Molecular farming of fluorescent virus-based nanoparticles for optical imaging in plants, human cells and mouse models.

The application of plant virus-derived nanostructures in materials science, biomedical research and engineering has recently been promoted by the development of fluorescence-labeled viruses for optical imaging in tissue culture and preclinical animal models. Most studies reported thus far have focused on the application of viruses that have been chemically modified with organic dyes. In this investigation, we sought to develop and study genetically-engineered virus-based biomaterials that incorporate green or red fluorescent proteins. The genetic introduction of such imaging moieties is advantageous because post-harvest modifications are not required, thus minimizing the number of manufacturing steps and maximizing the yields of each fluorescent probe. Specifically, we engineered the filamentous plant virus Potato virus X (PVX) to display green fluorescent protein (GFP) or mCherry as N-terminal coat protein (CP) fusions, producing a 1 : 3 fusion protein to CP ratio. The infection of Nicotiana benthamiana plants with the recombinant GFP-PVX and mCherry-PVX particles was documented by fluorescence imaging, structural analysis and genetic characterization to determine the stability of the chimeras and optimize the molecular farming protocols. We also demonstrated the application of fluorescent mCherry-PVX filaments as probes for optical imaging in human cancer cells and a preclinical mouse model. Cell viability assays and histological analysis following the administration of mCherry-PVX indicated the biocompatibility and rapid tissue clearance of the particles. Such particles could therefore be functionalized with additional cancer-specific detection ligands to provide tools for molecular imaging, allowing the investigation of molecular signatures, disease progression/recurrence and the efficacy of novel therapies.

Generation and characterization of functional recombinant antibody fragments against tomato yellow leaf curl virus replication-associated protein.

Tomato yellow leaf curl virus (TYLCV) is a complex of geminivirus species prevalent in the tropics and sub-tropics, which causes severe diseases in economically important crops such as tomato. Conventional strategies for disease management have shown little success and new approaches based on genetic engineering need to be considered. We generated two single-chain variable fragment antibodies (scFv-ScRep1 and scFv-ScRep2) that bound strongly to continuous epitopes within the TYLCV replication-associated protein (Rep). The TYLCV-Ir C1 gene (encoding Rep) was expressed as glutathione-S-transferase (GST) and maltose-binding protein (MBP) fusions. Purified MBP-Rep was used to immunize mice allowing the construction of naïve and pre-immunized scFv phage display libraries. Immunoassays showed that scFv-ScRep1 recognized an N-terminal epitope of Rep, whereas scFv-ScRep2 recognized a more central epitope. This is the first successful production of scFv antibodies against a geminivirus Rep, the initial step in the production of transgenic plants with resistance to TYLCV.

Genetic stability of recombinant potato virus X virus vectors presenting foreign epitopes.

We investigated the genetic stability of recombinant potato virus X vectors presenting beet necrotic yellow vein virus (BNYVV) epitopes. Following N-terminal PVX coat protein (CP) fusion of the BNYVV epitopes, we inoculated Nicotiana benthamiana plants with recombinant (r)PVX and carried out five serial passages through systemically-infected plants. RT-PCR investigation of the BNYVV epitope sequences revealed the accumulation of several point mutations and deletions, predominantly affecting positively-charged residues. A comparison of the isoelectric point (pI) values and charges of the wild type and rCPs showed that the initial high rCP pI values had changed to values closer to that of the wild-type CP.

Zygocactus virus X-based expression vectors and formation of rod-shaped virus-like particles in plants by the expressed coat proteins of Beet necrotic yellow vein virus and Soil-borne cereal mosaic virus.

Expression vectors were constructed from 35S promoter-containing full-length cDNA clones of Zygocactus virus X (ZVX). The expression of foreign genes was driven by the ZVX coat protein (cp) subgenomic promoter. It was successful only when the variable region downstream of the conserved putative promoter region GSTTAAGTT(X(12-13))GAA was retained. Most of the ZVX cp gene, except for a short 3' part, was replaced by the corresponding sequence of the related Schlumbergera virus X (SVX) and its cp subgenomic promoter to enable encapsidation of the transcribed RNA by an SVX/ZVX hybrid cp. Vector-expressed cp of Beet necrotic yellow vein virus (BNYVV) assembled in Chenopodium quinoa, Tetragonia expansa and Beta vulgaris leaves into particles resembling true BNYVV particles. The virus produced from these constructs retained its ability to express BNYVV cp in local infections during successive passages on C. quinoa. This ability was lost, however, in the rarely occurring systemic infections.

Expression of multiple foreign epitopes presented as synthetic antigens on the surface of Potato virus X particles.

We describe the construction of recombinant Potato virus X (PVX) vectors expressing two different epitopes, ep4 and ep6, from Beet necrotic yellow vein virus (BNYVV). The seven-amino-acid epitopes were expressed as N-terminal coat protein fusions and were displayed on the surface of PVX particles. Particle assembly into full virions was successful even though no wild type coat protein subunits were present, and the epitopes could be detected in crude extracts and purified virus preparations with appropriate antibodies. A construct containing both epitope sequences in tandem was also prepared. The resulting PVX particles could be detected by antibodies against ep4 and ep6, either individually or simultaneously, showing that both epitopes were accessible. In addition mixed infections with PVX vectors containing the individual ep4 and ep6 sequences were carried out. This resulted in the formation of PVX particles displaying ep4 alone, ep6 alone, or both epitopes. These experiments demonstrate for the first time that PVX can be utilized to present multiple epitopes, either tandemly on every coat protein subunit or as heteromultimeric assemblies, both of which could be useful vaccination strategies. The production of epitope-presenting viruses in which every coat protein subunit contains a foreign epitope allows the high-level expression of defined numbers of foreign antigen sites, making such viruses useful standards for immune detection.

Transgene Pflanzen als orale Impfstoffe Grüne Revolution in der Medizin?: Grüne Revolution in der Medizin?

With the methods of molecular biotechnology it is possible today to produce therapeutical effectiv proteins in plants. So called edible vaccines can be used for active immunization as well as for passive immunization. For an efficient production of recombinant proteins a procedure was developed, in which only the leaves are treated with genetically modified bacteria instead of the time consuming regeneration of transgenic plants. With an orally applicated vaccine a complete systematic immune response can be induced, but in comparison to a parenteral injection, a 100 times higher dosis of the antigen is necessary. The immunogenity of antigens produced in plants was confirmed in several animal experiments. Clinical studies with recombinant antibodies of transgenic plants against the caries pathogen Streptococcus mutans were successful. Future studies will focus on improved production rates, the search for suitable plant species and the phenomenon of oral tolerance.

Tagging potato leafroll virus with the jellyfish green fluorescent protein gene.

A full-length cDNA corresponding to the RNA genome of Potato leafroll virus (PLRV) was modified by inserting cDNA that encoded the jellyfish green fluorescent protein (GFP) into the P5 gene near its 3' end. Nicotiana benthamiana protoplasts electroporated with plasmid DNA containing this cDNA behind the 35S RNA promoter of Cauliflower mosaic virus became infected with the recombinant virus (PLRV-GFP). Up to 5% of transfected protoplasts showed GFP-specific fluorescence. Progeny virus particles were morphologically indistinguishable from those of wild-type PLRV but, unlike PLRV particles, they bound to grids coated with antibodies to GFP. Aphids fed on extracts of these protoplasts transmitted PLRV-GFP to test plants, as shown by specific fluorescence in some vascular tissue and epidermal cells and subsequent systemic infection. In plants agroinfected with PLRV-GFP cDNA in pBIN19, some cells became fluorescent and systemic infections developed. However, after either type of inoculation, fluorescence was mostly restricted to single cells and the only PLRV genome detected in systemically infected tissues lacked some or all of the inserted GFP cDNA, apparently because of naturally occurring deletions. Thus, intact PLRV-GFP was unable to move from cell to cell. Nevertheless, PLRV-GFP has novel potential for exploring the initial stages of PLRV infection.

Towards molecular farming in the future: moving from diagnostic protein and antibody production in microbes to plants.

Molecular farming of pharmaceuticals in plants has the potential to provide almost unlimited amounts of recombinant proteins for use in disease diagnosis and therapy. Transgenic plants are attracting interest as bioreactors for the inexpensive production of large amounts of safe, functional, recombinant macromolecules, such as blood substitutes, vaccines and antibodies. In some cases, the function of expressed recombinant proteins can be rapidly analysed by expression in microbes or by transient expression in intact or virally infected plants. Protein production can be increased by upscaling production in fermenters, using yeast- or plant-suspension cells or by using transient-expression systems. Stable transgenic plants can be used to produce leaves or seeds rich in the recombinant protein for long-term storage or direct processing. This demonstrates the promise for using plants as bioreactors for the molecular farming of recombinant therapeutics, diagnostics, blood substitutes and antibodies. We anticipate that this technology has the potential to greatly benefit human health by making safe recombinant pharmaceuticals widely available.

Towards molecular farming in the future: pichia pastoris-based production of single-chain antibody fragments.

This review article focuses on the use of the methylotrophic yeast Pichia pastoris as a recombinant protein-expression system. P. pastoris is a useful system for the expression of milligram-to-gram quantities of a protein, which can be scaled up to fermentation to meet greater demands. Compared with mammalian cells, Pichia do not require a complex growth medium or culture conditions, they are as easy to manipulate genetically as Escherichia coli and have a eukaryotic protein-synthesis pathway. They seem suited to laboratory-scale production of recombinant proteins for in-house use or, in some cases, molecular farming of recombinant products. This review article focuses on the use of P. pastoris, describes a fermentation production run of a single-chain antibody fragment and includes a discussion of fermentation as a production strategy.

Towards molecular farming in the future: transient protein expression in plants.

Molecular farming in plants can be achieved by stable or transient expression of a recombinant protein. Transient expression of recombinant proteins in plants can rapidly provide large amounts of the proteins for detailed characterization. It is fast, flexible and can be carried out at field scale using viral vectors, but it lacks the increases in production volume that can be achieved easily with stable transgenic crops. This review article focuses on discussing the applications of transient expression using viral vectors, biolistic methods or agroinfiltration.

Genome properties of beet virus Q, a new furo-like virus from sugarbeet, determined from unpurified virus.

Based solely on the information that beet virus Q (BVQ) contains tubular particles, the entire nucleotide sequence of its tripartite genome was determined from unpurified virus in ca. 40 ml crude sap from locally infected Chenopodium quinoa. A starting sequence for RNA 1 was generated using primers corresponding to highly conserved helicase domains in the respective RNAs of furo-, pomo-, peclu-, hordei- and tobraviruses, and was extended by a walking random-primed cDNA approach. The similarity of the 3' ends of furoviral RNAs allowed starting sequences for BVQ RNAs 2 and 3 to be obtained once the 3' end of RNA 1 was known. BVQ RNA 1 encodes a protein with a methyltransferase-like, a variable and a helicase-like region, and for a readthrough protein which, in addition, contains an RNA-dependent RNA polymerase region. RNA 2 carries the coat protein gene, a coat protein read-through protein gene and two additional ORFs which may have arisen by deletions from an originally larger readthrough domain. RNA 3 carries a triple gene block resembling that of several other rod-shaped viruses. The 5' UTRs of the three RNAs have the potential to form a series of hairpins with C-A and C-C mismatches resembling those found in tymoviral RNAs. The 3' ends can be folded into tRNA-like structures which are preceded by a long hairpin-like structure and an upstream pseudoknot domain. BVQ belongs to the recently proposed genus Pomovirus; it shows evolutionary relationships to furoviruses in sensu stricto, peclu-, hordei-, tobra-, tymo-, tobamo-, carla- and potexviruses.

Beet soil-borne virus RNA 2: similarities and dissimilarities to the coat protein gene-carrying RNAs of other furoviruses.

The complete sequence of the 3454 nt of RNA 2 of the Ahlum isolate of beet soil-borne furovirus (BSBV) has been determined starting with two short stretches of cloned cDNA. Unknown parts of the sequence were amplified by means of RT-PCR techniques using combinations of specific and random primers. BSBV RNA 2 is more similar in its genetic organization to potato mop top virus (PMTV) RNA 3 than to any other furoviral RNA, although it is more than 1100 nt longer. Its 3'-end, unlike that of PMTV RNA 3, has the potential to fold into a tRNA-like structure. It contains one large open reading frame for a readthrough protein with a molecular mass of 104 kDa (104K protein) which is interrupted internally by an amber stop codon terminating the coding region for a protein of 19 kDa (19K), most likely the viral coat protein (CP). The readthrough domain of the 104K protein is much larger than that of PMTV, but the N- and C-proximal portions of these domains are similar for the two viruses. No serological relationships were found between the particles of the two viruses, although more than 50% of the amino acid sequences of the putative CPs are identical.

Detection and characterization of a distinct type of beet necrotic yellow vein virus RNA 5 in a sugarbeet growing area in Europe.

A fifth beet necrotic yellow vein virus (BNYVV) RNA species has been detected in Europe in sugarbeet infected with P-type BNYVV. Very little sequence variation was found between two European sources of this RNA 5*, but considerable differences were detected between these two European sources on the one hand and the four Japanese sources recently analysed by Kiguchi et al. on the other. The BNYVV RNA 5-encoded 26 K proteins share a stretch of six amino acids (FRGPGN) with the BNYVV RNA 3-encoded 25 K protein which may be of interest in view of the reported interactions between the two RNAs in pathogenicity.

Detection and Characterization of a Distinct Type of Beet Necrotic Yellow Vein Virus RNA 5 in a Sugarbeet Growing Area in Europe.

A fifth beet necrotic yellow vein virus (BNYVV) RNA species has been detected in Europe in sugarbeet infected with P-type BNYVV. Very little sequence variation was found between two European sources of this RNA 5*, but considerable differences were detected between these two European sources on the one hand and the four Japanese sources recently analysed by Kiguchi et al. on the other. The BNYVV RNA 5-encoded 26K proteins share a stretch of six amino acids (FRGPGN) with the BNYVV RNA 3-encoded 25K protein which may be of interest in view of the reported interactions between the two RNAs in pathogenicity.

Expression of single-chain antibody fragments (scFv) specific for beet necrotic yellow vein virus coat protein or 25 kDa protein in Escherichia coli and Nicotiana benthamiana.

The coding sequences for the variable regions of heavy and light chains of monoclonal antibodies (mAbs) to beet necrotic yellow vein virus (BNYVV) coat protein (cp) or the 25 kDa nonstructural protein (P25) were cloned into the pCOCK vector and expressed as single-chain antibody fragments (scFv) in Escherichia coli. For expression in higher plants the scFv were targeted either to the secretory pathway by including the sequences encoding the pectate lyase B (PelB) or the phytohemagglutinin (PHA) signal peptides in the vector constructs or they were targeted to the cytoplasm by omitting a signal peptide-encoding sequence from the constructs. The scFv were detected mainly in plants in which the PHA signal peptide had been used for targeting demonstrating for the first time the usefulness of this peptide for enabling scFv expression in plants. The scFv were not secreted into the culture fluids of suspension cultures, but were retained in the cells. The amount of expression of scFv in the best expressing plants was at least as high as in bacterial culture supernatants. In a dot blot immunoassay, 0.4 ng BNYVV cp or 0.8 ng P25 were detected by the respective scFv either from E. coli or from plants. The majority of the 21 plants expressing cp-specific scFv had near-normal growth whereas the three plants expressing P25-specific scFv grew poorly and did not form roots.

Beet soil-borne virus RNA 3--a further example of the heterogeneity of the gene content of furovirus genomes and of triple gene block-carrying RNAs.

The complete nucleotide sequence of RNA 3 of the Ahlum isolate of beet soil-borne virus (BSBV) was determined from cDNAs obtained with immunocaptured virus particles and denatured preparations of dsRNA. BSBV RNA 3 is unique among the plant virus RNAs studied so far in containing apparently only the coding sequences of a triple gene block (TGB). The derived amino acid sequences of the three putative TGB-encoded proteins showed the highest level of sequence similarities with those of the corresponding proteins of potato mop top furovirus (PMTV) followed by those of peanut clump furovirus and barley stripe mosaic hordeivirus. Progressively fewer similarities were found with the TGB-encoded proteins of beet necrotic yellow vein virus (uncertain classification), potato X potexvirus, and potato M carlavirus. The 3'-terminal 78 nucleotides of BSBV RNA 3 can be folded into a tRNA-like structure and a high degree of sequence similarity exists between the 122 3'-terminal nucleotides of BSBV RNA 3 and PMTV RNA 2. In other regions, however, no pronounced sequence similarities were found between the two RNAs, and PMTV RNA 2 contains an additional putative gene for a cysteine-rich protein downstream of the TGB. The two viruses are unrelated serologically. BSBV RNA 3 adds a further variant to the heterogeneity of the gene content of furovirus genomes and of triple gene block-carrying RNAs.

Restriction fragment length polymorphism analysis of reverse transcription-PCR products reveals the existence of two major strain groups of beet necrotic yellow vein virus.

Beet necrotic yellow vein virus (BNYVV)-infected sugarbeets were obtained from many parts of Europe and also from some sites in Asia and the U.S.A. Reverse transcription (RT)-PCR products of more than 1 kbp were obtained for four different regions of the viral genome which may be particularly important with respect to the pathogenic properties of the virus, i.e. for the coat protein and the 42K protein-encoding regions on RNA 2 and for major parts of RNAs 3 and 4. Restriction fragment length polymorphism (RFLP) patterns obtained with these PCR products revealed the existence of two major strain groups of BNYVV, named type A and type B. The A type was detected in Greece, the former Yugoslavia, Slovakia, parts of Austria, Italy, Spain, parts of France, Belgium, The Netherlands and England as well as in Asia (Turkey, Kazachstan, China and Japan) and the U.S.A. The B type occurs in Germany and parts of France. Mixed infections were detected at the borderline regions between areas of the A and B types. Comparisons of published and newly determined nucleotide sequences of the respective parts of the BNYVV genome indicate that the percentage of nucleotide differences between the A and the B type is approximately 3% for the respective regions of RNAs 2 and 3 and approximately 1.5% for RNA 4. Nucleotide sequences appear to be remarkably stable within each of the two strain groups. The majority of the nucleotide differences between the A and B types occur in the third triplet position. The amino acid changes in the coat protein area are outside the four previously determined antigenic regions that are accessible on the surface of the virus particles and are involved in the formation of continuous and presumably also discontinuous epitopes. This may explain why serological differences between the two strain groups have not been found.

Location, size, and complexity of epitopes on the coat protein of beet necrotic yellow vein virus studied by means of synthetic overlapping peptides.

Five regions on the coat protein of BNYVV which had been shown previously to be involved in the formation of continuous epitopes were further analyzed by means of synthetic overlapping peptides. It was found that at least some of these regions may encompass several overlapping epitopes (or parts thereof). Four monoclonal antibodies (MAbs) which were known to be specific for the C-terminus of BNYVV coat protein (amino acids 182-188 = RTSPPGQ) were found to react with different sets of peptides which had either the sequence RTS, RTSP, RTSPP, or PPGQ in common. Two other MAbs which also had been shown previously to be specific for the C-terminus of BNYVV coat protein failed to react with overlapping decapeptides. Two epitopes which were previously located in the areas of amino acids 115-125 and 125-140 could now be located more precisely on the sequences SANVRRD (amino acids 115-121) and AESSG (amino acids 128-132), respectively. Replacement studies with alanine showed that not all amino acids within these sequences are equally important for antibody binding. On the other hand, amino acids outside these sequences may strongly influence the reactivity of epitopes. The accessibility of amino acid sequences on the particles of BNYVV is discussed.

Nucleotide sequence of the coat protein gene of pelargonium leaf curl virus and comparison of the deduced coat protein amino acid sequence with those of other tombusviruses.

The sequence of 1,787 nucleotides (nts) in the genomic RNA of pelargonium leaf curl virus (PLCV) was determined. It included the entire coat protein (cp) gene (nts 585 to 1,754), 558 nts of the 3' end of the putative RNA polymerase gene, 26 nts of an intercistronic region between the two genes and 33 nts downstream of the stop codon of the cp gene. The cp gene was cloned into the expression vector pET8c and expressed in E. coli. The deduced cp amino acid sequence of PLCV was compared with those of five other tombusviruses. The closer the degree of serological relatedness between two viruses, the more similarity was found in their cp amino acid sequences not only in the protruding domains, but also in their random and shell domains and in the arm regions. Nucleic acid hybridization tests, cp amino acid comparisons and serological tests all suggest the same order of sequence for the relationships in the tombusvirus group.

Epitope mapping on fragments of beet necrotic yellow vein virus coat protein.

The location of five SDS-stable epitopes on the coat protein (CP) of beet necrotic yellow vein virus was determined by reacting Escherichia coli-expressed free CP, as well as fusion proteins (FP) containing fragments of the CP, with polyclonal and monoclonal antibodies on Western blots. Epitope 1, which has previously been found to be exposed on only one extremity of the virus particle, was located in the region between amino acids (aa) 1 and 7, i.e. on the N terminus of the CP. It was blocked when the N terminus of the CP was linked to a portion of the beta-galactosidase sequence in an FP. Epitope 3, which has previously been found to be exposed on the opposite extremity of the particle, was located in the region between aa 37 and 59. Epitope 4, which is exposed along the entire length of the particle, occurs on the C terminus of CP (aa 183 to 188). Two previously unknown epitopes were identified in the regions between aa 115 and 125 and 125 and 140, respectively. The former was located on the same extremity of the particle as epitope 3, the latter became accessible only after denaturation of the particle. Nothing is known about the probably non-adjacent aa sequences that participate in the formation of the two SDS-labile epitopes (epitopes 2 and 5) which are found on one extremity and along the entire length of the particle, respectively.