WO2001007613A2 - Method for enhancing rna or protein production using non-native 5' untranslated sequences in recombinant viral nucleic acids - Google Patents

Abstract

The present invention provides a method for enhancing the production of RNAs or proteins in a plant host using either non-native 5' untranslated sequences or artificial leader sequences. Preferably, commercially useful proteins, polypeptides, or fusion products thereof are produced, such as, enzymes, antibodies, hormones, pharmaceuticals, vaccines, pigments, anti-microbial polypeptides, and the like. The non-native 5' untranslated enhancers may also be effective in many different types of transcription or translation systems, such as bacterial and animal systems.

Description

METHOD FOR ENHANCING RNA OR PROTEIN PRODUCTION USING NON- NATIVE 5' UNTRANSLATED SEQUENCES IN RECOMBINANT VIRAL

NUCLEIC ACIDS

The present Application claims pπonty to U S Application Seπal Nos 09/359.299, 09 359,304. 09 359,301 , 09/359.305. all filed on July 21. 1999

FIELD OF INVENTION This invention relates generally to the field of molecular biology and viral genetics Specifically, this invention relates to using non-native 5^' untranslated sequences to enhance protein or RNA production by recombinant viral nucleic acids

BACKGROUND OF THE INVENTION Plant proteins and enzymes have long been exploited for many purposes, from viable food sources to biocatalytic reagents, or therapeutic agents Duπng the past decade, the development of transgenic and transfected plants and improvement in genetic analysis have brought renewed scientific significance and economical incentives to these applications The concepts of molecular plant breeding and molecular plant farming, wherein a plant system is used as a bioreactor to produce recombinant bioactive mateπals, have received great attention

Foreign genes can be expressed in plant hosts either by permanent insertion into the genome or by transient expression using virus-based vectors. Each approach has its own distinct advantages Transformation for permanent expression needs to be done only once, whereas each generation of plants needs to be inoculated with the transient expression vector Virus-based expression systems, in which the foreign mRNA is greatly amplified by virus replication, can produce very high levels of proteins in leaves and other tissues Viral vector-produced protein can also be directed to specific subcellular locations, such as endomembrane, cytosol, or organelles, or it can be attached to macromolecules, such as viπons, which aids purification of the protein

In order for plant-based molecular breeding and farming to gam widespread acceptance in commercial areas, it is necessary to develop methods for increasing the production of bioactive species produced in plants Factors influencing the production of bioactive species include transcπption and translation activities The mechanisms by which eukaryotes and prokaryotes initiate translation are known to have certain features in common and to differ in others Eukaryotic messages are functionally monocistromc, translation initiates at the 5' end and is stimulated by the presence of a cap structure (m'G" ppp G . . .) at this end (Shatkin, Cell 9:645 (1976)). Prokaryotic messages can be polycistronic, can initiate at sites other than the 5' terminus, and the presence of a cap does not lead to translational stimulation. Both eukaryotes and prokaryotes begin translation at the codon AUG, although prokaryotes can also use GUG. Translation in both is stimulated by certain sequences near the start codon. For prokaryotes, it is the so-called Shme-Dalgarno sequence (a puπne rich region 3-10 nucleotides upstream from the initiation codon). For eukaryotes, it is a purine at the -3 position and a G residue in the+4 position (where the A of the AUG start codon is designated +1), plus other sequence requirements involved in finer tuning. This is part of the "relaxed" version of the scanning model (Kozak, Nuc. Acids. Res. 13:857 (1984)) whereby a 40S ribosomal sub-unit binds at the 5' end of the eukaryotic mRNA and proceeds to scan the sequence until the first AUG, which meets the requirements of the model, is encountered, at which point a 60S sub-unit joins the 40S sub-unit, eventually resulting in protein synthesis. For sequence requirements related to initiation codon, see publications by Kozak: Cell 15:1109-1123 (1978), Nuc. Acid. Res. 9:5233-5266 (1981) and Cell 44:283-292 (1986).

One of the most widely studied RNA viruses is the Tobacco Mosaic Virus (TMV). Recently, U.S. Patent No. 5,891,665 issued to Wilson, describes how native 5' untranslated sequences of TMV, i.e. the omega region, act as enhancers of translation of mRNA. The omega region was previously shown to be related to ribosome association. Shivprasad et al., Virology 255:312-323 (1999) also demonstrated that the presence of a 3' native nontranslated region affects foreign gene expression in TMV-based vectors.

This invention describes the use of non-native 5' untranslated sequences to enhance RNA or protein production. Previously, short sequences (4 to 6 base pairs) that mimic the 5' leader of the coat subgenomic RNA was expected to give optimal expression of foreign genes. For example, the highly expressed TMV-U1 coat subgenomic RNA contains an extremely short 3 bp untranslated leader (AAU). In this invention, the use of non-native sequences at the 5 'untranslated region causes an increase in RNA or protein production. These non-native 5' untranslated sequences act as enhancers of RNA or protein production. Since viral genome is extremely streamlined (Dawson et al., Adv. Virus Res. 38:307-342 (1990)), it is not obvious to include non-native 5' untranslated sequences in the recombinant viral nucleic acids that will lead to an increase in RNA or protein production. SUMMARY OF THE INVENTION The present inv ention prov ides a method ior enhancing production of RNAs or proteins m plant hosts using either non-native 5^' untranslated sequences or artificial leader sequences m recombinant \ iral nucleic acids These foreign sequences may encode commerciallv useful proteins, polypeptides, or fusion products thereof, such as enzymes, antibodies hormones, pharmaceuticals, vaccines, pigments, antimicrobial polypeptides, and the like These enhancer sequences may be hgated upstream of an appropπate mRNA or used in the form of a cDNA expression vector The non-native enhancers may also be effective in many different types of transcπption or translation systems, such as bacteπal and animal systems

BRIEF DESCRIPTION OF THE FIGURES Figure 1 Rice α-amylase expression vector, TTO1A 103L This plasmid contains the TMV-Ul 126-, 183-. and 30-kDa ORFs, the ToMV coat protein gene (ToMVcp), the SP6 promoter, the πce α-amylase cDNA pOS103, and part of the pBR322 plasmid The TAA stop codon the 30-kDa ORF is underlined The TMV-Ul subgenomic promoter located within the minus strand of the 30-kDa ORF controls the expression of α-amylase The putative transcπption start point (tsp) of the subgenomic RNA is indicated with a peπod

0

Figure 2 Nucleotide sequences of (a) TT01 A 103L and (b) the 5' untranslated leader in

TTO1A 103

Figure 3 GFP expression vector, TTOSA1 APE pBAD #5 This plasmid contains the

TMV-Ul 126-, 183-, and 30-kDa ORFs, the ToMV coat protein gene (ToMVcp), the SP6 promoter, the πce α-amylase cDNA pOS103 5' untranslated leader, GFP, and part of the pBR322 plasmid The TAA stop codon in the 30-kDa ORF is underlined The TMV-Ul subgenomic promoter located withm the minus strand of the 30-kDa ORF controls the expression of α-amylase The putative transcπption start point (tsp) of the subgenomic

RNA is indicated with a peπod ( )

Figure 4 Nucleotide sequence of TTOSA1 APE

Figure 5 38C13 single chain antibody expression vector. NHL RV This plasmid contains the TMV-Ul 126-, 183-, and 30-kDa ORFs, the ToMV coat protein gene

(ToMVcp), the SP6 promoter, the πce α-amylase cDNA pOS103 5' untranslated leader and signal peptide ORF, munne 38C13 ScFv, and part of the pBR322 plasmid The TAA stop codon in the 30-kDa ORF is underlined. The TMV-Ul subgenomic promoter located within the minus strand of the 30-kDa ORE controls the expression of α-amylase. The putative transcription start point (tsp) of the subgenomic RNA is indicated with a period

(•)•

Figure 6. Nucleotide sequence of BA46 expression vector TTUDABP.

Figure 7. Nucleotide sequence of the hemoglobin expression vector RED1.

DETAILED DESCRIPTION OF THE INVENTION The present invention describes the use of non-native 5' untranslated sequences to enhance RNA or protein production in bacterial, plant or animal hosts. The non-native enhancer sequences may derive from viruses from same or different taxonomic groups. They may also contain sequences from non-viral sources, such as from bacteria, fungi, plants, animals, or other sources. The non-native 5' untranslated sequences typically have less than about 90%, e.g. less than about 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of sequence homology relative to the native viral sequences. In some embodiments of the instant invention, the 5 ' non-native untranslated sequence is a new sequence from a different taxonomic viral group, a non-viral source, a random, or a semi-random sequence inserted into any nucleotide position before the initiation codon of the viral genome.

The non-native 5' untranslated sequences also encompass analogs of naturally occurring nucleotides. Such analogs include, but are not limited to, phosphoramidates, peptide-nucleic acids, phosphorothioates, methylphosphonates, and the like. In addition to having non-naturally occurring backbones, analogs of naturally occurring polynucleotides may comprise nucleic base analogs, e.g., 7-deazaguanosine, 5-methyl cytosine, inosine, and the like. Descriptions of these analogs and their synthesis can be found, among other places, in U.S. patents 4,373,071; 4,401,796; 4,415,732; 4,458,066; 4,500,707; 4,668,777; 4,973,679; 5,047,524; 5,132,418; 5,153,319; 5,262,530; and 5,700,642.

In some embodiments of the invention, the non-native enhancer sequences may be generated by in vitro mutagenesis, recombination or a combination thereof. In vitro methods, including, but not limited to, chemical treatment, oligonucleotide mediated mutagenesis, error-prone PCR, combinatorial cassette mutagenesis, DNA shuffling, random-priming recombination, restriction enzyme fragment induced template switching, staggered extension process, among others. In some embodiments of the instant invention, a library containing sequence variants of the enhancer sequences may be expressed in plant hosts to select the enhancer sequences that confer optimal level of RNA or protein production A more detailed discussion of methods for generating libraπes of nucleic acid sequence vaπants and selecting desired RNA or protein production level is presented in three U S Patent Application Nos 09/359.300 and 09/359,304, 09/359,301 and 09/359,305

The non-native 5^" untranslated sequences or the inserted non-native sequences may be of vaπous lengths Preferably, the size of non-native nucleic acid sequence or the inserted non-native sequences is from about 5 to 1,000 base pairs (bp), e g less than about 500, 200, 100, 50, 20, 10. etc

I Recombinant plant viral nucleic acids

The construction of viral vectors may use a vaπety of methods known in the art In preferred embodiments of the instant invention, the viral vectors are deπved from the RNA plant viruses A vaπety of plant virus families may be used, such as Bromovmdae, Bunyavmdae, Comoviπdae, Gemmivindae, Potyviπdae, and Tombusviπdae, among others Within the plant virus families, vaπous genera of viruses may be suitable for the instant invention, such as alfamovirus, llarvirus, bromovirus, cucumovirus, tospovirus, carlavirus, cauhmovirus, closterovirus, comovirus, nepovirus, dianthovirus, furovirus, hordeivirus, luteovirus, necrovirus, potexvirus, potyvirus, rymovirus, bymovirus, oryzavirus, sobemovirus, tobamovirus, tobravirus, carmovirus, tombusvirus, tymovirus, umbravirusa, and among others

Withm the genera of plant viruses, many species are particular preferred They include alfalfa mosaic virus, tobacco streak virus, brome mosaic virus, broad bean mottle virus, cowpea chlorotic mottle virus, cucumber mosaic virus, tomato spotted wilt virus, carnation latent virus, caulflower mosaic virus, beet yellows virus, cowpea mosaic virus, tobacco πngspot virus, carnation πngspot virus, soil-bome wheat mosaic virus, tomato golden mosaic virus, cassava latent virus, barley stπpe mosaic virus, barley yellow dwarf virus, tobacco necrosis virus, tobacco etch virus, potato virus X, potato virus Y, πce necrosis virus, ryegrass mosaic virus, barley yellow mosaic virus, πce ragged stunt virus, Southern bean mosaic virus, tobacco mosaic virus, nbgrass mosaic virus, cucumber green mottle mosaic virus watermelon strain, oat mosaic virus, tobacco rattle virus, carnation mottle virus, tomato bushy stunt virus, turnip yellow mosaic virus, carrot mottle virus, among others. In addition. RNA satellite viruses, such as tobacco necrosis satellite may also he_≤mpkjyeoX

A given plant virus may contain either DNA or RNA. which may be either single- or double-stranded. One example of plant viruses containing double-stranded DNA includes, but not limited to. caulimoviruses such as cauliflower mosaic virus (CaMV). Representative plant viruses which contain smgle-stranded DNA are cassava latent virus, bean golden mosaic virus (BGMV), and chloris striate mosaic virus. Rice dwarf virus and wound tumor virus are examples of double-stranded RNA plant viruses. Single-stranded RNA plant viruses include tobacco mosaic virus (TMV), turnip yellow mosaic virus (TYMV), rice necrosis virus (RNV) and brome mosaic virus (BMV). The single-stranded RNA viruses can be further divided into plus sense (or positive-stranded), minus sense (or negative-stranded), or ambisense viruses. The genomic RNA of a plus sense RNA virus is messenger sense, which makes the naked RNA infectious. Many plant viruses belong to the family of plus sense RNA viruses. They include, for example, TMV, BMV, and others. RNA plant viruses typically encode several common proteins, such as replicase/polymerase proteins essential for viral replication and mRNA synthesis, coat proteins providing protective shells for the extracellular passage, and other proteins required for the cell-to-cell movement, systemic infection and self-assembly of viruses. For general information concerning plant viruses, see Matthews, Plant Virology, 3^rd Ed., Academic Press, San Diego (1991).

Selected groups of suitable plant viruses are characterized below. However, the invention should not be construed as limited to using these particular viruses, but rather the method of the present invention is contemplated to include all plant viruses at a minimum.

TOBAMOVIRUS GROUP

Tobacco Mosaic virus (TMV) is a member of the tobamoviruses. The TMV virion is a tubular filament, and comprises coat protein sub-units arranged in a single right- handed helix with the single-stranded RNA intercalated between the turns of the helix. TMV infects tobacco as well as other plants. TMV is transmitted mechanically and may remain infective for a year or more in soil or dried leaf tissue.

The TMV virions may be inactivated by subjection to an environment with a pH of less than 3 or greater than 8, or by formaldehyde or iodine. Preparations of TMV may be obtained from plant tissues bv (NH₄),SO₄ precipitation, followed by differential c entri fu s. atϊδn

Tobacco mosaic virus (TMV) is a positive-stranded ssRNA virus whose genome is 6395 nucleotides long and is capped at the 5 '-end but not polvadenylated The genomic RNA can ser e as mRN'λ for protein of a molecular weight of about 130,000 (130K) and another produced bv read-through of molecular weight about 180.000 (180K) However, it cannot function as a messenger for the synthesis of coat protein Other genes are expressed duπng infection by the formation of monocistronic. 3'-cotermιnal subgenomic mRNAs, including one (LMC) encoding the 17 5K coat protein and another (I₂) encoding a 3 OK protein The 3 OK protein has been detected in infected protoplasts as descπbed m Miller, J , Virology 132 71 (1984), and it is involved in the cell-to-cell transport of the virus in an infected plant as descπbed by Deom et al , Science 237 389 (1987) The functions of the two large proteins are unknown, however, they are thought to function m RNA replication and transcπption

Several double-stranded RNA molecules, including double-stranded RNAs corresponding to the genomic, I₂ and LMC RNAs, have been detected in plant tissues infected with TMV These RNA molecules are presumably intermediates in genome replication and/or mRNA synthesis processes which appear to occur by different mechanisms

TMV assembly apparently occurs in plant cell cytoplasm, although it has been suggested that some TMV assembly may occur in chloroplasts since transcnpts of ctDNA have been detected in puπfied TMV viπons Initiation of TMV assembly occurs by interaction between πng-shaped aggregates (" discs") of coat protein (each disc consisting of two layers of 17 subunits) and a unique internal nucleation site in the RNA, a hairpm region about 900 nucleotides from the 3 '-end in the common strain of TMV Any RNA, including subgenomic RNAs containing this site, may be packaged into viπons The discs apparently assume a helical form on interaction with the RNA, and assembly (elongation) then proceeds in both directions (but much more rapidly m the 3'- to 5'- direction from the nucleation site)

Another member of the Tobamoviruses, the Cucumber Green Mottle Mosaic virus watermelon strain (CGMMV-W) is related to the cucumber virus (Nozu et al , Virology 45 577 (1971)) The coat protein of CGMMV-W interacts with RNA of both TMV and CGMMV to assemble viral particles in vitro (Kuπsu et al , Virology 70 214 (1976)) Several strains of the tobamovirus group are divided into two subgroups, on the basis of the location of the origin of assembly. Subgroup I, which includes the vulgare, OM, and tomato strain, has an oπgin of assembly about 800-1000 nucleotides from the 3'- end of the RNA genome, and outside the coat protein cistron (Lebeuπer et al, Proc. Natl. Acad. Sci_. USA 74: 149 (1977); and Fukuda et al, Viroloev 101 :493 (1980)). Subgroup II, which includes CGMMV-W and cowpea strain (Cc) has an origin of assembly about 300- 500 nucleotides from the 3 '-end of the RNA genome and within the coat protein cistron. The coat protein cistron of CGMMV-W is located at nucleotides 176-661 from the 3 '-end. The 3 ' noncoding region is 175 nucleotides long. The origin of assembly is positioned within the coat protein cistron (Meshi et al., Virology 127:54 (1983)).

BROME MOSAIC VIRUS GROUP

Brome Mosaic virus (BMV) is a member of a group of tripartite, single-stranded, RNA-containing plant viruses commonly referred to as the bromoviruses. Each member of the bromoviruses infects a narrow range of plants. Mechanical transmission of bromoviruses occurs readily, and some members are transmitted by beetles. In addition to BMV, other bromoviruses include broad bean mottle virus and cowpea chlorotic mottle virus.^!

Typically, a bromovirus virion is icosahedral, with a diameter of about 26 μm, containing a single species of coat protein. The bromovirus genome has three molecules of linear, positive-sense, single-stranded RNA, and the coat protein mRNA is also encapsidated. The RNAs each have a capped 5 '-end, and a tRNA-like structure (which accepts tyrosine) at the 3 '-end. Virus assembly occurs in the cytoplasm. The complete nucleotide sequence of BMV has been identified nnd characterized as described by Ahlquist et al., J. Mol. Biol. 153:23 (19?^" .

RICE ! ROSIS VIRUS Rice Necrosis virus is a member of the Potato Virus Y Group or Potyviruses. The Rice Necrosis virion is a flexuous filament comprising one type of coat protein (molecular weight about 32,000 to about 36,000) and one molecule of linear positive-sense single- stranded RNA. The Rice Necrosis virus is transmitted by Polymyxa oraminis (a eukaryotic intracellular parasite found in plants, algae and fungi). GEMΓNIVIRUSES

G m ιvιιii555^~are^~argrσu -ol^πratt ^mg^ plant-viruses - with viπons of unique morphologv Each viπon consists of a pair of lsometnc particles (incomplete icosahedral), composed of a single type of protein (with a molecular weight of about 2 7-3 4X10⁴ Each geminivirus viπon contains one molecule of circular, positive- sense, smgle-stranded DNA In some gemmiviruses (ι e , Cassava latent virus and bean golden mosaic virus) the genome appears to be bipartite, containing two smgle-stranded DNA molecules

POTYVIRUSES

Potyviruses are a group of plant viruses which produce polyprotem A particularly preferred potyvirus is tobacco etch, virus (TEV) TEV is a well characteπzed potyvirus and contains a positive-strand RNA genome of 9 5 kilobases encoding for a single, large polyprotem that is processed by three virus-specific prote ases The nuclear inclusion protein " a" protemase is involved m the maturation of several replication-associated proteins and capsid protein The helper component-protemase (HC-Pro) and 35-kDa protemase both catalyze cleavage only at their respective C-termini The proteolytic domain in each of these proteins is located near the C-terminus The 35-kDa protemase and HC-Pro deπve from the N-terminal region of the TEV polyprotem

The selection of the genetic backbone for the viral vectors of the instant invention may depend on the plant host used The plant host may be a monocotyledonous or dicotyledonous plant, plant tissue, or plant cell Typically, plants of commercial interest, such as food crops, seed crops, oil crops, ornamental crops and forestry crops are preferred For example, wheat, πce, corn, potato, barley, tobacco, soybean canola, maize, oilseed rape, lilies, grasses, orchids, mses, onions, palms, tomato, the legumes, or Arabidopsis, can be used as a plant host Host plants may also include those readily infected by an infectious virus, such as Nicotiana, preferably, Nicotiana benthamiana, or Nicotiana clevelandu

One feature of the present invention is the use of plant viral nucleic acids which compπse one or more non-native nucleic acid sequences capable of being transcπbed in a plant host These nucleic acid sequences may be native nucleic acid sequences that occur m a host plant Preferably, these nucleic acid sequences are non-native nucleic acid sequences that do not normally occur in a host plant For example, the plant viral vectors may contain sequences from more than one virus, including viruses from more than one taxonomic group The plant viral nucleic acids may also contain sequences from non-viral^" sources, such as foreign genes, regulatory sequences, fragments thereof from bacteπa, fungi, plants, animals or other sources These foreign sequences may encode commercially useful proteins, polypeptides, or fusion products thereof, such as enzymes, antibodies, hormones, pharmaceuticals, vaccines, pigments, anti-microbial peptides and the like Or they may be sequences that regulate the transcπption or translation of viral nucleic acids, package viral nucleic acid, and facilitate systemic infection in the host, among others

Examples of enzymes that may be produced using the instant invention include, but are not limited to, glucanase, chymosin, proteases, polymerases, sacchaπdases, deyhdrogenases, nucleases, glucose oxidase, α-amylase, oxidoreductases (such as fungal peroxidases and laccases), xylanases, phytases, cellulases, hemicellulases, and hpases This invention may also be used to produce enzymes such as, those used in detergents, rennm, horseradish peroxidase, amylases from other plants, soil remediation enzymes, and other such mdustπal proteins

Examples of proteins that may be produced using the instant invention include, but are not limited to, blood proteins (e g , serum albumin, Factor VII, Factor VIII (or modified Factor VIII), Factor IX, Factor X, tissue plasminogen factor, tissue plasminogen activator (t-PA), Protein C, von Willebrand factor, antithrombin III, and erythropoietm (EPO), urokmase, prourokmase, epoetm-α, colony stimulating factors (such as granulocyte colony-stimulatmg factor (G-CSF), macrophage colony-stimulatmg factor (M- CSF). and granulocyte macrophage colony-stimulating factor (GM-CSF)), cytokmes (such as mterleukms or mterferons), mtegπns. addressms, selectms, homing receptors, surface membrane proteins (such as, surface membrane protein receptors), T cell receptor units, immunoglobulms, soluble major histocompatibility complex antigens, structural proteins (such as collagen, fibπn, elastm, tubulin, actm, and myosm), growth factor receptors, growth factors, growth hormone, cell cycle proteins, vaccines, fibπnogen, thrombin, cytokmes, hyaluromc acid and antibodies

In some embodiments of the instant invention, the plant viral vectors may compπse one or more additional native or non-native subgenomic promoters which are capable of transcπbmg or expressing adjacent nucleic acid sequences the plant host These non- native subgenomic promoters are inserted into the plant viral nucleic acids without destroying the biological function of the plant viral nucleic acids using known methods in the art. For example, the CaMVpromδteTcan be^~used when^_plant^~cells^~are^"Krbe transfected. The subgenomic promoters are capable of functioning in the specific host plant. For example, if the host is tobacco, TMV, tomato mosaic virus, or other viruses containing subgenomic promoter may be utilized. The inserted subgenomic promoters should be compatible with the TMV nucleic acid and capable of directing transcription or expression of adjacent nucleic acid sequences in tobacco. It is specifically contemplated that two or more heterologous non-native subgenomic promoters may be used. The non- native nucleic acid sequences may be transcribed or expressed in the host plant under the control of the subgenomic promoter to produce the products of the nucleic acids of interest.

In some embodiments of the instant invention, the recombinant plant viral nucleic acids may be further modified by conventional techniques to delete all or part of the native coat protein coding sequence or put the native coat protein coding sequence under the control of a non-native plant viral subgenomic promoter. If it is deleted or otherwise inactivated, a non-native coat protein coding sequence is inserted under control of one of the non-native subgenomic promoters, or optionally under control of the native coat protein gene subgenomic promoter. Thus, the recombinant plant viral nucleic acid contains a coat protein coding sequence, which may be native or a normative coat protein coding sequence, under control of one of the native or non-native subgenomic promoters. The native or non-native coat protein gene may be utilized in the recombinant plant viral nucleic acid. The non-native coat protein, as is the case for the native coat protein, may be capable of encapsidating the recombinant plant viral nucleic acid and providing for systemic spread of the recombinant plant viral nucleic acid in the host plant.

In some embodiments of the instant invention, recombinant plant viral vectors are constructed to express a fusion between a plant viral coat protein and the foreign genes or polypeptides of interest. Such a recombinant plant virus provides for high level expression of a nucleic acid of interest. The location(s) where the viral coat protein is joined to the amino acid product of the nucleic acid of interest may be referred to as the fusion joint. A given product of such a construct may have one or more fusion joints. The fusion joint may be located at the carboxyl terminus of the viral coat protein or the fusion joint may be located at the amino terminus of the coat protein portion of the construct. In instances where the nucleic acid of interest is located internal with respect to the 5' and 3' residues of the nucleic acid sequence encoding for the viral coat protein, there are two fusion joints.

That is. the nucleic acid of interest may be located 5', 3', upstream, downstream or within the coat protein. In some embodiments of such recombinant plant viruses, a " leaky" start or stop codon may occur at a fusion joint which sometimes does not result in translational termination.

In some embodiments of the instant invention, nucleic sequences encoding reporter protein(s) or antibiotic/herbicide resistance gene(s) may be constructed as carrier protein(s) for the polypeptides of interest, which may facilitate the detection of polypeptides of interest. For example, green fluorescent protein (GFP) may be simultaneously expressed with polypeptides of interest. In another example, a reporter gene, β-glucuronidase (GUS) may be utilized. In another example, a drug resistance marker, such as a gene whose expression results in kanamycin resistance, may be used.

Since the RNA genome is typically the infective agent, the cDNA is positioned adjacent a suitable promoter so that the RNA is produced in the production cell. The RNA is capped using conventional techniques, if the capped RNA is the infective agent. In addition, the capped RNA can be packaged in vitro with added coat protein from TMV to make assembled virions. These assembled virions can then be used to inoculate plants or plant tissues. Alternatively, an uncapped RNA may also be employed in the embodiments of the present invention. Contrary to the practiced art in scientific literature and in issued patent (Ahlquist et al., U.S. Patent No. 5,466,788), uncapped transcripts for virus expression vectors are infective on both plants and in plant cells. Capping is not a prerequisite for establishing an infection of a virus expression vector in plants, although capping increases the efficiency of infection. In addition, nucleotides may be added between the transcription start site of the promoter and the start of the cDNA of a viral nucleic acid to construct an infectious viral vector. One or more nucleotides may be added. In some embodiments of the present invention, the inserted nucleotide sequence may contain a G at the 5 '-end. Alternatively, the inserted nucleotide sequence may be GNN, GTN, or their multiples, (GNN)_X or (GTN)_X.

In some embodiments of the instant invention, more than one nucleic acid is prepared for a multipartite viral vector construct. In this case, each nucleic acid may require its own origin of assembly. Each nucleic acid could be prepared to contain a subgenomic promoter and a non-native nucleic acid. Alternatively, the insertion of a non- native nucleic acid into the nucleic acid of a monopartite virus may result in the creation of two nucleic acids (1 e the nucleic acid necessary for the creation of a bipartite viral vector) This vvoul Tbe advantageous when iTϊslIesϊraEfte to keep the replication and transcπption or expression of the nucleic acid of interest separate from the replication and translation of some of the coding sequences of the native nucleic acid

The recombinant plant viral nucleic acid may be prepared by cloning a viral nucleic acid If the viral nucleic acid is DNA. it can be cloned directly into a suitable vector using conventional techniques One technique is to attach an on gin of replication to the viral DNA which is compatible with the cell to be transfected In this manner, DNA copies of the chimenc nucleotide sequence are produced in the transfected cell If the viral nucleic acid is RNA, a DNA copy of the viral nucleic acid is first prepared by well-known procedures For example, the viral RNA is transcπbed into DNA using reverse transcnptase to produce subgenomic DNA pieces, and a double-stranded DNA may be produced using DNA polymerases The cDNA is then cloned into appropπate vectors and cloned into a cell to be transfected In some instances, cDNA is first attached to a promoter which is compatible with the production cell The recombinant plant viral nucleic acid can then be cloned into any suitable vector which is compatible with the production cell Alternatively, the recombinant plant viral nucleic acid is inserted in a vector adjacent a promoter which is compatible with the production cell In some embodiments, the cDNA ligated vector may be directly transcπbed into infectious RNA in vitro and inoculated onto the plant host The cDNA pieces are mapped and combined in proper sequence to produce a full-length DNA copy of the viral RNA genome, if necessary

Those skilled in the art will understand that these embodiments are representative only of many constructs suitable for housing libraπes of sequence vanants All such constructs are contemplated and intended to be within the scope of the present invention The invention is not intended to be limited to any particular viral constructs but specifically contemplates using all operable constructs A person skilled in the art will be able to construct the plant viral nucleic acids based on molecular biology techniques well known m the art Suitable techniques have been descπbed in Sambrook et al (2nd ed ), Cold Spπng Harbor Laboratory, Cold Spnng Harbor (1989), Methods in En∑γmol (Vols 68, 100, 101, 1 18, and 152-155) (1979, 1983, 1986 and 1987), and DNA Clonιng, O M Clover, Ed , IRL Press, Oxford (1985), Walkey, Applied Plant Virology, Chapman & Hall (1991), Matthews, Plant Virology, 3^rd Ed , Academic Press, San Diego (1991), Turpen et al . Jof Virological Metho s. 42 227-240 (1993), U S Patent Nos 4,885,248, 5,173,410.

5,3

5,866.785, 5,889,190. and 5.589,367, U S Patent Application No 08/324,003 Nucleic acid manipulations and enzyme treatments are earned out accordance with manufacturers' recommended procedures in making such constructs

Viral nucleic acids containing non-native 5' untranslated sequence or artificial leader sequence can be transfected as populations or individual clones into host 1) protoplasts, 2) whole plants, or 3) plant tissues, such as leaves of plants (Dijkstra et al , Practical Plant Virology Protocols and Exercises, Spπnger Verlag (1998), Plant Virology Protocol From Virus Isolation to Transgemc Resistance in Methods in Molecular Biology. ol 81. Foster and Taylor, Ed , Humana Press (1998)) The plant host may be a monocotyledonous or dicotyledonous plant, plant tissue, or plant cell Typically, plants of commercial interest, such as food crops, seed crops, oil crops, ornamental crops and forestry crops are preferred For example, wheat, πce, corn, potato, barley, tobacco, soybean canola, maize, oilseed rape, lilies, grasses, orchids, mses, onions, palms, tomato, the legumes, or Arabidopsis, can be used as a plant host Host plants may also include those readily infected by an infectious virus, such as Nicotiana, preferably, Nicotiana henthamiana, or Nicotiana clevelandu

In some embodiments of the instant invention, the delivery of the plant virus expression vectors into the plant may be affected by the inoculation of in vitro transcπbed RNA, inoculation of v ons, or internal inoculation of plant cells from nuclear cDNA, or the systemic infection resulting from any of these procedures In all cases, the co-infection mav lead to a rapid and pervasive systemic expression of the desired nucleic acid sequences in plant cells The systemic infection of the plant by the foreign sequences may be followed by the growth of the infected host to produce the desired product, and the isolation and puπfication of the desired product, if necessary The growth of the infected host is in accordance with conventional techniques, as is the isolation and the puπfication of the resultant products

The host can be infected with a recombinant viral nucleic acid or a recombinant plant virus by conventional techniques Suitable techniques include, but are not limited to, leaf abrasion, abrasion in solution, high velocity water spray, and other injury of a host as well as imbibing host seeds with water containing the recombinant viral RNA or recombinant plant virus More specifically, suitable techniques include (a) Hand Inoculations Hand inoculations are pertormed using a neutral pH low

— mftlamy phosphate buffer with the addition nf r.elite nr carborundum usually aoout 1%) One to four drops of the preparation is put onto the upper surface of a leaf and genth rubbed

(b) Mechanized Inoculations of Plant Beds Plant bed inoculations are performed b\ spraying (gas-propelled) the vector solution into a tractor-dπven mower while cutting the leaves Alternatively, the plant bed is mowed and the vector solution sprayed immediately onto the cut leaves

(c) High Pressure Spray of Single Leaves Single plant inoculations can also be performed by spraying the leaves with a narrow, directed spray (50 psi, 6-12 inches from the leaf) containing approximately 1% carborundum in the buffered vector solution

(d) V acuum Infiltration Inoculations may be accomplished by subjecting a host organism to a substantially vacuum pressure environment m order to facilitate infection

(e) High Speed Robotics Inoculation Especially applicable when the organism is a plant, individual organisms may be grown m mass array such as in microtiter plates Machinery such as robotics may then be used to transfer the nucleic acid of interest

(f) Ballistics (High Pressure Gun) Inoculation Single plant inoculations can also be performed by particle bombardment A ballistics particle delivery system (BioRad Laboratoπes, Hercules, (A) can be used to transfect plants such as N benthamiana as descπbed previously (Νagar et al , Plant Cell,

7 705-719 (1995))

An alternative method for introducing viral nucleic acids into a plant host is a technique known as agromfection or Agrobacterium-mzdiated transformation (also known as Agro-mfection) as descπbed by Gπmsley et al Nature 325 177 (1987) This technique makes use of a common feature of Agrobacterium which colonizes plants by transfemng a portion of their DΝA (the T-DΝA) into a host cell, where it becomes integrated into nuclear DΝA The T-DΝA is defined by border sequences which are 25 base pairs long, and any DΝA between these border sequences is transferred to the plant cells as well The insertion of a recombinant plant viral nucleic acid between the T-DΝA border sequences results in transfer of the recombinant plant viral nucleic acid to the plant cells, where the recombinant plant viral nucleic acid is replicated, and then spreads systemically through

(Gardner et al. Plant Mol. Biol. 6:221 (1986); CaV (Gπmsley et al, Proc. Natl. Acad. Sci. USA 83:3282 (1986)); MSV (Gπmsley et al, Nature 325:177 (1987)), and Lazarowitz, S., Nuc I. Acids Res. 16:229 (1988)) digitaria streak virus (Donson et al, Virology 162:248 (1988)), wheat dwarf virus (Hayes et al, J. Gen. Virol. 69:891 (1988)) and tomato golden mosaic virus (TGMV) (Elmer et al, Plant Mol. Biol. 10:225 (1988) and Gardiner et al, EMBO J. 7:899 (1988)). Therefore, agro-infection of a susceptible plant could be accomplished with a virion containing a recombinant plant viral nucleic acid based on the nucleotide sequence of any of the above viruses. Particle bombardment or electrosporation or any other methods known in the art may also be used.

In some embodiments of the instant invention, infection may also be attained by placing a selected nucleic acid sequence into an organism such as E. coli, or yeast, either integrated into the genome of such organism or not, and then applying the organism to the surface of the host organism. Such a mechanism may thereby produce secondary transfer of the selected nucleic acid sequence into a host organism. This is a particularly practical embodiment when the host organism is a plant. Likewise, infection may be attained by first packaging a selected nucleic acid sequence in a pseudovirus. Such a method is described in WO 94/10329. Though the teachings of this reference may be specific for bacteria, those of skill in the art will readily appreciate that the same procedures could easily be adapted to other organisms. II. Recombinant bacterial or animal viral nucleic acids

One skilled in the art will appreciate that the viral nucleic acids may also be derived from a variety of bacterial or animal viruses, such as Ml 3, 0X174, MS2, T4, lamda, T7, Mu, alphavirus, rhinovirus, poliovirus, polyomavirus, simian virus 40, and adenovirus, among others. Selected groups of bacterial viruses are discussed in Brock et al, Biology of Microorganisms, pp. 263-284, Prentice-Hall Inc., Upper Saddle River, NJ (1997). Selected groups of animal viruses are discussed in Flint et al, Principles of Virology, ASM Press, Washington, D.C. (2000). A subset of the animal viruses is described below. However, the invention should not be construed as limited to using these particular viruses, but rather the method of the present invention is contemplated to include all animal viruses at a minimum. Recombinant viral nucleic acids comprising non-native 5 'untranslated sequences may be obtained using conventional molecular biology techniques (Sambrook et al (2nd ed ), Cold Spπng Harbor Laboratory, Cold s rin Haibor ( 1989 — Methods for producing recombinant protein or polypeptide in bacterial or animal hosts are also known to those skilled the art (Sambrook et al (2nd ed ), Cold Spπng Harbor Laboratory, Cold Spπng Harbor (1989))

ALPHAVIRUSES

The alphaviruses are a genus of the viruses of the family Togavmdae Almost all of the members of this genus are transmitted by mosquitoes, and may cause diseases in man or animals Some of the alphaviruses are grouped into three serologicallly defined complexes The complex-specific antigen is associated with the El protein of the virus, and the species-specific antigen is associated with the E2 protein of the virus

The Semhki Forest virus complex includes Bebaru virus, Chikungunya Fever virus, Getah virus, Mayaro Fever virus, O'nyongnyong Fever virus, Ross River virus, Sagiyama virus, Semhki Forest virus and Una virus The Venezuelan Equine Encephalomyelitis virus complex includes Cabassou virus, Everglades virus, Mucambo virus, Pixuna virus and Venezuelan Equme Encephalomyelitis virus The Western Equine Encephalomyelitis virus complex includes Aura virus, Fort Morgan virus, Highlands J virus, Kyzylagach virus, Smdbis virus, Western Equine Encephalomyelitis virus and Whataroa virus

The alphaviruses contain an icosahedral nucleocapsid consisting of 180 copies of a single species of capsid protein complexed with a plus-stranded mRNA The alphaviruses mature when preassembled nucleocapsid is surrounded by a hpid envelope containing two virus-encoded integral membrane glycoprotems, called El and E2 The envelope is acquired when the capsid, assembled in the cytoplasm, buds through the plasma membrane The envelope consists of a hpid bilayer deπved from the host cell

The mRNA encodes a glycoprotein which is cotranslationally cleaved into nonstructural proteins and structural proteins The 3' one-third of the RNA genome consists of a 26S mRNA which encodes for the capsid protein and the E3, E2, K6 and El glycoprotems The capsid is cotranslationally cleaved from the E3 protein It is hypothesized that the ammo acid tπad of His, Asp and Ser at the COOH terminus of the capsid protein compπses a senne protease responsible for cleavage Hahn et al , Proc Natl Acad Sci USA 82 4648 (1985) Cotranslational cleavage also occurs between E2 and K proteins Thus, two proteins PE2 which consists of E3 and E2 pnor to cleavage and an El protein compπsing K6 and El are formed These proteins are cotranslationallv_inserted into the endoplasmic reticulum of the host ceil, glvcosvlated and transported via the Golεi apparatus to the plasma membrane where thev can be used for budding \t the point of viπon maturation the E3 and E2 proteins are separated The El and E2 proteins are incorporated into the hpid envelope

It has been suggested that the basic amino-terminal half of the capsid protein stabilizes the interaction of capsid with genomic RNA or interacts with genomic RNA to initiate a encapsidation, Strauss et al in the To^gavmdae and Flavivmdaei^, Ed S Schlesmger & M Schlesmger, Plenum Press, New York, pp 35-90 (1980) These suggestions imply that the ongin of assembly is located either on the unencapsidated_genomic RNA or at the ammo-terminus of the capsid protein It has been suggested that E3 and K6 function as signal sequences for the insertion of PE2 and El, respectively, into the endoplasmic reticulum

Work with temperature sensitive mutants of alphaviruses has shown that failure of cleavage of the structural proteins results in failure to form mature viπons Lmdquist et al Virology Hi 10 (1986) characteπzed a temperature sensitive mutant of Sindbis virus, t 20 Temperature sensitivity results from an A-U change at nucleotide 9502 The t_s lesion present cleavage of PE2 to E2 and E3 and the final maturation of progeny viπons at the nonpermissive temperature Hahn et al supra, reported three temperature sensitive mutations m the capsid protein which prevents cleavage of the precursor polyprotem at the nonpermissive temperature The failure of cleavage resulted in no capsid formation and very little envelope protein

Defective interfeπng RNAs (DI particles) of Sindbis virus are helper-dependent deletion mutants which interfere specifically with the replication of the homologous standard virus Perrault, J , Mwrobwl Immunol 93 151 (1981) DI particles have been found to be functional vectors for introducing at least one foreign gene into cells Levis, R , Proc Natl Acad Sci USA 84 4811 (1987)

It has been found that it is possible to replace at least 1689 internal nucleotides of a DI genome with a foreign sequence and obtain RNA that will replicate and be encapsidated Deletions of the DI genome do not destroy biological activity The disadvantages of the system are that DI particles undergo apparently random rearrangements of the internal RNA sequence and size alterations Monroe et al , J Virology 49-865 (1984) Expression of a gene inserted into the internal sequence is not as high as expected Lev is et al supra found that replication of the inserted gene was excellent but translaUUll W as-lo — Thi could he the rpcnlt of romppTiTion wit_no] particles for translation sites and/or also from disruption of the gene due to reaπangement through several passages

Two species of mRNA are present m alphavirus-iniected cells A 42S mRNA region, which is packaged into nature viπons and functions as the message for the nonstructural proteins, and a 26S mRNA, which encodes the structural polypeptides the 26S mRNA is homologous to the 3' third of the 42S mRNA It is translated into a 130K Dolyprotem that is cotranslationally cleaved and processed into the capsid protein and two glycosylated membrane proteins, El and E2

The 26S mRNA of Eastern Equine Encephalomyelitis (EEE) strain 82V-2137 was cloned and analyzed by Chang et al , J Gen Virol 68 2129 (1987) The 26S mRNA region encodes the capsid proteins, E3, E2, 6K and El The amino terminal end of the capsid protein is thought to either stabilize the interaction of capsid with mRNA or to interact with genomic RNA to initiate encapsidation

Uncleaved E3 and E2 proteins called PE2 is inserted into the host endoplasmic reticulum dunng protein synthesis The PE2 is thought to have a region common to at least five alphaviruses which interacts with the viral nucleocapsid dunng morphogenesis

The 6K protein is thought to function as a signal sequence involved in translocation of the El protein through the membrane The El protein is thought to mediate virus fusion and anchonng of the El protein to the virus envelope

RHΓNQVΓRUSES

The rhino viruses are a genus of viruses of the family Picomaviπdae The rhmoviruses are acid-labile, and are therefore rapidly inactivated at pH values of less than about 6 The rhmoviruses commonly infect the upper respiratory tract of mammals

Human rhmoviruses are the major causal agents of the common cold, and many serotypes are known Rhmoviruses may be propagated in vaπous human cell cultures, and have an optimum growth temperature of about 33°C Most strains of rhmoviruses are stable at or below room temperature and can withstand freezing Rhmoviruses can be inactivated by citπc acid, tincture of iodine or phenol/alcohol mixtures

The complete nucleotide sequence of human rhmovirus 2 (HRV2) has been sequenced The genome consists of 7102 nucleotides with a long open reading frame of 6450 nucleotides which is initiated 611 nucleotides from the 5'-end and stops 42

been identified

Rhmovirus RNA is smgle-stranded and positive-sense The RNA is not capped, but is joined at the 5 '-end to a small virus-encoded protein, vmon-protem genome-linked (VPg) Translation is presumed to result m a single polyprotem which is broken by proteolytic cleavage to yield individual virus proteins An icosahedral viral capsid contains 60 copies each of 4 virus proteins VPl, VP2, VP3 and VP4 and sunounds the RNA genome Medappa, K., Virology 44 259 (1971)

Analysis of the 610 nucleotides preceding the long open reading frame shows several short open reading frames However, no function can be assigned to the translated proteins since only two sequences show homology throughout HRV2, HRV14 and the 3 sterotypes of pohovirus These two sequences may be cntical m the life cycle of the virus They are a stretch of 16 bases beginning at 436 m HRV2 and a stretch of 23 bases beginning at 531 in HRV2. Cutting or removing these sequences from the remainder of the sequence for non-structural proteins could have an unpredictable effect upon efforts to assemble a mature viπon

The capsid proteins of HRV2: VP4, VP2, VP3 and VPl begin at nucleotide 611, 818, 1601 and 2311, respectively. The cleavage point between VPl and P2A is thought to be around nucleotide 3255 Skern et al , Nucleic Acids Research 13.2111 (1985).

Human rhmovirus type 89 (HRV89) is very similar to HRV2. It contains a genome of 7152 nucleotides with a single large open reading frame of 2164 condons Translation begins at nucleotide 619 and ends 42 nucleotides before the poly(A) tract. The capsid structural proteins, VP4, VP2, VP3 and VPl are the first to be translated Translation of VP4 begins at 619. Cleavage cites occur at

VP4/VP2 825 determined

VP2/VP3 1627 determined

VP3/VP1 2340 determined VP1/P2-A 3235 presumptive

Duechler et al , Proc Natl Acad Sci USA 84-2605 (1987) POLIOVIRUSES

— Pohoviruscs are the causal agents of poliomyelitis in man, and are one of three_groups of enteroviruses Entero viruses are a genus of the family Picornaviπdae (also the family of rhmoviruses) Most enteroviruses replicate pπmaπly m the mammalian gastrointestinal tract, although other tissues may subsequently become infected Many enteroviruses can be propagated in pπmaπly cultures of human or monkey kidney cells and in some cell lines (e g HeLa, Vero, WI-e8) Inactivation of the enteroviruses may be accomplished ith heat (about 50°C), formaldehyde (3%), hydrochloπc acid (0 IN) or chlonne (ca 0 3-0 5 ppm free residual Cl₂)

The complete nucleotide sequence of pohovirus PV2 (Sab) and PV3 (Sab) have been determined They are 7439 and 7434 nucleotide in length, respectively There is a s_ingle long open reading frame which begins more than 700 nucleotides from the 5^'-end Pohovirus translation produces a single polyprotem which is cleaved by proteolytic processing Kitamura et al Nature 291 547 ( 1981 )

It is speculated that these homologous sequences in the untranslated regions play an essential role in viral replication such as

1 viral-specific RNA synthesis,

2 viral-specific protein synthesis, and

3 packaging

Toyoda, H et al , J Mol Biol 174 561 (1984)

The structures of the serotypes of pohovirus have a high degree of sequence homology Their coding sequences code for the same proteins in the same order Therefore, genes for structural proteins are similarly located In PV1 , PV2 and PV3, the polyprotem begins translation near the 750 nucleotide The four structural proteins VP4, VP2, VP3 and VPl begin at about 745, 960, 1790 and 2495, respectively, with VPl ending at about 3410 They are separated in vivo by proteolytic cleavage, rather than by stop/start codons

SIMIAN VIRUS 40 Simian virus 40 (SV40) is a virus of the genus Polyomavirus, and was onginally_isolated from the kidney cells of the rhesus monkey The virus is commonly found, in its latent form, in such cells Simian virus 40 is usually non-pathogenic in its natural host Simian virus 40 virions are made by the assembly of three structural proteins, VPl ,

-VP2 and VP3. Girard et al, Biochem. Biophys. Res. Co mun, 40:97 (1970); Prives et al,

Proc_. Natl. Acad. Sci. USA 7X302 (1974); and Jacobson et al, Proc. Natl Acad. Sci. USA 73:2742-2746 (1976). The three corresponding viral genes are organized in a partially overlapping manner. They constitute the late genes portion of the genome. Tooze, J., Molecular Biology of Tumor Viruses Appendix A The SV40 Nucleotide Sequence, 2nd Ed_. Part 2, pp. 799-829 (1980), Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Capsid proteins VP2 and VP3 are encoded by nucleotides 545 to 1601 and 899 to 1601, respectively, and both are read in the same frame. VP3 is therefore a subset of VP2. Capsid protein VPl is encoded by nucleotides 1488-2574. The end of the VP2-VP3 open reading frame therefore overlaps the VPl by 113 nucleotides but is read in an alternative frame. Tooze, J., supra. Wychowski et al, J. Virology 61 :3862 (1987).

ADENOVIRUSES

Adenovirus type 2 is a member of the adenovirus family or adenovirus. This family of viruses are non-enveloped, icosahedral, linear, double-stranded DNA-containing viruses which infect mammals or birds.

The adenovirus virion consists of an icosahedral capsid enclosing a core in which the DNA genome is closely associated with a basic (arginine-rich) viral polypeptide VII. The capsid is composed of 252 capsomeres: 240 hexons (capsomers each sunounded by 6 other capsomers) and 12 pentons (one at each vertex, each sunounded by 5 'peripentonal' hexons). Each penton consists of a penton base (composed of viral polypeptide III) associated with one (in mammalian adenoviruses) or two (in most avian adenoviruses) glycoprotein fibres (viral polypeptide IV). The fibres can act as haemagglutinins and are the sites of attachment of the virion to a host cell-surface receptor. The hexons each consist of three molecules of viral polypeptide II; they make up the bulk of the icosahedron. Various other minor viral polypeptides occur in the virion.

The adenovirus dsDNA genome is covalently linked at the 5 '-end of each strand to a hydrophobic 'terminal protein', TP (molecular weight about 55,000 Da); the DNA has an inverted terminal repeat of different length in different adenoviruses. In most adenoviruses examined, the 5 '-terminal residue is dCMP.

During its replication cycle, the virion attaches via its fibres to a specific cell- surface receptor, and enters the cell by endocytosis or by direct penetration of the plasma membrane Most of the capsid proteins are removed in the cvtoplasm The viπon core enters the nucleus, where the uncoatmg is completed to release viral DNA almost free σf vinon polypeptides Virus gene expression then begins The viral dsDNA contains genetic information on both strands Early genes (regions Ela, Elb, E2a, E3, E4) are expressed before the onset of viral DNA replication Late genes (regions LI, L2. L3, L4 and L5) are expressed only after the initiation of DNA synthesis Intermediate genes (regions E2b and Iva₂) are expressed in the presence or absence of DNA synthesis Region Ela encodes proteins involved m the regulation of expression of othei early genes, and is also involved in transformation The RNA transcπpts are capped (with m⁷G⁵ppp⁵N) and polyadenylated m the nucleus before being transfened to the cytoplasm for translation

Viral DNA replication requires the terminal protein, TP, as well as virus-encoded DNA polymerase and other viral and host proteins TP is synthesized as an 80K precursor, pTP, which binds covalently to nascent replicating DNA strands pTP is cleaved to the mature 55K TP late in vinon assembly, possibly at this stage, pTP reacts with a dCTP molecule and becomes covalently bound to a dCMP residue, the 3' OH of which is believed to act as a pπmer for the initiation of DNA synthesis Late gene expression, resulting m the synthesis of viral structural proteins, is accompanied by the cessation of cellular protein synthesis, and virus assembly may result m the production of up to 10⁵ vmons per cell

In order to provide a clear and consistent understanding of the specification and the claims, including the scope given herein to such terms, the following definitions are given

5' untranslated sequences sequences at the 5' end of a viral genome up to the initiation codon

Coat protein (capsid protein) an outer structural protein of a virus

Gene a discrete nucleic acid sequence responsible for a discrete cellular product

Host a cell, tissue or organism capable of replicating a vector or viral nucleic acid and which is capable of being infected by a virus containing the viral vector or viral nucleic acid This term is intended to include prokaryotic and eukaryotic cells, organs, tissues, organisms, or in vitro extracts thereof, where appropnate

Infection the ability of a virus to transfer its nucleic acid to a host or introduce viral nucleic acid into a host, wherein the viral nucleic acid is replicated, viral proteins are synthesized, and new viral particles assembled Movement protein a noncapsid protein required for cell-to-cell movement of RNA rcphcons or viruses m plants

Non-native (foreign) any sequence that does not normally occur in the virus or its host or does not occur at its normal location in the viral or its host genome

Open Reading Frame a nucleotide sequence of suitable length in which there are no stop codons

Plant Cell the structural and physiological unit of plants, consisting of a protoplast and the cell wall

Plant Tissue any tissue of a plant in planta or in culture This term is intended to include a whole plant, plant cell, plant organ, protoplast, cell culture, or any group of plant cells organized into a structural and functional unit

Promoter the 5 '-flanking, non-coding sequence adjacent to a coding sequence which is involved in the initiation of transcπption of the coding sequence.

Protoplast an isolated cell without cell walls, having the potency for regeneration into cell culture or a whole host

Subgenomic mRNA promoter, a promoter that directs the synthesis of an mRNA smaller than the full-length genome in size.

Vector: a self-replicating nucleic acid molecule that contains non-native sequences and which transfers nucleic acid segments between cells.

Viπon. a particle composed of viral nucleic acid, viral coat protein (or capsid protein)

Virus, an infectious agent composed of a nucleic acid encapsulated in a protein. EXAMPLES OF THE PREFERRED EMBODIMENTS

The following examples further illustrate the present invention. These examples are intended merely to be illustrative of the present invention and are not to be construed as being limiting

EXAMPLE 1 Construction of the nee alpha-amylase expression vector TTO1A 103

Unique Xhol, Avrll sites were inserted into the nee α-amylase OS 103 cDNA by polymerase chain reaction (PCR) mutagenesis using oligonucleotides: 5'-GCC TCG AGT GCA CCA TGC AGG TGC TGA ACA CCA TGG TG-3' (upstream) (SEQ. ID. No 1) and 5'-TCC CTA GGT CAG ATT TTC TCC CAG ATT GCG TAG C-3' (downstream) (SEQ. ID. No. 2). The 1.4-kb Xhol. Avrll OS 103 PCR fragment was subcloned into

^' pTTOlA, creating plasmid TTO1A 103. Plasmid TTO1Λ 103 has been deposited in

American Type Culture Collection (assigned PTA-333)

Construction of the rice alpha-amylase expression vector TTOl A 103L

Unique Xhol, Avrll sites were inserted into the rice α-amylase pOS103 cDNA by polymerase chain reaction (PCR) mutagenesis using oligonucleotides: 5'-CTC TCG AGA TCA ATC ATC CAT CTC CGA AGT GTG TCT GC-3' (upstream) (SEQ. ID. No. 3) and 5'-TCC CTA GGT CAG ATT TTC TCC CAG ATT GCG TAG C-3' (downstream) (SEQ. ID. No. 4). The 1.4-kb Xhol, Avrll OS103 PCR fragment was subcloned into pTTOlA (Kumagai et al., Proc. Natl Acad. Sci. USA 92:1679-1683 (1995)), creating plasmid TTO1A 103L (Figure 1). Plasmid TTOl A 103L has been deposited in American Type Culture Collection (assigned PTA-327). In Figure 1, the nucleic acid sequence is designated as SEQ. ID. No. 5 and the amino acid sequence as SEQ. ID. No. 6.

In vitro transcriptions, inoculations, and analysis of transfected plants

N. benthamiana plants were inoculated with in vitro transcripts of p»I-digested TTO1A 103, TTO1A 103L as described in Kumagai et al, Proc. Natl. Acad. Sci. USA 92:1679-1683 (1995). Virions were isolated from N. benthamiana leaves infected with TTO1A 103L transcripts and stained with 2% aqueous uranyl acetate. Transmission electron micrographs were taken using a Zeiss™ CEM902^® instrument. Purification, immunological detection, and in vitro assay of α-amylase

Ten days after inoculation, total soluble protein was isolated from 10 g of upper, noninoculated N. benthamiana leaf tissue transfected with TTOl A 103L. The leaves were frozen in liquid nitrogen and ground in 20 ml of 10 mM 2-mercaptoethanol/lO mM Tris- bis propane, pH 6.0. The suspension was centrifuged and the supernatant, containing recombinant α-amylase, was bound to a POROS 50 HQ^® ion exchange column (PerSeptive Biosystems™). The α-amylase was eluted with a linear gradient of 0-1.0 M ΝaCl in 50 mM Tris-bis propane pH 7.0. The α-amylase eluted in fraction 16, 17 and its enzyme activity was analyzed (Sigma™ Kit #576-3). Fractions containing cross-reacting material to α-amylase antibody were concentrated with a Centriprep-30^® (Amicon™) and the buffer was exchanged by diafiltration (50 mM Tris-bis propane, pH 7.0). The sample was then loaded on a POROS HQ/M^® column (Perceptive Biosystems™), eluted with a linear gradient of 0-1 0 M NaCl in 50 mM Tπs-bis propane pH 7 0. and assayed for α- amylase activity Fractions containing cross-reacting matenal lu α-amylase antibody were- concentrated with a Centπprep-30^δ and the buffer was exchanged by diafiltration (20 mM Sodium Acetate/HEPES^/MES, pH 6 0) The sample was finally loaded on a POROS HS/M^£ column (Perceptive Biosystems™), eluted with a linear gradient of 0-1 0 M NaCl in 20 mM Sodium Acetate/HEPES/MES. pH 6 0, and assayed for α-amylase activity Total soluble plant protein concentrations were determined using bovme serum albumin as a standard The proteins were analyzed on a 0.1 % SDS/10% polyacrylamide gel and transfened by electroblottmg for 1 hr to a nitrocellulose membrane The blotted membrane was incubated for 1 hr with a 2000-fold dilution of anti-α-amylase antiserum. Using standard protocols, the antisera was raised in rabbits against S cerevisiae expressed πce α-amylase The enhanced chemiluminescence horseradish peroxidase-lmked, goat anti-rabbit IgG assay (Cappel Laboratoπes™) was performed according to the manufacturer's (Amersham™) specifications The blotted membrane was subjected to film exposure times of up to 10 sec The quantity of total recombinant α-amylase in an extracted leaf sample was determined (using a 1-sec exposure of the blotted membrane) by companng the crude extract chemiluminescent signal to the signal obtained from known quantities of α-amylase Shorter and longer chemiluminescent exposure times of the blotted membrane gave the same quantitative results.

Compansion of N benthamiana transfected with TTOl A 103 and N benthamiana transfected with TTOl A 103L

Tobamo viral vectors have been developed for the expression of heterologous proteins in plants. The πce α-amylase gene (OS 103) was placed under the transcnptional control of a tobamovirus subgenomic promoter in TTOl A 103L, a RΝA viral vector. One to two weeks after inoculation, transfected Nicotiana benthamiana plants accumulated glycosylated α-amylase to levels of at least 5% total soluble protein. The 46 kDa recombinant enzyme was punfied and its structural and biological properties were analyzed. The πce α-amylase 5' untranslated leader enhanced the production of recombinant enzyme m transfected plants. It is possible that there is synergy between the 5' leader and 3'-untranslated region (UTR) of the recombinant tobamovirus. The highly expressed viral coat subgenomic RΝA has a 5' cap (m7GpppΝ) and terminates with a tRNA-like structure instead of a poly(A) tail. The 3'-UTR has two domains which contains five RNA pseudoknots. The tobacco etch viral (TEV) 5' leader and poly(A) tail are synergistic regulators of translation in transfected plants and animal cells. In the^"present embodiment, a modified α-amylase cDNA was placed under the control of the

TMV-Ul coat protein subgenomic promoter. The 34 bp rice α-amylase 5' untranslated leader can help to enhance the initiation of translation, the stability of viral sequences, and the synthesis of subgenomic RNA. There was at least a one hundred fold increase in the accumulation of α-amylase in plants transfected with constructs containing the 34 bp rice α-amylase 5' untranslated leader (5'-G ATC AAT CAT CCA TCT CCG AAG TGT GTC TGC AGC-3' (SEQ. ID. No. 7), see Figure 2A) compared to plants transfected with TTOl A 103, a construct that contains only a 5 bp leader (5'-GG TGC-3', see Figure 2B). In Figure 2A, the nucleic acid sequence is designated as SEQ. ID. No. 8 and the amino acid sequence as SEQ. ID. No. 9. In Figure 2B, the nucleic acid sequence is designated as SEQ. ID. No. 10 and the amino acid sequence as SEQ. ID. No. 11.

EXAMPLE 2 Construction of cvtoplasmic expression vector containing the rice α-amylase 5' untranslated leader

TTOSA1 APE pBAD was designed to express GFP in the cytoplasm. Using PCR mutagenesis, the Sphl site in the 126K replicase open reading frame (ORF) of TTOl A was removed using oligonucleotide 5'-CGT CCA GGT TGG GCA TAC AGC AGT GTA CAT ATG C-3' (SEQ. ID. No. 12) and a unique Pmel site was inserted at the 3' end of tomato mosaic virus cDNA (fruit necrosis strain F; ToMV-F) using oligonucleotide 5'-CGG GGT ACC GTT TAA ACT GGG CCC CAA CCG GGG GTT CCG GG-3' (SEQ. ID. No. 13). A 1.4 Kb Xhol, Avrll fragment from TTOl A 103L containing the rice α-amylase OS 103 cDNA (O'Neill et al, Mol. Gen. Genet. 221:235-244 (1990)) was inserted, creating plasmid TTOSA1 APE 103L. A unique Sphl site (start codon) and a unique Avrll site (adjacent to the stop codon) was inserted in the jellyfish Aequorea victoria GFP cDNA by PCR mutagenesis using oligonucleotides GFP MIS 5'-TAA GCA TGC TGA AAG GAG AAG AAC TTT TCA CTG GAG TT-3' (upstream) (SEQ. ID. 14) and GFP K238 5'-TAC CTA GGA GAT ATC CTT GTA TAG TTC ATC CAT GCC ATG TGT-3' (downstream) (SEQ. ID. 15), subcloned into TTOSA1 APE 103L, creating plasmid TTOS A 1 APE pBAD #5 (Figure 3). EXAMPLE 3

Construction of secretion v ector containing the nee alpha-amylase 5' untranslated leader

Using polymerase chain reaction (PCR) mutagenesis. the Sphl site m the 126K replicase open reading frame (ORF) of TTOl A was removed using oligonucleotide 5' CGT CCA GGT TGG GCA TAC AGC AGT GTA CAT ATG C 3' (SEQ ID No 16), and a unique Pmel site was inserted at the 3' end of tomato mosaic virus cDNA (ToMV) using oligonucleotide 5'-CGG GGT ACC GTT TAA ACT GGG CCC CAA CCG GGG GTT CCG GG-3' (SEQ ID No 17) Unique Xhol, Avrll sites were inserted into the πce α- amylase OS 103 cDNA by PCR mutagenesis using oligonucleotides: 5'-CTC TCG AGA TCA ATC ATC CAT CTC CGA AGT GTG TCT GC-3' (upstream) (SEQ. ID No. 18) and 5' TCC CTA GGT CAG ATT TTC TCC CAG ATT GCG TAG C 3' (downstream) (SEQ ID. No 19) and subcloned into the Sphl, Pmel modified tobamoviral vector, creating plasmid TTOSAl APE 103L In order to clone the 5^' untranslated leader adjacent to a modified α -amylase signal peptide ORF, we utilized a plasmid, TTOAB4, that contained a unique Sphl site that was introduced into the πce α-amylase signal peptide ORF of OS 103 by PCR mutagenesis using oligonucleotides 5'-GCC TCG AGT GCA CCA TGC AGG TGC TGA ACA CCA TGG TG-3' (upstream) (SEQ. ID. No 20) and 5'- GAG CAT GCC GGC TGT CAA GTT GGA GGA GAG GCC-3' (downstream) (SEQ. ID. No. 21). An Ncol fragment from TTO1A 103L containing part of the TMV-Ul 30K ORF, 5' untranslated leader, and six codons of the πce α-amylase was subcloned into TTOAB4, creating plasmid TTO1/TTOAB4 Finally, the Sphl, Kpnl a -amylase ORF/ToMV 3' end containing fragment from TTOSAl APE 103L was subcloned into TTO1/TTOAB4 creating plasmid TTOSAl APE AB4 103L (TTOSAl APE) (Figure 4). In Figure 4, the nucleic acid sequence is designated as SEQ ID No. 22 and the ammo acid sequence as SEQ. ID No. 23.

EXAMPLE 4 Construction of secretion vector containing the nee alpha- amylase 5' untranslated leader and non-Hodgkin's lvmphoma (NHL) single chain antibody cDNA

Autonomously replicating RNA viral vectors were developed for the production and secretion of heterologous proteins m plants. These constructs were denved from hybπd fusions of two tobamoviruses and contained additional subgenomic promoters for expression of foreign genes. A sequence encoding a modified πce α-amylase signal peptide (OS 103) was fused to a single chain Fv (scFv) open reading frame in the

-tnH*inr i™l vpotor TTO Λ 1 A PF fMrCormirk et πl Prnr Natl Ara Sri. USA 96:703-

708 (1999)).

Construction of the single chain antibody expression vector NHL

PCR primers specific for murine 38C13 sequences (GenBank accession nos. X14096-X14099) were used to amplify the 38C13 scFv coding sequence. 38C13 scFv insert was then cloned in-frame with the sequence encoding a rice -amylase signal peptide into TTOSAl APE AB4 103L, a modified TTO1A vector containing a hybrid fusion of TMV and tomato mosaic virus. The resulting plasmid was named NHL RV (Figure 5).

Expression, and Purification of 38C13 scFv from transfected N. benthamiana

Infectious RΝA transcripts were made in vitro and directly applied to plants. High-level expression and accumulation of the single chain antibody occuned within ten days post inoculation. The interstitial fluid containing the scFv was isolated using vacuum infiltration and the secreted protein was purified to homogeneity by affinity chromatography. Infected N. benthamiana plants contained high levels of secreted scFv protein in the extracellular compartment. The material reacted with an anti-idiotype antibody by Western blotting, ELISA, and affinity chromatography, suggesting that the plant-produced 38C13 scFv protein was properly folded in solution. Mice vaccinated with the affinity-purified 38C13 scFv generated >10 μg/ml anti-idiotype immunoglobulins. These mice were protected from challenge by a lethal dose of the syngeneic 38C13 tumor, similar to mice immunized with the native 38C13 IgM -keyhole limpet hemocyanin conjugate vaccine. This rapid production system for generating tumor-specific protein vaccines may provide a viable strategy for the treatment of non-Hodgkin's lymphoma.

EXAMPLE 5 Construction of an artificial leader using a modified TMV coat ORF

During replication of the tobamoviral vectors, a small amount of negative strand RΝA is synthesized. The native subgenomic promoter is located on the minus strand and controls the expression of foreign genes. Although deletion analysis of sequences surrounding the TMV coat protein transcriptional start site revealed that the major portion of the subgenomic promoter was upstream of the coat AUG, a small portion of the promoter may reside downstream of the start codon. In order to address this issue, an artificial leader was ronsTmr.tpd hy mutating the TMV coat protein start codon ATG to

AGA by site-directed mutagenesis. Foreign gene inserted downstream of the artificial leader sequence (5'-TCTTACAGTATCACTACTCCATCTCAGTTCGTGTTCTTGTCA- 3') (SEQ. ID. No. 24) at several unique cloning sites, showed increased genetic stability and led to a higher level of when compared with virus constructs lacking the leader sequence.

EXAMPLE 6 Construction of secretion vector containing an artificial leader and a human BA46 gene In several cloning steps a secretion vector was constructed that contains a hybrid virus, TTU51, consisting of TMV-Ul and tobacco mild green mosaic virus (TMGMV; U5 strain) and the sequence encoding a modified rice α-amylase signal peptide. In this plasmid the Sphl site in the 126K replicase open reading frame was removed using oligonucleotide 5'-CGT CCA GGT TGG GCA TAC AGC AGT GTA CAT ATG C-3' (SEQ. ID. No. 25), and a 1-Kb Avrll-Kpnl TMGMV 3' end from TTU51 was attached. Unique Sphl, Avrll sites were inserted into human BA46 cDNA (Couto et al, DNA Cell Biology 15:281-286 (1996)) by polymerase chain reaction (PCR) mutagenesis using oligonucleotides: 5'-CTC GAG GCA TGC TCC TGG ATA TCT GTT CCA AAA ACC-3' (upstream) (SEQ. ID. No. 26) and 5' GAC CGG TCC TAG GTT AAC AGC CCA GCA GCT CCA GGC GCA GGG C 3' (downstream) (SEQ. ID. No. 27) and subcloned into the tobamoviral secretion vector, creating plasmid TTUDABP (Figure 6). Infectious RNA transcripts were made in vitro and directly applied to plants. One week after transfection, recombinant human BA46 was detected in systemically infected tissue using an anti- BA46 antibody. In Figure 6, the nucleic acid sequence is designated as SEQ. ID. No. 28 and the amino acid sequence as SEQ. ID. No. 29.

EXAMPLE 7 Construction of β-globin expression vector

The hemoglobin expression vector, RED1 (Figure 7), was constructed in several subcloning steps. A unique Sphl site was inserted in the start codon for the human β- globin and an Xbal site was placed downstream of the stop codon by polymerase chain reaction (PCR) mutagenesis by using oligonucleotides 5' CAC TCG AGA GCA TGC TGC ACC TGA CTC CTG AGG AGA AG 3' (upstream) (SEQ. ID. No. 30) and 5'-CGT CTA GAT TAG TGΛ TAC TTG TGG GCC AGC GCA TTA GC-3' (downstream) (SEQ. ID. No. 31). The 452 bp Sphl-Xbal hemoglobin fragment was subcloned into the Sphl- Avrll site of a modified tobamoviral vector, TTU51D. This construct consisits of a 1020 bp fragment from the tobacco mild green mosaic virus (TMGMV; U5 strain) containing the viral subgenomic promoter, coat protein gene, and the 3' end that was isolated by PCR using TMGMV pnmers 5'-GGC TGT GAA ACT CGA AAA GGT TCC GG-3' (upstream) (SEQ. ID. No. 32) and 5'-CGG GGT ACC TGG GCC GCT ACC GGC GGT TAG GGG AGG-3' (downstream) (SEQ. ID. No. 33). In this vector, an artificial 40 bp 5' untranslated coat protein leader was fused to a hybrid cDNA encoding rice α-amylase signal peptide and human β-globin. The heterologous gene was under the control of the tobacco mosaic virus (TMV-Ul) coat protein subgenomic promoter. Infectious RNA transcripts were made in vitro and directly applied to plants. One week after transfection, recombinant human β-globin was detected in systemically infected tissue using an anti-hemoglobin antibody. In Figure 7, the nucleic acid sequence is designated as SEQ. ID. No. 34 and the amino acid sequence as SEQ. ID. No. 35.

EXAMPLE 8 cDNA library construction in a recombinant viral nuclei acid vector cDNA libraries can be constructed or obtained from a variety of private or public sources such as the Arabidopsis Biological Resource Center (ABRC). The cDNA libraries can be digested with appropriate restriction enzymes and the inserts can be modified by adding linker adapters with cohesive ends, and directly cloned into recombinant viral nucleic acid vectors containing non-native 5' untranslated leader sequences. Bacterial cells can be transformed with the viral based cDNA library. DNA that is isolated from the cells can be used to make infectious RNA that is directly applied to plants. The viral constructs causing changes in the phenotype or biochemical properties of the transfected plants can be characterized by nucleic acid sequencing. Selected leaf disc from the transfected plants can be taken for biochemical analysis such as MALDI-TOF. A recombinant viral nucleic acid expression vector library containing non-native 5' untranslated leaders would be especially useful in detecting tranfected plants that are over- expressing foreign proteins. EXAMPLE 9 Use of inserted non-naiive sequences to enhance the expression of foreign genes-m transfected plants

Insertion of foreign gene sequences into virus expression vectors can result in arcangements of sequences that interfere with normal virus function and thereby, establish a selection landscape that favors the genetic deletion of the foreign sequence. Such events are adverse to the use of such expression vectors to stably express gene sequences systemically in plants. A method that would allow sequences to be identified that may insulate functional virus sequences from the potential adverse effects of insertion of foreign gene sequences would greatly augment the expression potential of virus expression vectors. In addition, identification of such " insulating" sequences that simultaneously enhanced the translation of the foreign gene product or the stability of the mRNA encoding the foreign gene would be quite helpful. The example below demonstrates how libraries of random sequences can be introduced into virus vectors flanking foreign gene sequences. Upon analysis, a subset of introduced sequences allowed a foreign gene sequence that was previously prone to genetic deletion to remain stabily in the virus vectors upon serial passage. The use of undefined sequences to enhance the stability of foreign gene sequences can be extrapolated to the use of undefined sequences to enhance the translation of foreign genes and the stability of coding mRNAs by those skilled in the art.

Undefined sequences can also be used to enhance and extend the expression of foreign genes in a viral vector. To test this hypothesis random sequences of N20 were cloned in-between the TMV subgenomic promoter and the gene sequence for either human growth hormone (hGH) or a ubiquitin-hGH fusion gene. In this experiment the site of random nucleotide insertion was following a Pad (underlined) restriction enzyme site in the virus vector. This sequence is known as a leader sequence and has been derived from the native leader and coding region from the native TMV UI coat protein gene. In this leader, the normal coat protein ATG has been mutated to a Aga sequence (underlined in GTTTTAAATAgaTCTTACAGTATCACTACTCCATCTCAGTTCGTGTTCTTGTCAJ TAATTAA ATG ... (hGH GENE)) (SEQ. ID. No. 36). A particular subset of this leader sequence (TCTTACAGTATCACTACTCCATCTCAGTTCGTGTTCTTGTCA) (SEQ. ID. No. 37) has been known to increase genetic stability and gene expression when compared with virus construct lacking the leader sequence. The start site of subgenomic RNA synthesis is tound at the GTTTT An oligonucleotide RL-1

(GTTTTAAATAGATCTTAC N(20)TTAATTAAGGCC ) (SEQ ID No 38) was used with a pπmer homologous to the NcollApal region of the TMV genome to amplify a portion of the TMV movement protein The population of sequences were cloned into the Apal and Pad sites of the p30B hGH vector Vectors containing the undefined sequences leading the hGH genes were transcπbed and inoculated onto Nicotiana benthamiana plants Fourteen days post inoculation, systemic leaves were ground and the plant extracts were inoculated onto a second set of plants Following the onset of virus symptoms in the second set of plants, Western blot analysis was used to detect if hGH or Ubiq-hGH fusions were present m the seπally mocuated plants Several vanants containing novel sequences in the non-translated leader sequence were associated with viruses that expressed higher than control levels of hGH or Ubiquitin hGH fusion proteins in plants inoculated with in vitro synthesized transcnpts or upon senal passage of virus The sequence sunoundmg the leader was determined and compared with that of the control virus vectors p30B #5 HGH GTTTTAAATAGATCTTAC— TATAACATGAATAGTCATCG (SEQ ID No

39) p30B #5 HGH GTTTTAAATAGATCTTAC—TATACCATGAATTAGTACCG (SEQ ID No

40) p30B #6 U iqHGH GTTTTAAATAGATCTTAC— ACTCGGTTGAGATAAAACTAAACTA (SEQ

ID No 41) p30B #2 HGH GTTTTAAATAGATCTTAC—TCCGACGTATAGTCACCACG (SEQ ID No

42) p30B HGH GTTTTAAATAGATCTTAC—

AGTATCACTACTCCATCTCAGTTCGTGTTCT (SEQ ID No 43) p30BUbιqHGH GTTTTAAATAGATCTTAC—

AGTATCACTACTCCATCTCAGTTCGTGTTCT (SEQ ID No 44)

*********************** p30B #5 HGH - — TTAATTAAAATGGGA— (SEQ ID No 45) p30B #5 HGH - — TTATTTAAAATGGGAAAAATGGCTTCTCTATTTGCCACATTTTTA

(SEQ ID No 46) p30B #6 UbiqHGH - — TTAATTAAAATGGGAAAAΛTGGCTCTCTTATTGGCCCCATTTTTA

(SEQ ID No 47) p30B #2 HGH - — TTAATTAAAAATGCAGATTTTCGTCAAGACTTTGACCGGG (SEQ

ID No 48) p30B HGH TGTCATTAATTAAAATGGGAAAAATGGCTTCTCTATTTGCCACATTTTTA

(SEQ ID No 49) p30B UbiqHGH TGTCATTAATTAAAATGCAGATTTTCGTCAAGACTTTGACCGGT (SEQ ID

No 50)

****************

* indicates sequences that are identical in all viruses

— indicates end of defined pπmer and start of N(20) region of the oligonucleotide that was introduced during PCR amplification

The result was that undefined leader constructs transcnbed were passageable as virus The nature of the random leaders indicates that each are unique and that multiple solutions are readily available to solve RNA based stability problems. Likewise, such random sequence iniroduciiuiis could also increase the translational efficiency.

In order to select for undefined sequences that may increase the translational efficiency of foreign genes or increases the stability of the mRNA encoding the foreign gene derived from a virus expression vector, a selectable marker could be used to discover which of the undefined sequences yield the desired function. The amount of the GFP protein conelates with the level of fluorescence seen under long wave UV light and the amount of herbicide resistance gene product conelates with survival of plant cells or plants upon treatment with the herbicide. Therefore introduction of undefined sequences sunounding the GFP or herbicide resistance genes and then screening for individual viruses that either express the greatest level of fluorescence or cells that survive the highest amount of herbicide. In this manner the cells with the viruses with the highest foreign gene activity would be then purified and characterized by sequencing and more thorough analysis such as Northern and Western blotting to access the stability of the mRNA and the abundance of the foreign gene of interest.

EXAMPLE 10 Use of the untranslated non-native 5' sequence to enhance the ratio of coat protein fusion protein /coat protein production

U.S. Patent No. 5,618,699 issued to Hamamoto et al. describes virion particles comprising a TMV coat protein and a fusion protein of the TMV coat protein and a foreign protein. Such virion particles are produced by inoculating a plant with a viral vector comprising a foreign gene linked downstream of a TMV coat protein via a nucleotide sequence which occassionally causes readthrough. Such a " leaky stop codon" sequence results in mostly upstream coat protein gene expression, but occasionally results in readthrough expression of both the upstream coat protein and the downstream foreign protein. These coat proteins and coat protein fusion proteins will self-assemble around the vector nucleic acid, resulting in a virion particle having a coat protein subunits interspersed with coat protein fusion proteins. Because of steric hindrance, it is preferable to be able to modulate the level of coat protein fusion proteins interspersed with the coat proteins. The leaky stop codon thus useful, as it only allows readthrough transcription from the coat protein gene into the foreign protein gene a small percentage of the time.

An alternative way of modulating ratio of coat protein expression relative to coat protein fusion protein expression, is to construct a viral vector comprising both a coat protein coding sequence and a coat protein fusion protein coding sequence, with each naving its own subgenomic pioniuiei', and with a 5' untranslated, non native sequence of — the present invention operably placed upstream of the coat protein In this manner, the ratio of the production of coat protein fusion gene v s that of the coat protein are increased

Although the foregoing invention has been descπbed in some detail by way of illustration and example for purposes of claπty of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims

All publications, patents, patent applications are herein incorporated by reference m their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety

Claims

WE CLAIM

1 A recombinant viral nucleic acid compnsmg

(a) a first sequence which compπses a non-native 5'-untranslated sequence, and

(b) a second sequence which is downstream of and operatively linked to said first sequence, wherein the amount of RNA or protein produced from said second sequence is increased compared to the amount produced m the absence of said first sequence

2 The recombinant viral nucleic acid according to claim 1 , wherein said recombinant viral nucleic acid is denved from an RNA plant virus

3 The recombinant viral nucleic acid according to claim 1 , wherein said recombinant viral nucleic acid is denved from a smgle-stranded, positive sense RNA plant virus

4 The recombinant viral nucleic acid according to claim 1 , wherein said recombinant viral nucleic acid is denved from an animal virus

5 The recombinant viral nucleic acid according to claim 1 , wherein said recombinant viral nucleic acid is denved from a bactenal virus

6 The recombinant viral nucleic acid according to claim 1 wherein said non- native 5 '-untranslated sequence is obtained by in vitro mutagenesis, recombination, or a combination thereof

7 The recombinant viral nucleic acid according to claim 1 wherein said non- native 5 '-untranslated sequence is constructed by moving the ATG start codon downstream to a new site, thus creating an artificial leader sequence

8 The recombinant viral nucleic acid according to claim 1 wherein said second sequence compnses a non-native coding sequence The recombinant viral nucleic acid according to claim 8 wherein said non- native coding sequence encodes a fusion protein between a coat protein and a non-native protein or polypeptide.

10. The recombinant viral nucleic acid according to claim 8 wherein said non- native coding sequence encodes a product selected from the group consisting of enzymes, antibodies, hormones, pharmaceuticals, vaccines, pigments, and anti-microbial polypeptides.

11. A vector comprising claim 1.

12. The vector of claim 11 which is a plasmid.

13. An isolated host cell transformed with claim 1.

14. The recombinant viral nucleic acid according to claim 1, wherein said first or second sequence further comprising a promoter sequence.

15. An expression vector comprising claim 14.

16. The expression vector of claim 15 which is a plasmid.

17. An isolated host cell transformed with claim 14.

18. A recombinant viral nucleic acid comprising a non-native sequence inserted in any nucleotide position 5' to the initiation codon of said recombinant viral nucleic acid, wherein the amount of RNA or protein produced from said recombinant viral nucleic acid is increased compared to the amount produced in the absence of said non-native sequence.

19. The recombinant viral nucleic acid according to claim 18, wherein said recombinant viral nucleic acid is derived from an RNA plant virus.

20. The recombinant viral nucleic acid according to claim 18, wherein said recombinant viral nucleic acid is derived from a single stranded, positive sense RNA plant virus. 21 The recombinant viral nucleic acid according to claim 18 wherein said recombinant viral nucleic acid is denved from an animal virus

22 The recombinant viral nucleic acid according to claim 18, wherein said recombinant viral nucleic acid is denved from a bactenal virus

23 The recombinant viral nucleic acid according to claim 18 wherein said recombinant viral nucleic acid compnses a non-native coding sequence

24 The recombinant viral nucleic acid according to claim 23 wherein said non- native coding sequence encodes a fusion protein between a coat protein and a non-native protein or polypeptide

25 The recombinant viral nucleic acid according to claim 23 wherein said non- native sequence encodes a product selected from the group consisting of enzymes, antibodies, hormones, pharmaceuticals, vaccines, pigments, and anti-microbial polypeptides

26 A vector compnsmg claim 18

27 The vector of claim 26 which is a plasmid

28 An isolated host cell transformed with claim 18

29 The recombinant viral nucleic acid according to claim 18, wherein said recombinant viral nucleic acid further compnses a promoter sequence

30 An expression vector compnsmg claim 29

31 The expression vector of claim 30 which is a plasmid

32 An isolated host cell transformed with claim 29

33 A method for enhancing the production of a protein m a host compnsmg the steps of expressing in said host a recombinant viral nucleic acid compnsmg (a) a first sequence which compπses a non-nativ e 5 '-untranslated sequence, and

(b) a second sequence hich is downstream of and operatively linked to said first sequence, wherein said second sequence compπses a coding sequence encoding said protein

34 The method according to claim 33 wherein said protein is a fusion protein with a coat protein

35 A method for enhancing the production of a protein m a host compnsmg the steps of expressing in said host a recombinant viral nucleic acid compnsmg

(a) a non-native sequence inserted in any nucleotide position 5' to the initiation codon of said recombinant viral nucleic acid and a coding sequence encoding said protein

36 The method according to claim 35 wherein said protein is a fusion protein with a coat protein