligonucleotides are designed R3f (Seq Id no.7) : GGGATCCGTAATCTTACCACTGTG, which is homologous to nucleotides 1 to 17 of CMV RNA3 and R3r (Seq Id no.8): GGGATCCTGGTCTCCTTTTGGAGGCC, which is complementary to sequences 2198 to 2216 of CMV RNA3. Both primers contain BamHl sites to enable further cloning of the amplified DNA molecule. Purified CMV RNA is subjected to the Gen Amp RNA PCR, using oligonucleotides R3f and R3r. The resulting PCR fragment is digested with BamHl, isolated from an agarose gel and cloned into BamHl linearized pSK+, yielding the recombinant plasmid pSK-RNA3. This plasmid has the additional G residue present at the 3' end of the minus strand of CMV RNA 3 (Collmer, C.W. & Kaper, J.M., Virology 145: 249-259, 1985).

Example 4 : Molecular cloning of the Arabidops±s thaliana Act2 promoter, the Agrobacterium tvαnefaciens , the nopalin synthase terminator and the Hepatitis delta antigenomic ribozyme Two oligonucleotides are designed A2f (Seq Id no.9): GGGGCGCGCCGCATGCCTGCAGGTCG, homologous to the 5' end of the Act2 promoter and A2r (Seq Id. No. 10) : GGGGCGGCCGCTTTTTATGAGCTGCAAAC complementary to sequences flanking the translational start codon. (Y-Q An et al., The Plant Journal (1996) 10:107-121) The oligonucleotides contain recognition sites of Ascl or Notl, which are absent in CMV RNA-derived cDNA, and the Act2 promoter sequence: This enables further cloning of the amplified DNA molecules. Genomic DNA was isolated from A. thaliana plants using standard procedures which was then subjected to PCR using oligonucleotides A2f and A2r. The resulting PCR fragment is digested with Ascl and Notl that cleave within the A2f and A2r oligonucleotide sequences respectively and is cloned into plasmid pSK+, yielding recombinant plasmid pSK-Act2p.

Two oligonucleotides are designed Nlf (Seq Id no.11): GGGCGGCCGCATCGTTCAAACATTTGG, complementary to the 5' end of the nopaline synthase (NOS) terminator with a Notl site and Nlr (Seq Id no.12) :

GGTTAATTAAGTAACATAGATGACACCGCG complementary to sequences at the 3' end of the NOS terminator with a Pacl site (Depicker et al . , J. of Mol. And Appl . Gen. 1:561-573, 1982) . Genomic DNA was isolated from A. tumefaciens plants, which was subjected to PCR using oligonucleotides Nlf and Nlr. The resulting PCR fragment is digested with Notl and Pacl that cleave within the Nlf and Nlr oligonucleotide sequences respectively and is cloned downstream of the Act2 promoter in pSK-Act2p, yielding recombinant plasmid pSK-Act2p-NOSt .

Two primers are designed; Hlf (Seq Id no.13) : GGGCTGCAGGGGTCGGCATGGCATCTCCACCTCCTCGCGGTCCGACCTGGGCATCCG

, homologous to the sequences corresponding with the 5' half of the Hepa ti tis del ta antigenomic ribozyme and containing a Pstl cloning site (Perotta & Been, 1990) and Hlr (Seq Id no. 14) :

GGGCTGCAGCTCCCTTAGCCATCCGAGTGGACGTGCGTCCTCCTTCGGATGCCCAGG TCGG, complementary to the 3' half of the ribozyme also containing a Pstl restriction site. The 20 3' -terminal nucleotides of both primers are complementary to each other . Hlf and Hlr are annealed and subjected to PCR. The resulting DNA fragment is digested with Pstl, isolated from an agarose gel and cloned in Pstl linearized pSK+ yielding plasmid pSK-HDR. The PCR fragment comprising the NOS terminator is cloned downstream of the Hepa ti tis delta antigenomic ribozyme in pSK-HDR yielding pSK-HDR-NOSt .

Example 5 : Molecular cloning of the human erythropietin (EPO) gene

Twelve oligonucleotides denoted El to E12 are designed, on the basis of the published human EPO amino acid sequence (Jacobs et al . , Nature 313:806-810, 1985), in which the codon usage has been optimised for N. benthamiana . The nucleotides of the sense and coding DNA strand are in small letters, whereas those of the antisense and non-coding DNA strand are presented in capital letters.

El (Seq ID no. 15) : gggccatgggtgttcatgaatgtccagcttggctgtggctgctgctgtctctgctgt ctctgccactggg

E2 (Seq Id. no.16) :

GAACTCTACTATCACAAATCAGTCTTGGTGGAGCACCCAGAACTGGCAGACCCAGTG

GCAGAGACAGCAG E3 (Seq Id no.17) : gatttgtgatagtagagttctggaaagataσctgctggaagctaaggaagctgaaaa tattacaacaggt

E4 Seq Id no.18) :

TTTGTATCTGGAACTGTAATATTTTCATTCAGAGAACAATGTTCAGCACAACCTGTT

GTAATATTTTCAG

E5 (Seq Id no.19) : attacagttcσagatacaaaggttaatttttacgcttggaagagaatggaagttggt cagσaggctgttg

E6 (Seq Id no . 20) : CTGACCTCTCΑGAACΛGCTTCAGAC^GCAGAGCClAGACCCTGCCy y CTTC^ACAGC CTGCTGACCAACT

E7 (Seq Id no . 21) : aagctgttctgagaggtcaggctctgcttgttaattcttσtσagccttgggaaccac ttcagctgσatgt

E8 (seq Id no.22) :

GAGCCCTAAGC^GTGTTGTC^GAGATCTCAGACCACTAACAGCCTTATCAACATGCA GCTGAAGTGGTTC

E9 (SEQ ID NO .23) : gacaacactgcttagggctctgggtgctcagaaggaagctattagtccaccagatgctg cttctgctgct

E10 (SEQ ID NO.24) :

TAAACTCTAAACAGCTTTCTAAATGTATCAGCAGTAATAGTTCTCAGTGGAGCAGCAGAA GCAGCATCTG Ell ( SEQ ID NO . 25 ) : agaaagctgtttagagtttaσtcaaattttctgagaggtaagctgaagctgtatactgg t

E12 (SEQ ID NO. 26) :

CCCCCATGGCTATCTATCACCTGTTCTACAAGCCTCACCAGTATACAGCTTCAGCT

All 12 oligonucleotides are annealed by mixing, heating for 1 minute at 95 degrees Celsius and subsequent cooling down to room temperature for 5 minutes . Second strand cDNA is synthesised by taq polymerase for 3 minutes at 55 degrees Celsius and the gaps are ligated by T4 DNA ligase. The annealed DNA fragment is subsequently re- amplified using oligonucleotides El and E12, digested with restriction enzyme Ncol, gel-purified and cloned in pSK+ linearized with Ncol, yielding pSK-EPO (Seq Id no.27) .

gggccatgggtgttcatgaatgtccagcttggctgtggctgctgctgtctctgctgt ctctgccactgggtctgccagttctgggtgctccaccaagactgatttgtgatagta gagttctggaaagatacctgctggaagctaaggaagctgaaaatattacaacaggtt gtgctgaacattgttctctgaatgaaaatattacagttccagatacaaaggttaatt

^' tttacgcttggaagagaatggaagttggtcagcaggctgttgaagtttggcagggtc tggctctgctgtctgaagctgttctgagaggtcaggctctgcttgttaattcttctc agccttgggaaccacttcagctgcatgttgataaggctgttagtggtctgagatctc tgacaacactgcttagggctctgggtgctcagaaggaagctattagtccaccagatg ctgcttctgctgctccactgagaactattactgctgatacatttagaaagctgttta gagtttactcaaattttctgagaggtaagctgaagctgtatactggtgaggcttgta gaacaggtgatagatagccatggggg

SEQ ID 27. Nucleotide sequence of the synthetic EPO coding region Example 5 : Construction of a plant transformation vecto , which codes for the CMV Pi & P2 polymerase proteins

The DNA fragments harbouring the CMV Pland P2 genes are isolated from pSK-Pl and pSK-P2 and cloned in pSK-Act2p- NOSt using Notl yielding pSK-Act2p-Pl-N0St and pSK-Act2p- P2-N0St respectively. The gene cassette comprising CMV PI is isolated by digestion of pSK-Act2p-Pl-N0St, with Ascl and Pacl and cloned in binary vector pBIN-plus (Van Engelen et al . , 1984, Plant Mol. Biol. 26: 1701-1710 digested Ascl and Pacl yielding pBIN-Pl. pSK-Act2-P2-N0St is digested with Ascl and oligonucleotide CGCGAT is ligated to the 3' termini, yielding a plasmid with a Pacl 3' overhang. The plasmid is digested with Pacl and the P2 containing DNA fragmented is ligated into pBIN-Pl digested with Pacl, yielding pBIN-PlP2. Plasmid pBIN-P12P2b is transferred to Λgrojacteriϊim tumefaciens strain LBA4404 using triparental mating.

Example 6: Construction of a plant transformation vector, which encodes a minus-sense CMV RNA 3 molecule

A recombinant DNA vector is produced by fusion PCR, comprising respectively the CaMV 35S promoter, a cDNA molecule representing the 3' trailer of CMV RNA 3, an Ncol cloning site, a cDNA representing the intercistronic region of CMV RNA3, an Sphl cloning site, a cDNA molecule representing the 5' leader sequence of CMV RNA3, the Hepa titis delta antigenomic ribozyme and the NOS terminator.

To produce this DNA vector 10 oligonucleotides are designed, denoted R3a to R3j R3a (Seq Id no. 28) GGGCGCGCCGATCCGTCAACATGG

R3b (Seq Id no . 29) CCAAAAGGAGACCACCTCTCCAAATGAAATG

R3c (Seq Id no. 30) CATTTCATTTGGAGAGGTGGTCTCCTTTTGG

R3d (Seq Id no. 31) GGGGCATGCGTTCCCAGAATCCTCCC

R3e (Seq Id no. 32) GGGGCATGCCTCGACTCAATTCTACG

R3f (Seq Id no. 33) GGGCCATGGTGCGTATTAGTATATAAG

R3g (Seq Id no. 34) GGGCCATGGCTCGGGAAATCTAACAC

R3h (Seq ID no. 35) GATGCCATGCCGACCCCGTAATCTTACCACTG

R3i (Seq Id no. 36) CAGTGGTAAGATTACGGGGTCGGCATGGCATC 3j (Seq Id no. 37) GGGTTAATTAAGTAACATAGATGACACC

pRT103 (Tδpfer et al., 1987, Nucl . Ac. Res. 15:5890) is subjected to PCR using oligonucleotides R3a & R3b. pSK- RNA3 is subjected to PCR using oligonucleotide combinations R3c & R3d, R3e & R3f and R3g & R3h respectively and finally, pSK-HDR-NOSt (See example 4) is subjected to PCR using oligonucleotides R3i & R3j All 5 amplified DNA fragments with correct sizes are gel- purified and annealed by mixing, heating for 1 minute at 95 degrees Celsius and subsequent cooling down to room temperature for 5 minutes. Second strand cDNA is synthesised by taq polymerase for 3 minutes at 55 degrees Celsius and the gaps are ligated by T4 DNA ligase. The annealed DNA fragment is subsequently re-amplified using oligonucleotides R3a and R3j, digested with the restriction enzymes Ascl and Pacl, gel-purified and cloned in pNEBl93 (New England Biolabs) linearized with Ascl and Pacl, yielding pNEB-RNA3-Ncol-Sphl (Seq Id 38) .

Two oligonucleotides are designed CPf (Seq Id no. 39) : GTCGAGGCATGCACAAATCTTCTGAATCAAC and CPr (seq Id no.40) : GGAACGCATGCTCAGACTGGGAGTACTCTAG. pSK-RNA3 is subjected to PCR using CPf & CPr. The resulting PCR fragment is digested with Sphl that cleaves within the CPf and CPr oligonucleotide sequences respectively and is cloned in minus-sense orientation in pNEB-RNA3-Ncol-Sphl, linearized with Sphl, yielding recombinant plasmid pNEB- RNA3-Ncol.

Two oligonucleotides are designed P3f (Seq Id no. 41) : CGAGCCATGGCTTTCCAAGGTACCAG and P3r (Seq Id no. 42) : CGCACCATGGTGCCTAAAGACCGTTAAC . pSK-RNA3 is subjected to PCR using P3f & P3r. The resulting PCR fragment is digested with Ncol that cleaves within the P3f and P3r oligonucleotide sequences respectively and is cloned in minus-sense orientation in pNEB-RNA3-Ncol, linearized with Ncol, yielding recombinant plasmid pNEB-RNA3mod. The gene cassette comprising CMV RNA3 is isolated by digestion of pNEB-RNA3 with Ascl and Pacl and cloned in pBIN-plus digested Ascl and Pacl, yielding pBIN-RNA3mod. Plasmid pBIN-RNA3mod is transferred to A. tumefaciens strain LBA4404 using triparental mating.

GGCGCGCCGATCCGTCAACATGGTGGAGCACGACACTCTCGTCTACTCCAAGAATAT CAAAGATACAGTCTCAGAAGACCAAAGGGCTATTGAGACTTTTCAACAAAGGGTAAT ATCCGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTCATCAAAAGGAC AGTAGAAAAGGAAGGTGGCACCTACAAATGCCATCATTGCGATAAAGGAAAAGGCTA TCGTTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGA GCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTG ATATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCGCAAGACCCTT CCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGTGGTCTCCTTTTGGAGCCCCCA CGAAAGTGGGGGGGCACCCGTACCCTGAAACTAGCACGTTGTGCTAGAGGTACACGG CCCGAAGTCCTTCCGAAGAAACCTAGGAGATGGTTTCAAAGGCGCCTCAGAGATTTG TAAATCTACTGGCGTGGATTTCTCCACGACTGACCATTTTAGCCGTAAGCTGGATGG ACAACCCGTTCACCGTGAGATCGGAGGGAGGATTCTGGGAACGCATGCCTCGACTCA ATTCTACGACGTAAAAGAGAAAACACAGCACACACACACTCTTTATACAATCAGTAG ACAATAACGCAATCTCGCGGAGATGATGTAGGCTTACTAAAACCAGATGTGTTCCCT TCTCAACACGGCATCGCGTCACAGACGTCTACTGTATCAACTCACACAGGACACTAT AGATATAATATTATGTACAGACTCATAATACTTATATACTAATACGCACCATGGCTC GGGAAATCTAACACACTGTACACAAAAGGTAAAATATGTGCGGACAGCACGACTCAA ATGTGCGGACAACACGACTCGACACACACACGCACACACACAGTGGTAAGATTACGG GGTCGGCATGGCATCTCCACCTCCTCGCGGTCCGACCTGGGCATCCGAAGGAGGACG CACGTCCACTCGGATGGCTAAGGGAGCTGCAGATCGTTCAAACATTTGGCAATAAAG TTTCTTAAGATTGAATCCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTT GAATTACGTTAAGCATGTAATAATTAACATGTAATGCATGACGTTATTTATGAGATG GGTTTTTATGATTAGAGTCCCGCAATTATACATTTAATACGCGATAGAAAACAAAAT ATAGCGCGCAACCTAGGATAAATTATCGCGCGCGGTGTCATCTATGTTACTTAATTA A

Seq Id 38: Nucleotide sequence of pNEB-RNA3-Ncol-Sphl

Example 7 : Production of Nicotiana benthamiana plants expressing CMV Pi and P2

Transformation of IV. benthamiana leaf disk explants with the recombinant A. tumefaciens strain containing binary vector pBIN-P12P2b is performed essentially according Horsh et al . To demonstrate that pBIN-RNA3mod is infectious to the transgenic plants and that the recombinant molecule moves systemically through polymerase expressing plants, progeny SI plants are screened by the Agrobacterium tumefaciens transient assay (ATTA) using the recombinant A. tumefaciens strain containing binary vector pBIN-RNA3mod. Double sandwich ELISA is performed 14 days post inoculation according Gielen et al . , Euphytica 88: 139-149, 1996. The CP levels in the transgenic plant cells are 10% of total soluble protein. 10 individual plants from each of the lines showing characteristic CMV symptoms and accumulating high amounts of CMV CP are further grown and seeds harvested. 30 S2 progeny plants of each line are checked by Southern analysis for transgene copy numbers. Homozygous lines are collected and used in further experiments.

Example 8 : Construction of CMV RNA3-based plant transformation vectors , encoding the CMV P2b RNA silencing suppressor and the human erythropoietin genes

The DNA fragment comprising the human EPO coding sequence is isolated from pSK-EPO (See example 5) by digestion with respectively Sphl and cloned in pNEB-RNA3-Ncol-Sphl digested with Sphl, yielding pNEB-RNA3-EP0-Nco. The DNA fragment comprising CMV P2b coding sequence is isolated from pSK-P2b (See example 2) by digestion with respectively Ncol and cloned in pNEB-RNA3-EP0-Nco digested with Ncol, yielding pNEB-RNA3-EPO-P2b.

The gene cassette comprising CMV RNA3 with the EPO and P2b genes is isolated by digestion of pSK-RNA3-EPO-P2b with Ascl and Pacl and cloned in pBIN-plus digested Ascl and Pacl, yielding pBIN-RNA3-EPO-P2b. This recombinant binary vector is transferred to A. tumefaciens strain LBA4404 using triparental mating.

Example 9 : Production of N. benthamiana plants expressing minus-sense CMV RNA 3 molecules comprising the human erythropoietin gene and the CMV P2b silencing suppressor gene.

Transformation of N. benthamiana leaf disk explants with the recombinant A. tumefaciens strain containing binary vector pBIN-RNA3-EPO-P2b is performed essentially according Horsh et al . The resulting transgenic plants are analysed by Northern blotting for the expression of recombinant minus sense CMV RNA sequences. The plants accumulating the highest amounts of transgenic RNA molecules are crossed with a homozygous plant transformed with pBIN-P12P2b (See example 7) . Progeny FI seedlings are analysed by Western blotting using a polyclonal antiserum against human EPO, for the expression of EPO. The EPO content of plants harbouring pBIN-RNA3-EPO-P2b is 5 % of total soluble protein content.

Example 10 Molecular cloning of the Cucumber mosaic virus PI , P2 polymerase genes , RNA 3 and the tomato spotted wilt virus NSs RNA silencing suppressor gene

A cucumber mosaic virus (CMV) subgroup 1 isolate was collected from lily and maintained in Nicotiana benthamiana by mechanical passaging. Virus particles were purified from systemically infected N. benthamiana plants following the procedure of Francki et al [(1979) CMI/AAB Descr. Of plant viruses 213]. Tomato spotted wilt virus (TSWV) strain Br-01, a Brasilian isolate from tomato, was maintained in Nicotiana rustica plants by mechanical passaging. Virus was isolated according to Tas et al [(1977) Neth. J. Plant Pathol 83: 61-72].

Approximately 100 μg of purified virus in a volume of 250 μl was extracted with phenol, then with a mixture of phenol & chloroform and finally with chloroform. RNA was precipitated with ethanol and collected by centrifugation. The pellets were dissolved in 20 μl of water.

The sequences of the CMV PI and P2 genes were isolated using RNA-based PCR. Oligonucleotides Plf (Seq Id no.l): GGGCGGCCGCAATTCCTATGGCGACGTCC andPlr (Seq Id no.2) : GGGCGGCCGCCTAGGCACGAGCAACAC were designed, which are homologous and complementary respectively to the RNA sequences on RNA 1 flanking the translational start and stop codons of the PI protein. Oligonucleotides Plf (Seq Id no. 3) : GGGCGGCCGCTTTCTCATGGCTTTCCCC and P2r (Seq Id no.4) GGGCGGCCGCTCAGGCTCGGGTAACTCC were designed, which are homologous and complementary respectively to the RNA sequences on RNA 2 flanking the translational start and stop codons of the P2 protein.

To enable further cloning of the amplified PI and P2 cDNA molecules, Seq Ids 1 - 4 oligonucleotides contain a Notl recognition site, which is absent in cDNA sequences derived from CMV RNA 1 and RNA 2-. Purified CMV RNA was subjected to RT-PCR using oligonucleotides Seq Id 1 and Seq Id 2 yielding the PI cDNA. Similarly, using oligonucleotides Seq Id 3 and Seq Id 4 in an RT-PCR reaction yielded the P2 cDNA. The resulting PCR fragments were directly ligated into the pGEM-T vector (Promega), yielding the recombinant plasmids pHB5 and pHB7, respectively.

A full length CMV RNA3 DNA construct was produced using RNA-based PCR. Two oligonucleotides were designed R3f (Seq Id no.7): GGGATCCGTAATCTTACCACTGTG, which is homologous to nucleotides 1 to 17 of CMV RNA3 and R3r (Seq Id no. 8): GGGATCCTGGTCTCCTTTTGGAGGCC, which is complementary to nucleotides 2198 to 2216 of CMV RNA3. Both oligonucleotides contain BamHl sites to enable further cloning of the amplified DNA molecule. Purified CMV RNA was subjected to the Gen Amp RNA PCR, using oligonucleotides R3f and R3r. The resulting PCR fragment was digested with BamHl and cloned into BamHl linearized pSK+, yielding the recombinant plasmid pSK-RNA3. This plasmid has the additional G residue present at the 3' end of the minus strand of CMV RNA 3 to improve replication (Collmer, C.W. & Kaper, J.M. , Virology 145: 249-259, 1985) . HB-NSs-F(ll) (Seq Id no.43) :

GGGCGGCCGCTTCAAGTGTTTATGAGTCGATC and HB-NSs-R(12) (Seq Id no.44): GGGCGGCCGCTTATTTTGATCCTGAAGCATACGCTTC were designed, which are homologous and complementary respectively to the TSWV S RNA sequences the flanking the translational start and stop codons of the NSs protein. Purified TSWV RNA was subjected to RNA-based PCR using oligonucleotides HB-NSs-F(ll) and HB-NSs-R(12) yielding the NSs cDNA. The resulting PCR fragment was digested with Notl and directly ligated into the pGEM-T vector (Promega), yielding the recombinant plasmid pHBll.

Construction of plant transformation vectors, which code for the CMV PI & P2 polymerase proteins, a minus-sense CMV RNA 3 molecule harbouring the green fluorescence protein gene and the TSWV NSs protein

Two oligonucleotides were designed HB-Act-F(3) (Seq Id no. 45): GGGGCGCGCCGCATGCCTGCAGGTCG, homologous to the 5' end of the Act2 promoter and HB-Act-R(4) (seq Id no. 46) : GGGGCGGCCGCTTTTTATGAGCTGCAAAC complementary to sequences flanking the translational start codon. (Y-Q An et al., The Plant Journal (1996) 10:107-121). The oligonucleotides contain recognition sites of Ascl or Notl, which are absent in CMV RNA-derived cDNA, and the Act2 promoter sequence: This enables further cloning of the amplified DNA molecules. Genomic DNA was isolated from A. thaliana plants using standard procedures, which was subjected to PCR using oligonucleotides HB-Act- F(3)and HB-Act-R(4) . The resulting PCR fragment was digested with Ascl and Notl and cloned into plasmid pGEM- T vector (Promega) , yielding recombinant plasmid pHB3. Two oligonucleotides were designed HB-nos-F(l) (Seq Id. no. 47) : GGGCGGCCGCATCGTTCAAACATTTGG, complementary to the 5' end of the nopaline synthase (NOS) terminator with a Notl site and HB-nos-R(l) (seq Id no. 48) : GGTTAATTAAGTAACATAGATGACACCGCG complementary to sequences at the 3'end of the NOS terminator with a Pad site (Depicker et al . , J. of Mol. And Appl. Gen. 1:561-573, 1982). Genomic DNA was isolated from A. tumefaciens, which was subjected to PCR using oligonucleotides HB-nos- F(l) and HB-nos-R(l) . The resulting PCR fragment was cloned into plasmid pGEM-T vector (Promega) , resulting in plasmid pHBl.

Subsequent digestion of pHBl with Notl and Pad and of pHB3 with Ascl and Notl yielded two fragments that were simultaneously cloned between the Ascl and Pad sites of a pSK+ vector previously supplied with these sites. The resulting plasmid was denoted as pHB4.

Two primers were designed; Hlf (Seq Id no.13) :

GGGCTGCAGGGGTCGGCATGGCATCTCCACCTCCTCGCGGTCCGACCTGGGCATCCG

, homologous to the sequences corresponding with the 5' half of the Hepa titis delta antigenomic ribozyme and containing a Pstl cloning site (Perotta & Been, 1990) and Hlr (Seq Id. no 14) :

GGGCTGCAGCTCCCTTAGCCATCCGAGTGGACGTGCGTCCTCCTTCGGATGCCCAGG TCGG, complementary to the 3' half of the ribozyme also containing a Pstl restriction site. The 20 3' -terminal nucleotides of both primers are complementary to each other. Hlf and Hlr were annealed and subjected to PCR to fill in 5 Overhanging sequences. The resulting DNA fragment was digested with Pstl and cloned in Pstl linearized pSK+ yielding plasmid pSK-HDR. The PCR fragment comprising the NOS terminator was cloned downstream of the Hepa ti tis delta antigenomic ribozyme in pSK-HDR yielding pSK-HDR-NOSt .

A recombinant DNA vector was produced by fusion PCR, comprising respectively the CaMV 35S promoter, a cDNA molecule representing the (antisense of the) 3' trailer of CMV RNA 3, an Ncol cloning site, a cDNA representing the (antisense) intercistronic region of CMV RNA3, an Sphl cloning site, a cDNA molecule representing the (antisense of the) 5' leader sequence of CMV RNA3, the Hepatitis delta antigenomic ribozyme and the NOS terminator.

To produce this DNA vector 10 oligonucleotides are designed, denoted R3a to R3j

R3a (Seq Id no .28) GGGCGCGCCGATCCGTCAACATGG

R3b (Seq Id no .29) CCAAAAGGAGACCACCTCTCCAAATGAAATG

R3c (Seq Id no .30) CATTTCATTTGGAGAGGTGGTCTCCTTTTGG

R3d (Seq Id.no .31) GGGGCATGCGTTCCCAGAATCCTCCC

R3e (Seq Id.no .32) GGGGCATGCCTCGACTCAATTCTACG

R3f (Seq Id.no .33) GGGCCATGGTGCGTATTAGTATATAAG

R3g (Seq Id no .34) GGGCCATGGCTCGGGAAATCTAACAC

R3h (Seq Id no .35) GATGCCATGCCGACCCCGTAATCTTACCACTG

R3i (Seq Id no .36) CAGTGGTAAGATTACGGGGTCGGCATGGCATC

R3j (Seq Id no .37) GGGTTAATTAAGTAACATAGATGACACC

pRT103 (Tδpfer et al., 1987, Nucl . Ac. Res. 15:5890) was subjected to PCR using oligonucleotides R3a & R3b. pSK- RNA3 was subjected to PCR using oligonucleotide combinations R3c & R3d, R3e & R3f and R3g & R3h respectively and finally, pSK-HDR-NOSt was subjected to PCR using oligonucleotides R3i & R3j . All 5 amplified DNA fragments with correct sizes were gel-purified and annealed by mixing, heating for 1 minute at 95 degrees Celsius and subsequent cooling down to room temperature for 5 minutes. Second strand cDNA was synthesised by taq polymerase for 3 minutes at 55 degrees Celsius and the gaps were ligated by T4 DNA ligase. The annealed DNA fragment was subsequently re-amplified using oligonucleotides R3a and R3j , digested with the restriction enzymes Ascl and Pad and cloned in pNEBl93 (New England Biolabs) linearized with Ascl and Pad, yielding pNEB-RNA3-NcoI-SphI .

Two oligonucleotides were designed CPf (Seq Id no.41): GTCGAGGCATGCACAAATCTTCTGAATCAAC and CPr (Seq Id no.42): GGAACGCATGCTCAGACTGGGAGTACTCTAG. pSK-RNA3 was subjected to PCR using CPf & CPr. The resulting PCR fragment was digested with Sphl that cleaves within the CPf and CPr oligonucleotide sequences respectively and was cloned in minus-sense orientation in pNEB-RNA3-NcoI-SphI, linearized with Sphl, yielding recombinant plasmid pNEB- RNA3-NcoI.

Two oligonucleotides were designed P3f (Seq Id no.43): CGAGCCATGGCTTTCCAAGGTACCAG and P3r (Seq Id no. 44) : CGCACCATGGTGCCTAAAGACCGTTAAC . pSK-RNA3 was subjected to PCR using P3f & P3r. The resulting PCR fragment was digested with Ncol that cleaves within the P3f and P3r oligonucleotide sequences respectively and was cloned in minus-sense orientation in pNEB-RNA3-NcoI, linearized with Ncol, yielding recombinant plasmid pNEB-RNA3mod.

The coding sequence of the enhanced green fluorescent protein (GFP) was PCR-amplified using purified DNA of pEGFP (Clontech) as a template with oligonucleotides PGf: and PGr, both having Ncol restriction sites flanking the translational start and stop codons of the EGFP gene. The DNA fragment was digested with Ncol and cloned in the Ncol restriction site of pSK+, yielding pSK-GFP. The DNA fragment harbouring the GFP gene was isolated from pSK-GFP and cloned in pSK-Act2p-NOSt using Notl yielding pSK-Act2p-GFP-NOSt . Plasmid pSK-Act2-GFP-NOSt was digested with Ascl and Pad and cloned in binary vector pBIN-plus (Van Engelen et al . , 1984, Plant Mol. Biol. 26: 1701-1710) digested with Ascl and Pad yielding pBIN-GFP.

Plasmid pSK-GFP was digested with Ncol and the DNA fragment comprising the GFP gene was ligated in the minus sense orientation in the Ncol restriction site of pNEB- RNA3mod, yielding pNEB-RNA3-GFP. The gene cassette comprising CMV RNA3 with the GFP gene was isolated by digestion of pNEB-RNA3-GFP with Ascl and Pad and cloned in likewise digested pBIN-plus, yielding pBIN-RNA3-GFP.

The DNA fragments harbouring the CMV PI and P2 genes were isolated from pSK-Pl and pGSK-P2 and cloned in pSK-Act2p- NOSt using Notl yielding pSK-Act2p-Pl-NOSt and pSK-Act2p- P2-NOSt respectively. The gene cassette comprising CMV PI was isolated by digestion of pSK-Act2p-Pl-N0St, with Ascl and Pad and cloned in binary vector pBIN-plus digested with Ascl and Pad yielding pBIN-Pl. pSK-Act2-P2-NOSt was digested with Ascl and Pad and cloned in binary vector pBIN-plus digested with Ascl and Pad yielding pBIN-P2.

The DNA fragment harbouring the TSWV NSs gene was isolated from pHBll and cloned in pSK-Act2p-N0St using Notl, yielding pSK-Act2p-NSs-NOSt . pSK-Act2-NSs-NOSt was digested with Ascl and Pad and cloned in binary vector pBIN-plus digested with Ascl and Pad yielding pBIN-NSs.

Plasmids pBIN-Pl, pBIN-P2, pBIN-GFP, pBIN-RNA3-GFP and pBIN-NSs were transferred to Agrobacterium tumefaciens strain LBA4404 using triparental mating.

Transient expression of pBIN-Pl, pBIN-P2 , pBIN-GFP, pBIN- NSs and pBIN-RNA3-GFP in N. benthamiana leaves

To demonstrate that TSWV NS_S enhances transgene expression by RNA silencing suppression, the Agrobacterium tumefaciens transient assay (ATTA) was used. Constructs pBIN-GFP and pBIN-NSs were simultaneously infiltrated to N. benthamiana leaves and GFP expression was assayed using Western blot analysis. In the presence of TSWV NS_S protein, a seven-fold increase is observed in the production of GFP in plant cells. To demonstrate that pBIN-Pl, pBIN-P2 and pBIN-RNA3-GFP are functional, pBIN- Pl, pBIN-P2 and pBIN-RNA3-GFP were simultaneously infiltrated to IV. benthamiana leaves and GFP expression assayed. GFP can only be expressed in cells where PI and P2 replicate the RNA3-GFP molecule. Individual cells harbouring all three DNA constructs show green fluorescence. However, leaves infiltrated with pBIN-Pl, pBIN-P2 and pBIN-RNA3-GFP together with pBIN-NSs show ten times as many individual green fluorescent cells. This indicates that in 90% of the cells containing pBIN-Pl, pBIN-P2 and pBIN-RNA3-GFP, RNA3-GFP is silenced and that the NSs protein is able to prevent degradation of RNA3- GFP. Total protein extractions were quantified using the Bradford assay and all samples were normalized against the pBIN-GFP plus pBIN-plus leaf extract.

Claims

1. A method of producing at least a heterologous or exogenous target protein in a plant cell that comprises:

1) introducing into said plant cell a first isolated nucleic acid that comprises nucleic acid components that code for RNA dependent RNA polymerase proteins of a cucumovirus wherein each of said RNA dependent RNA polymerase nucleic acid components is operably linked to an exogenous promoter that drives expression in a plant cell; and

2) introducing into the said plant cell a second isolated nucleic acid that comprises nucleic acid operably linked to an exogenous promoter that drives expression in a plant cell wherein said nucleic acid encodes a recombinant cucumoviral RNA 3 molecule in the minus-sense orientation said minus-sense RNA 3 molecule comprising at least an RNA sequence that codes for a minus-sense RNA sequence of a target protein.

2. A method according to claim 1 wherein the first introduced nucleic acid of step 1) comprises a nucleic acid component in the plus-sense orientation that encodes a compatible viral RNA silencing suppressor protein.

3. A method according to claim 1 or claim 2 wherein the second isolated nucleic acid of step 2) comprises a nucleic acid component which encodes for the compatible viral RNA silencing suppressor protein in the minus-sense configuration.

4. A method according to any one of claims 1 to 3 wherein the RNA silencing suppressor protein is the CMV P2b or the TSWV NS_S.

5. A method according to claim 1 or claim 2 which comprises :

1) introducing into said plant cell a first isolated nucleic acid that .comprises nucleic acid components that code for RNA dependent RNA polymerase proteins of a cucumovirus wherein each of said RNA dependent RNA polymerase nucleic acid components is operably linked to an exogenous promoter that drives expression in a plant cell; and

2) introducing into the said plant cell a second isolated nucleic acid that comprises nucleic acid operably linked to an exogenous promoter that drives expression in a plant cell wherein said nucleic acid encodes i) a recombinant cucumoviral RNA 3 molecule in the minus-sense orientation said minus-sense RNA 3 molecule comprising at least an RNA sequence that codes for a minus-sense RNA sequence of a target protein and ii) a compatible viral RNA silencing suppressor protein wherein the nucleic acid encoding the said viral RNA silencing suppressor protein is in the minus-sense orientation.

6. A method according to any one of the preceding claims wherein the target protein is selected from a protein having the primary sequence of a human protein, the primary sequence of a non-human animal protein homologue, the primary sequence of a monoclonal antibody or an active fragment thereof, and the primary sequence of an industrial enzyme.

7. A method according to claim 6 wherein the target protein is selected from the group consisting of human insulin, preproinsulin, proinsulin, glucagon, interferons such as a-interferon, a-interferon, a-interferon, blood- clotting factors selected from Factor VII, VIII, IX, X, XI, and XII, fertility hormones including luteinising hormone, follicle stimulating hormone growth factors including epidermal growth factor, platelet-derived growth factor, granulocyte colony stimulating, prolactin, oxytocin, thyroid stimulating hormone, adrenocorticotropic hormone, calcitonin, parathyroid hormone, somatostatin, erythropoietin (EPO) , and enzymes such as a-glucocerebrosidase, haemoglobin, serum albumin, and collagen.

8. A method according to claim 7 wherein the target protein is selected from erythropoietin, insulin, preproinsulin, proinsulin, a-interferon, a-interferon, and a-interferon.

9. A method according to claim 8 wherein the target protein is erythropoietin.

10. A method according to any one of the preceding claims wherein the first isolated nucleic acid and/or the second isolated nucleic acid are derived from a cucumovirus selected from viruses from the families Nodaviridae,

To busviridae , Flaviviridae, Togaviridae, Bromoviridae and Closteroviridae .

11. A method according to claim 10 wherein the virus is a cucumovirus selected from the group tomato aspermy virus, cucumber mosaic virus of subgroup 1 or subgroup 2.

12. A method according to claim 10 or claim 11 wherein the virus is the cucumber mosaic virus of subgroup 1.

13. A method according to any one of the preceding claims wherein the exogenous promoter is selected from inducible, chemical-regulated, constitutive, and tissue specific promoters.

14. A method according to any one of claims 1 to 13 wherein the cucumoviral recombinant RNA 3 molecule is expressed transiently.

15. An isolated polynucleotide that encodes a functional cucumovirus RNA dependent RNA polymerase for use in a method according to any one of claims 1 to 14.

16. An isolated polynucleotide according to claim 15 wherein the RNA dependent RNA polymerase is from a cucumber mosaic virus, subgroup 1.

17. An isolated polynucleotide that encodes a recombinant cucumoviral RNA 3 molecule in the minus-sense orientation said minus-sense RNA 3 molecule comprising at least an RNA sequence that codes for a minus-sense RNA sequence of a target protein for use in a method according to any one of claims 1 to 14.

18. An isolated polynucleotide sequence according to any one of claims 15 to 17 comprising genomic DNA.

19. An isolated polynucleotide sequence according to any^' one of claims 15 to 17 comprising cDNA.

20. A nucleic acid vector suitable for transformation of a eucaryotic cell and including a polynucleotide according to any one of claims 15 to 19.

21. A nucleic acid vector according to claim 20 suitable for transformation of a plant or bacterial cell.

22. A nucleic acid vector suitable for transformation of a procaryotic cell and including a polynucleotide according to any one of claims 15 to 19.

23. A nucleic acid vector according to claim 22 suitable for transforming an Agrobacterium cell.

24. A host cell containing a heterologous polynucleotide or nucleic acid vector according to any one of claims 15 to 23.

25. A host cell according to claim 24 which is a plant or a bacterial cell.

26. A host cell according to claim 25 which is a bacterial cell.

27. A host cell according to claim 25 which is a plant cell.

28. A host cell according to any one of claims 24, 25 and 27 which is comprised in a plant, a plant part or a plant propagule, or an extract or derivative of a plant or in a plant cell culture.

29. A method of producing a cell according to any of claims 24 to 27, the method including incorporating said polynucleotide or nucleic acid vector into the cell by means of transformation.

30. A method according to claim 29 which includes regenerating a plant from a cell according to any of claims 24, 25 or 27 from one or more transformed cells.

31. A plant comprising a plant cell according to any one of claims 24, 25 or 27.

32. A method of producing a plant, the method including incorporating a polynucleotide or nucleic acid vector according to any of claims 15 to 21 into a plant cell and regenerating a plant from said cell.

33. Use of a polynucleotide according to any one of claims 15 to 19 in the production of a transgenic plant.

34. An isolated target polypeptide produced by the process according to any one of claims 1 to 14.