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Many virus capsids undergo exquisitely choreographed maturation processes in their host cells to produce infectious virions, and these remain poorly understood. As a tool for studying virus maturation, we transiently expressed the capsid protein of the insect virus Nudaurelia capensis omega virus (NωV) in Nicotiana benthamiana and were able to purify both immature procapsids and mature capsids from infiltrated leaves by varying the expression time. Cryo-EM analysis of the plant-produced procapsids and mature capsids to 6.6 Å and 2.7 Å resolution, respectively, reveals that in addition to large scale rigid body motions, internal regions of the subunits are extensively remodelled during maturation, creating the active site required for autocatalytic cleavage and infectivity. The mature particles are biologically active in terms of their ability to lyse membranes and have a structure that is essentially identical to authentic virus. The ability to faithfully recapitulate and visualize a complex maturation process in plants, including the autocatalytic cleavage of the capsid protein, has revealed a ~30 Å translation-rotation of the subunits during maturation as well as conformational rearrangements in the N and C-terminal helical regions of each subunit.

Poliovirus (PV) is the causative agent of poliomyelitis, a crippling human disease known since antiquity. PV occurs in two distinct antigenic forms, D and C, of which only the D form elicits a robust neutralizing response. Developing a synthetically produced stabilized virus-like particle (sVLP)-based vaccine with D antigenicity, without the drawbacks of current vaccines, will be a major step towards the final eradication of poliovirus. Such a sVLP would retain the native antigenic conformation and the repetitive structure of the original virus particle, but lack infectious genomic material. In this study, we report the production of synthetically stabilized PV VLPs in plants. Mice carrying the gene for the human PV receptor are protected from wild-type PV when immunized with the plant-made PV sVLPs. Structural analysis of the stabilized mutant at 3.6 Å resolution by cryo-electron microscopy and single-particle reconstruction reveals a structure almost indistinguishable from wild-type PV3.Despite the success of current vaccination against poliomyelitis, safe, cheap and effective vaccines remain sought for continuing eradication effort. Here the authors use plants to express stabilized virus-like particles of type 3 poliovirus that can induce a protective immune response in mice transgenic for the human poliovirus receptor.

Background: Hepatitis E virus (HEV) causes epidemics in developing countries and is primarily transmitted through the fecal-oral route. There have been recent reports on the zoonotic spread of the virus, and several animal species, primarily pigs, have been recognized as reservoirs of HEV. Because of its possible spread, there is an urgent need of a method for the cost-effective production of HEV proteins that can be used as diagnostic antigens for the serological detection of anti-HEV antibodies., Methods: The HEV open reading frame (ORF)2 protein was purified from plant tissue by using immobilized metal-anion chromatography (IMAC). The recombinant protein was used to develop an in-house ELISA for testing anti-HEV antibodies in both human and swine sera. Thirty-six serum samples collected from patients with serologically proven HEV infection with commercial kits were tested for anti-HEV IgG antibodies by using the plant-expressed protein. Forty-five serum samples collected from apparently healthy pigs in Bulgarian farms were also tested., Results: We confirmed the transient expression and purification of a truncated version of the HEV genotype 3 capsid protein in Nicotiana benthamiana and its usefulness as a diagnostic antigen. ELISA showed the presence of anti-HEV IgG antibodies in 29 of the 36 human samples. The in-house ELISA showed anti-HEV IgG antibodies in 34 of the 45 pigs., Conclusions: We describe a method for the production of HEV ORF2 protein in N. benthamiana and the usefulness of this protein for the serological detection of anti-HEV antibodies in both humans and swine., Competing Interests: George P. Lomonossoff declares that he is a named inventor on granted patent WO 29087391 A1, which describes the plant transient expression system used for the work described in this manuscript., (© The Korean Society for Laboratory Medicine)

Understanding capsid assembly following recombinant expression of viral structural proteins is critical to the design and modification of virus-like nanoparticles for biomedical and nanotechnology applications. Here, we use plant-based transient expression of the Bluetongue virus (BTV) structural proteins, VP3 and VP7, to obtain high yields of empty and green fluorescent protein (GFP)-encapsidating core-like particles (CLPs) from leaves. Single-particle cryo-electron microscopy of both types of particles revealed considerable differences in CLP structure compared to the crystal structure of infection-derived CLPs; in contrast, the two recombinant CLPs have an identical external structure. Using this insight, we exploited the unencumbered pore at the 5-fold axis of symmetry and the absence of encapsidated RNA to label the interior of empty CLPs with a fluorescent bioconjugate. CLPs containing 120 GFP molecules and those containing approximately 150 dye molecules were both shown to bind human integrin via a naturally occurring Arg-Gly-Asp motif found on an exposed loop of the VP7 trimeric spike. Furthermore, fluorescently labeled CLPs were shown to interact with a cell line overexpressing the surface receptor. Thus, BTV CLPs present themselves as a useful tool in targeted cargo delivery. These results highlight the importance of detailed structural analysis of VNPs in validating their molecular organization and the value of such analyses in aiding their design and further modification.

The ectodomain of the conserved influenza matrix protein M2 (M2e) is a promising target for the development of a universal influenza vaccines. Immunogenicity of M2e could be enhanced by its fusion to bacterial flagellin, the ligand for Toll-like receptor 5. Previously we reported the transient expression in plants of a recombinant protein Flg-4M comprising flagellin fused to 4 tandem copies of the M2e. The use of self-replicating recombinant vector based on the potato virus X allowed expression of Flg-4M in Nicotiana benthaminana leaves at a very high level, up to about 1 mg/g of fresh leaf tissue. Intranasal immunization of mice with Flg-4M induced M2e-specific serum antibodies and provided protection against lethal challenge with different strains of influenza A virus. Here we show that immunization with Flg-4M not only generates a strong immune response, but also redirects the response from the carrier flagellin toward the M2e epitopes. Significant IgG response to M2e was also developed in bronchoalveolar lavages of immunized mice. Protective activity of Flg-4M upon lethal influenza challenge correlated with a decrease of virus titers in lungs relative to the control. Overall these data show the potential for the development of a plant-produced M2e-flagellin universal influenza vaccine.

This chapter constitutes a practical guide to using the "pEAQ" vector series for transient or stable expression of one or more protein(s) in Nicotiana benthamiana plants. The pEAQ vectors are a series of small binary vectors designed for controlled expression of multiple proteins in plants. To achieve high levels of expression, an expression system based on translational enhancement by the untranslated regions of RNA-2 from cowpea mosaic virus (CPMV), named CPMV-HT, is used. The expression vector pEAQ-HT combines the user-friendly pEAQ plasmid with CPMV-HT to provide a system for high-level expression of proteins in plants.

The core protein of the hepatitis B virus, HBcAg, assembles into highly immunogenic virus-like particles (HBc VLPs) when expressed in a variety of heterologous systems. Specifically, the major insertion region (MIR) on the HBcAg protein allows the insertion of foreign sequences, which are then exposed on the tips of surface spike structures on the outside of the assembled particle. Here, we present a novel strategy which aids the display of whole proteins on the surface of HBc particles. This strategy, named tandem core, is based on the production of the HBcAg dimer as a single polypeptide chain by tandem fusion of two HBcAg open reading frames. This allows the insertion of large heterologous sequences in only one of the two MIRs in each spike, without compromising VLP formation. We present the use of tandem core technology in both plant and bacterial expression systems. The results show that tandem core particles can be produced with unmodified MIRs, or with one MIR in each tandem dimer modified to contain the entire sequence of GFP or of a camelid nanobody. Both inserted proteins are correctly folded and the nanobody fused to the surface of the tandem core particle (which we name tandibody) retains the ability to bind to its cognate antigen. This technology paves the way for the display of natively folded proteins on the surface of HBc particles either through direct fusion or through non-covalent attachment via a nanobody.

Plant expression systems based on nonreplicating virus-based vectors can be used for the simultaneous expression of multiple genes within the same cell. They therefore have great potential for the production of heteromultimeric protein complexes. This work describes the efficient plant-based production and assembly of Bluetongue virus-like particles (VLPs), requiring the simultaneous expression of four distinct proteins in varying amounts. Such particles have the potential to serve as a safe and effective vaccine against Bluetongue virus (BTV), which causes high mortality rates in ruminants and thus has a severe effect on the livestock trade. Here, VLPs produced and assembled in Nicotiana benthamiana using the cowpea mosaic virus-based HyperTrans (CPMV-HT) and associated pEAQ plant transient expression vector system were shown to elicit a strong antibody response in sheep. Furthermore, they provided protective immunity against a challenge with a South African BTV-8 field isolate. The results show that transient expression can be used to produce immunologically relevant complex heteromultimeric structures in plants in a matter of days. The results have implications beyond the realm of veterinary vaccines and could be applied to the production of VLPs for human use or the coexpression of multiple enzymes for the manipulation of metabolic pathways., (© 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.)

Bovine papillomavirus type 1 (BPV-1) is an economically important virus that induces tumourigenic pathologies in horses and cows. Given that the BPV-1 L1 major coat protein can self-assemble into highly immunogenic higher-order structures, we transiently expressed it in Nicotiana benthamiana as a prelude to producing a candidate vaccine. It was found that plant codon optimization of L1 gave higher levels of expression than its non-optimized counterpart. Following protein extraction, we obtained high yields (183 mg/kg fresh weight leaf tissue) of relatively pure L1, which had self-assembled into virus-like particles (VLPs). We found that these VLPs elicited a highly specific and strong immune response, and therefore they may have utility as a potential vaccine. This is the first report demonstrating the viable production of a candidate BPV vaccine protein in plants.

Recombinant human gastric lipase (hGL) was transiently expressed in Nicotiana benthamiana leaves using the CPMV-HT expression system. Expression levels of up to 0.5mg recombinant hGL per gram of infiltrated leaf tissue were obtained. Proteins expressed from two hGL constructs, wild type (wt-hGL) and with a Histidine tag at its C terminal (hGL-His), were purified from leaf tissue using Immobilized Lectin Affinity chromatography and Immobilized Metal Affinity chromatography. Both variants were glycosylated, enzymatically active, and had an apparent molecular weight similar to native hGL (approx. 50kDa). The recombinant hGLs were stable under acidic conditions and in the presence of gastric pepsin. Moreover, as found with the naturally occurring enzyme, the activity of recombinant hGL on the short chain lipid, tributyrin, was higher than on long chain Intralipid 30% emulsion. The maximum specific activity measured on tributyrin was 310 U/mg of protein and the maximum yield was 193 U/g of infiltrated leaf tissue. These results show that transient expression in plants can be used to produce active hGL that could be efficiently purified using established techniques. The approach provides a means of generating large quantities of hGL that could be of use for a number of applications both in vitro and in vivo., (Copyright © 2011 Elsevier Inc. All rights reserved.)

A series of vectors (the pEAQ series) based on cowpea mosaic virus has been developed which allows the rapid transient expression of high levels of foreign protein in plants without the need for viral replication. The plasmids are small binary vectors, which are introduced into plant leaves by agroinfiltration. They are modular in design and allow the insertion of multiple coding sequences on the same segment of T-DNA. These properties make the pEAQ vectors particularly suitable for use in situations, such as the investigation and manipulation of metabolic pathways, where the coexpression of multiple proteins within a cell is required., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Most human serum albumin (HSA) for medical applications is derived from human plasma due to the lack of suitable heterologous expression systems for recombinant HSA (rHSA). To determine whether plant cell cultures could provide an alternative source, we employed the hyper-translatable cowpea mosaic virus protein expression system (CPMV-HT) to stably express rHSA in tobacco Bright Yellow-2 (BY-2) cells. rHSA was stably produced with yield up to 11.88μg/ml in the culture medium, accounting for 0.7% of total soluble protein, in a 25-ml flask. Cultivation of transgenic cells in modified Murashige and Skoog medium with a pH of 8.0 improved the yield of rHSA two-fold, which may be the result of reduced proteolytic activity in the modified medium. A simple purification scheme was developed to purify the rHSA from culture medium, resulting in a recovery of 48.41% of the secreted rHSA. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and N-terminal sequence analysis of the purified rHSA revealed that plant cell-derived rHSA is identical to that of the plasma-derived HSA. Our results show that the CPMV-HT system, which was originally developed as a transient expression system for use in whole plants, can also be used for high-level expression of rHSA, a protein highly susceptible to proteolysis, in transgenic tobacco cells., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Endeavours to obtain elevated and prolonged levels of foreign gene expression in plants are often hampered by the onset of RNA silencing that negatively affects target gene expression. Plant virus-encoded suppressors of RNA silencing are useful tools for counteracting silencing but their wide applicability in transgenic plants is limited because their expression often causes harmful developmental effects. We hypothesized that a previously characterized tombusvirus P19 mutant (P19/R43W), typified by reduced symptomatic effects while maintaining the ability to sequester short-interfering RNAs, could be used to suppress virus-induced RNA silencing without the concomitant developmental effects. To investigate this, transient expression in Nicotiana benthamiana was used to evaluate the ability of P19/R43W to enhance heterologous gene expression. Although less potent than wt-P19, P19/R43W was an effective suppressor when used to enhance protein expression from either a traditional T-DNA expression cassette or using the CPMV-HT expression system. Stable transformation of N. benthamiana yielded plants that expressed detectable levels of P19/R43W that was functional as a suppressor. Transgenic co-expression of green fluorescent protein (GFP) and P19/R43W also showed elevated accumulation of GFP compared with the levels found in the absence of a suppressor. In all cases, transgenic expression of P19/R43W caused no or minimal morphological defects and plants produced normal-looking flowers and fertile seed. We conclude that the expression of P19/R43W is developmentally harmless to plants while providing a suitable platform for transient or transgenic overexpression of value-added genes in plants with reduced hindrance by RNA silencing., (Plant Biotechnology Journal © 2010 Society for Experimental Biology and Blackwell Publishing Ltd. No claim to original US government works.)

Background: The capacity of plants and plant cells to produce large amounts of recombinant protein has been well established. Due to advantages in terms of speed and yield, attention has recently turned towards the use of transient expression systems, including viral vectors, to produce proteins of pharmaceutical interest in plants. However, the effects of such high level expression from viral vectors and concomitant effects on host cells may affect the quality of the recombinant product., Methodology/principal Findings: To assess the quality of antibodies transiently expressed to high levels in plants, we have expressed and characterised the human anti-HIV monoclonal antibody, 2G12, using both replicating and non-replicating systems based on deleted versions of Cowpea mosaic virus (CPMV) RNA-2. The highest yield (approximately 100 mg/kg wet weight leaf tissue) of affinity purified 2G12 was obtained when the non-replicating CPMV-HT system was used and the antibody was retained in the endoplasmic reticulum (ER). Glycan analysis by mass-spectrometry showed that the glycosylation pattern was determined exclusively by whether the antibody was retained in the ER and did not depend on whether a replicating or non-replicating system was used. Characterisation of the binding and neutralisation properties of all the purified 2G12 variants from plants showed that these were generally similar to those of the Chinese hamster ovary (CHO) cell-produced 2G12., Conclusions: Overall, the results demonstrate that replicating and non-replicating CPMV-based vectors are able to direct the production of a recombinant IgG similar in activity to the CHO-produced control. Thus, a complex recombinant protein was produced with no apparent effect on its biochemical properties using either high-level expression or viral replication. The speed with which a recombinant pharmaceutical with excellent biochemical characteristics can be produced transiently in plants makes CPMV-based expression vectors an attractive option for biopharmaceutical development and production.

The core antigen of hepatitis B virus (HBcAg) has attracted considerable attention as a carrier for antigenic sequences for various diagnostic and vaccine applications. The hepatitis B core protein has been expressed in different expression systems. At present, for reasons of cost, scale, and safety, the plant-based expression systems are attracting increasing interest. The expression and assembly for the hepatitis B core protein were investigating in N. benthamiana plants using the new expression system based on deleted version of cowpea mosaic virus RNA-2. Analysis of HBcAg expression revealed that the core protein expressed in plants and could self-assemble into virus-like particles. Virus-like particles could be purified by differential and sucrose gradient centrifugation. This expression system has the advantage of biocontainment and can be used for the rapid production of HBcAg virus-like particles for immunological and vaccine applications.

The use of multiple copies of vectors based on either full-length or deleted versions of cowpea mosaic virus RNA-2 for the production of heteromeric proteins in plants was investigated. Co-infiltration of two full-length RNA-2 constructs containing different marker genes into Nicotiana benthamiana in the presence of RNA-1 showed that the two foreign proteins were efficiently expressed within the same cell in inoculated tissue. Furthermore, the proteins were co-localized to the same subcellular compartments, an essential prerequisite for heteromer formation. However, segregation of two separate RNA-2 molecules, and therefore expression of the two proteins, was observed on systemic spread of the recombinant viruses. Thus, efficient assembly of heteromeric proteins is likely to occur only in inoculated tissue. To determine the optimum approach for expression in inoculated tissue, the heavy and light chains of the blood group-typing immunoglobulin G (IgG) C5-1 were inserted into full-length and deleted versions of RNA-2, and the constructs were agroinfiltrated in the presence of RNA-1. The results obtained showed that full-size IgG molecules accumulated using both approaches, but that the levels were significantly higher when deleted RNA-2 vectors were used. The levels were also greatly enhanced by the inclusion of an endoplasmic reticulum retention signal at the C-terminus of the heavy chain. As the potential benefit of using full-length RNA-2 constructs, the ability to spread systemically, appears to be irrelevant to the production of heteromeric proteins, the use of deleted versions of RNA-2 is clearly advantageous, particularly as they offer the benefit of biocontainment.

We have developed combined transgene/virus vector systems for the expression of heterologous proteins in plants. The systems are based on the bipartite RNA plant virus, cowpea mosaic virus (CPMV), and involve the amplification of integrated copies of either full-length or deleted versions of RNA-2 carrying a foreign gene. In the case of plants transgenic for full-length versions of RNA-2 carrying the green fluorescent protein (GFP), amplification can be achieved by supplying RNA-1 either exogenously or by crossing. This allows either inducible or constitutive expression of the foreign gene and results in an infection that can be passaged to further plants. Replication of deleted versions of RNA-2 harbouring GFP requires the presence of both RNA-1 and a suppressor of gene silencing, a function which we show can be supplied by HcPro from potato virus Y. Replication of the deleted versions of RNA-2 can be achieved by supplying the suppressor and RNA-1 either exogenously or by crossing, showing that this system can also be used in an inducible and constitutive format. The use of deleted forms of RNA-2 has the advantage that no infectious virus is produced, providing an effective method of biocontainment. The CPMV-based systems have advantages over existing plant expression systems in terms of the expression levels obtainable and the simplicity and flexibility of use, and should be of great practical benefit in the development of plants as bioreactors.

The small (S) coat protein of Cowpea mosaic virus (CPMV) has been identified previously as a virus-encoded suppressor of post-transcriptional gene silencing (PTGS). Deletions within the C-terminal 24 aa of this protein affect the yield and systemic spread of the virus, suggesting that the C-terminal amino acids of the S protein, which are exposed on the surface of assembled virus particles, may be responsible for the suppressor activity. To investigate this, versions of CPMV RNA-2 with deletions at the C terminus of the S protein were tested for their ability to counteract PTGS in leaf-patch tests. The results showed that the C-terminal 16 aa of the S protein are particularly important for suppressing PTGS and that these amino acids are virus-specific and cannot be substituted by the equivalent sequence from the related virus Bean pod mottle virus.

Virus resistance in Nicotiana benthamiana plants containing a translatable Pea early browning virus (PEBV) 54K sequence from the 201K replicase gene has been reported previously. Resistant plants contain multiple transgene copies divided between two loci. Analysis of a genetic series containing the two loci in separate homozygous or heterozygous condition suggest that only one of the loci is necessary to induce the resistance. The resistance observed in R2 and R3 generations of lines containing both transgene loci in homozygous condition became less consistent in R4 and R5 generations. This inversely correlated with steady-state transgene transcript levels of the segregating populations. The use of recombinant Potato virus X vectors carrying PEBV 54K sequences showed that the resistance is based upon posttranscriptional gene silencing, is non-strand specific, and recognizes 3' located sequences within the PEBV 54K sequence.

The subcellular locations of two potyviral proteins, the coat (CP) and nonstructural cylindrical inclusion (CI) proteins of tobacco vein mottling virus (TVMV), during early stages in the development of systemic infections in plants, have been investigated. Ultrathin sections of newly emerged leaves in infected plants were treated with antibodies specific to these proteins and then with gold-labeled secondary antibodies and examined by electron microscopy. CI was detected near plasmodesmatal connections between mesophyll cells prior to the appearance of CP or any virus-induced features or effects. Further accumulation of CI was evident in the form of conical structures, many of which appeared to penetrate the cell wall and to be connected to cones in neighboring cells. Prior to its appearance in other parts of the cells, the viral CP was detected, often in linear arrays, near the vertices or inside the cones and in plasmodesmata. In situ hybridization analysis of similar tissue sections with a TVMV RNA-specific oligoribonucleotide probe revealed the presence of the viral RNA in plasmodesmata. These results lend support to the notion that the formation of specific structures by potyviral CI proteins is required for and plays a direct role in the intercellular passage of viral genetic material, in the form of virus particles or complexes containing viral CP and RNA, in infected plants.

Tobacco, Nicotiana tabacum L., supports cowpea mosaic virus (CPMV) replication and cell-to-cell movement, and thus may serve as a model system to study coat protein-mediated protection against CPMV. A chimeric gene consisting of the cauliflower mosaic virus 35S promoter, CPMV 60K coat proteins-precursor (CP-P) coding region, and the nopaline synthase polyadenylation signal was transferred to tobacco cv. Burley 21 via the Agrobacterium tumefaciens binary vector system. Gene integration and expression in the transgenic tobacco plants were confirmed by Southern and RNA dot blot analyses. Accumulation of CPMV 60K CP-P in transgenic plants, up to 2 micrograms/g of wet weight tissue, was detected by ELISA and Western blots. The results of Western blots and immunosorbent electron microscopy further indicated that CPMV CP-P neither undergoes autoproteolysis to generate the mature viral coat proteins nor assembles into virus-like capsids, suggesting that processing of the CP-P may be required for virus assembly. Because CPMV neither induces symptoms in tobacco nor moves systemically, evaluation of the reactions of the transgenic plants to virus inoculation was based on virus accumulation in the inoculated leaves. Results from such infectivity experiments did not differentiate between CP-P expressers and vector-transformed plants. The transgenic tobacco plants expressing CP-P should provide valuable material for investigating comovirus polyprotein processing and capsid assembly in vivo.

Nicotiana tabacum cv. Xanthi nn plants were transformed with nucleotides 3472-4916 of tobacco mosaic virus (TMV) strain U1. This sequence contains all but the three 3 terminal nucleotides of the TMV 54-kDa gene, which encodes a putative component of the replicase complex. These plants were resistant to infection when challenged with either TMV U1 virions or TMV U1 RNA at concentrations of up to 500 micrograms/ml or 300 micrograms/ml, respectively, the highest concentrations tested. Resistance was also exhibited when plants were inoculated at 100 micrograms/ml with the closely related TMV mutant YSI/1 but was not shown in plants challenged at the same concentrations with the more distantly related TMV strains U2 or L or cucumber mosaic virus. Although the copy number of the 54-kDa gene sequence varied in individual transformants from 1 to approximately 5, the level of resistance in plants was not dependent on the number of copies of the 54-kDa gene sequence integrated. The transformed plants accumulated a 54-kDa gene sequence-specific RNA transcript of the expected size, but no protein product was detected.