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Monoclonal antibodies have revolutionized therapies, but non-immunoglobulin scaffolds are becoming compelling alternatives owing to their adaptability. Their ability to be labeled with imaging or cytotoxic compounds and to create multimeric proteins is an attractive strategy for therapeutics. Focusing on HER2, a frequently overexpressed receptor in breast cancer, this study addresses some limitations of conventional targeting moieties by harnessing the potential of these scaffolds. HER2-binding Affimers were isolated and characterized, demonstrating potency as binding reagents and efficient internalization by HER2-overexpressing cells. Affimers conjugated with cytotoxic agent achieved dose-dependent reductions in cell viability within HER2-overexpressing cell lines. Bispecific Affimers, targeting HER2 and virus-like particles, facilitated efficient internalization of virus-like particles carrying enhanced green fluorescent protein (eGFP)-encoding RNA, leading to protein expression. Anti-HER2 affibody or designed ankyrin repeat protein (DARPin) fusion constructs with the anti-VLP Affimer further underscore the adaptability of this approach. This study demonstrates the versatility of scaffolds for precise delivery of cargos into cells, advancing biotechnology and therapeutic research., Competing Interests: The authors declare a potential conflict of interest. A patent application has been filed related to the work described in this manuscript. [Application number: LU505126, Inventors: George P. Lomonossoff, Sachin Shah, Keith Saunders, Darren C Tomlinson, Christian Tiede, Heather Martin, Ross Overman]. D.C.T. also is a named inventor on patent “scaffold protein derived from plant cystatins” Europe 14705565.1, US 14/768,032, Australia 2014217628. G.P.L. also sits as a chair on the advisory board for Leaf Expression Systems., (© 2024 The Authors. Published by Elsevier Inc.)

Plants are increasingly used for the production of high-quality biological molecules for use as pharmaceuticals and biomaterials in industry. Plants have proved that they can produce life-saving therapeutic proteins (Elelyso™-Gaucher's disease treatment, ZMapp™-anti-Ebola monoclonal antibodies, seasonal flu vaccine, Covifenz™-SARS-CoV-2 virus-like particle vaccine); however, some of these therapeutic proteins are difficult to bring to market, which leads to serious difficulties for the manufacturing companies. The closure of one of the leading companies in the sector (the Canadian biotech company Medicago Inc., producer of Covifenz) as a result of the withdrawal of investments from the parent company has led to the serious question: What is hindering the exploitation of plant-made biologics to improve health outcomes? Exploring the vast potential of plants as biological factories, this review provides an updated perspective on plant-derived biologics (PDB). A key focus is placed on the advancements in plant-based expression systems and highlighting cutting-edge technologies that streamline the production of complex protein-based biologics. The versatility of plant-derived biologics across diverse fields, such as human and animal health, industry, and agriculture, is emphasized. This review also meticulously examines regulatory considerations specific to plant-derived biologics, shedding light on the disparities faced compared to biologics produced in other systems.

The recent COVID-19 outbreak highlighted the need for lab-on-chip diagnostic technology fit for real-life deployment in the field. Existing bottlenecks in multistep analytical microsystem integration and upscalable, standardized fabrication techniques delayed the large-scale deployment of lab-on-chip solutions during the outbreak, throughout a global diagnostic test shortage. This study presents a technology that has the potential to address these issues by redeploying and repurposing the ubiquitous printed circuit board (PCB) technology and manufacturing infrastructure. We demonstrate the first commercially manufactured, miniaturised lab-on-PCB device for loop-mediated isothermal amplification (LAMP) genetic detection of SARS-CoV-2. The system incorporates a mass-manufactured, continuous-flow PCB chip with ultra-low cost fluorescent detection circuitry, rendering it the only continuous-flow μLAMP platform with off-the-shelf optical detection components. Ultrafast, SARS-CoV-2 RNA amplification in wastewater samples was demonstrated within 2 min analysis, at concentrations as low as 17 gc μL -1 . We further demonstrate our device operation by detecting SARS-CoV-2 in 20 human nasopharyngeal swab samples, without the need for any RNA extraction or purification. This renders the presented miniaturised nucleic-acid amplification-based diagnostic test the fastest reported SARS-CoV-2 genetic detection platform, in a practical implementation suitable for deployment in the field. This technology can be readily extended to the detection of alternative pathogens or genetic targets for a very broad range of applications and matrices. LoCKAmp lab-on-PCB chips are currently mass-manufactured in a commercial, ISO-compliant PCB factory, at a small-scale production cost of £2.50 per chip. Thus, with this work, we demonstrate a high technology-readiness-level lab-on-chip-based genetic detection system, successfully benchmarked against standard analytical techniques both for wastewater and nasopharyngeal swab SARS-CoV-2 detection.

We demonstrate a simple, low-energy method whereby tomato mosaic virus (ToMV) particles can be used to template the production of nanowires and particles consisting of alloys of gold (Au), platinum (Pt) and palladium (Pd) in various combinations. Selective nanowire growth within the inner channel of the particles was achieved using the polymeric capping agent polyvinylpyrrolidone (PVP K30 ) and the reducing agent ascorbic acid. The reaction conditions also resulted in the deposition of alloy nanoparticles on the external surface of the rods in addition to the nanowire structures within the internal cavity. The resulting materials were characterized using a variety of electron microscopic and spectroscopic techniques, which revealed both the structural and chemical composition of the alloys within the nanomaterials.

Sweet potato feathery mottle virus (SPFMV) and Sweet potato mild mottle virus (SPMMV) are members of the genera Potyvirus and Ipomovirus, family Potyviridae, sharing Ipomoea batatas as common host, but transmitted, respectively, by aphids and whiteflies. Virions of family members consist of flexuous rods with multiple copies of a single coat protein (CP) surrounding the RNA genome. Here we report the generation of virus-like particles (VLPs) by transient expression of the CPs of SPFMV and SPMMV in the presence of a replicating RNA in Nicotiana benthamiana. Analysis of the purified VLPs by cryo-electron microscopy, gave structures with resolutions of 2.6 and 3.0 Å, respectively, showing a similar left-handed helical arrangement of 8.8 CP subunits per turn with the C-terminus at the inner surface and a binding pocket for the encapsidated ssRNA. Despite their similar architecture, thermal stability studies reveal that SPMMV VLPs are more stable than those of SPFMV., (© 2023. The Author(s).)

We have investigated whether the presence of the origin of assembly sequence (OAS) of tobacco mosaic virus (TMV) is necessary for the specific encapsidation of replicating viral RNA. To this end TMV coat protein was expressed from replicating RNA constructs with or without the OAS in planta. In both cases the replicating RNA was specifically encapsidated to give nucleoprotein nanorods, though the yield in the absence of the OAS was reduced to about 60% of that in its presence. Moreover, the nanorods generated in the absence of the OAS were more heterogeneous in length and contained frequent structural discontinuities. These results strongly suggest that the function of the OAS is to provide a unique site for the initiation of viral assembly, leading to a one-start helix, rather than the selection of virus RNA for packaging., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing financial interests or personal relationships that could have appeared to influence the study reported in this paper., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

We have developed methods for the templated synthesis of palladium nanowires (Pd NWs) within the central channel of tobacco mosaic virus (TMV) nanorods of various lengths. We show that uniform 4 nm diameter Pd NWs can be produced by selective growth within these channels by including the capping reagent, poly(vinyl-pyrrolidone) (PVP 30K ) and reducing the metal precursor to metallic palladium with ascorbic acid. The length of the Pd NWs can be controlled either by varying the length of the nanorod templates and/or through alterations to the reaction conditions. We have also demonstrated bimetallic gold (Au)-palladium (Pd) in-situ metallization of TMV nanorods resulting in the production of Pd NWs 6 nm gold nanoparticles attached to their ends. The materials produced have many potential applications in the construction of nanoscale devices., Competing Interests: Declaration of competing interest The author declare that they do not have any conflict of interest., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The emergence of the SARS-CoV-2 coronavirus pandemic in China in late 2019 led to the fast development of efficient therapeutics. Of the major structural proteins encoded by the SARS-CoV-2 genome, the SPIKE (S) protein has attracted considerable research interest because of the central role it plays in virus entry into host cells. Therefore, to date, most immunization strategies aim at inducing neutralizing antibodies against the surface viral S protein. The SARS-CoV-2 S protein is heavily glycosylated with 22 predicted N -glycosylation consensus sites as well as numerous mucin-type O -glycosylation sites. As a consequence, O - and N -glycosylations of this viral protein have received particular attention. Glycans N -linked to the S protein are mainly exposed at the surface and form a shield-masking specific epitope to escape the virus antigenic recognition. In this work, the N -glycosylation status of the S protein within virus-like particles (VLPs) produced in Nicotiana benthamiana ( N. benthamiana ) was investigated using a glycoproteomic approach. We show that 20 among the 22 predicted N- glycosylation sites are dominated by complex plant N -glycans and one carries oligomannoses. This suggests that the SARS-CoV-2 S protein produced in N. benthamiana adopts an overall 3D structure similar to that of recombinant homologues produced in mammalian cells.

We have investigated the use of transient expression to produce virus-like particles (VLPs) of severe acute respiratory syndrome coronavirus 2, the causative agent of COVID-19, in Nicotiana benthamiana. Expression of a native form of the spike (S) protein, either alone or in combination with the envelope (E) and membrane (M) proteins, all of which were directed to the plant membranes via their native sequences, was assessed. The full-length S protein, together with degradation products, could be detected in total protein extracts from infiltrated leaves in both cases. Particles with a characteristic 'crown-shaped' or 'spiky' structure could be purified by density gradient centrifugation. Enzyme-linked immunosorbent assays using anti-S antibodies showed that threefold higher levels of VLPs containing the full-length S protein were obtained by infiltration with S alone, compared to co-infiltration of S with M and E. The S protein within the VLPs could be cleaved by furin in vitro and the particles showed reactivity with serum from recovering COVID-19 patients, but not with human serum taken before the pandemic. These studies show that the native S protein expressed in plants has biological properties similar to those of the parent virus. We show that the approach undertaken is suitable for the production of VLPs from emerging strains and we anticipate that the material will be suitable for functional studies of the S protein, including the assessment of the effects of specific mutations. As the plant-made material is noninfectious, it does not have to be handled under conditions of high containment., (© 2022 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

The production of designer-length tobacco mosaic virus (TMV) nanorods in plants has been problematic in terms of yields, particularly when modified coat protein subunits are incorporated. To address this, we have investigated the use of a replicating potato virus X-based vector (pEff) to express defined length nanorods containing either wild-type or modified versions of the TMV coat protein. This system has previously been shown to be an efficient method for producing virus-like particles of filamentous plant viruses. The length of the resulting TMV nanorods can be controlled by varying the length of the encapsidated RNA. Nanorod lengths were analyzed with a custom-written Python computer script coupled with the Nanorod UI user interface script, thereby generating histograms of particle length. In addition, nanorod variants were produced by incorporating coat protein subunits presenting metal-binding peptides at their C-termini. We demonstrate the utility of this approach by generating nanorods that bind colloidal gold nanoparticles., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Saunders, Thuenemann, Shah, Peyret, Kristianingsih, Lopez, Richardson and Lomonossoff.)

The current gold standard technique for SARS-CoV-2 diagnostics is hydrolysis probe-based RT-qPCR. Reliable testing requires reliable control reagents to monitor the efficiency of RNA extraction, reverse transcription and PCR amplification. Here we describe a custom RNA packaging system from the plant virus cowpea mosaic virus to produce virus-like particles that encapsidate specifically designed portions of the genome of SARS-CoV-2, the causative agent of COVID-19. These encapsidated mimics are highly stable particles which can be used either to spike patient swab samples for use as an in-tube extraction and reaction positive control in multiplex RT-qPCR, or alone as a side-by-side mock-positive control reagent. The selection of sequences in the packaged pseudogenomes ensures that these mimics are compatible with the most commonly used primer/probe combinations for SARS-CoV-2 diagnostics (including German Berlin Charité Hospital, American CDC, and Chinese CDC protocols). The plant transient expression system used to produce these encapsidated mimics is inherently low-cost, and sufficiently high-yielding that a single laboratory-scale preparation can provide enough positive control reagent for millions of tests., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Recent discoveries in the dynamics of genome replication and packaging in the plant virus Cowpea mosaic virus (CPMV) has led to the development of a novel method for specifically packaging an RNA molecule of choice into virus-like particles (VLPs) of CPMV. Thanks to modern gene synthesis and molecular cloning methods, the DNA sequence corresponding to an RNA sequence of interest can be cloned into a suitable expression plasmid for transient expression in plants. We describe here a method for ensuring that this RNA sequence will be packaged within VLPs of CPMV in plant cells by replication-dependent RNA packaging. This requires co-expression of the CPMV replication machinery alongside the CPMV coat protein precursor. These components are co-expressed in the leaves of the Nicotiana benthamiana plant and this co-expression results in the production of large quantities of VLPs that contain the RNA sequence of choice. These VLPs are easy to extract and purify from the plant tissue, and are stable for months in refrigerated conditions. These VLPs can then be used for a variety of different applications, such as RNA delivery or control reagents in RT-qPCR., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The fight against infectious diseases often focuses on epidemics and pandemics, which demand urgent resources and command attention from the health authorities and media. However, the vast majority of deaths caused by infectious diseases occur in endemic zones, particularly in developing countries, placing a disproportionate burden on underfunded health systems and often requiring international interventions. The provision of vaccines and other biologics is hampered not only by the high cost and limited scalability of traditional manufacturing platforms based on microbial and animal cells, but also by challenges caused by distribution and storage, particularly in regions without a complete cold chain. In this review article, we consider the potential of molecular farming to address the challenges of endemic and re-emerging diseases, focusing on edible plants for the development of oral drugs. Key recent developments in this field include successful clinical trials based on orally delivered dried leaves of Artemisia annua against malarial parasite strains resistant to artemisinin combination therapy, the ability to produce clinical-grade protein drugs in leaves to treat infectious diseases and the long-term storage of protein drugs in dried leaves at ambient temperatures. Recent FDA approval of the first orally delivered protein drug encapsulated in plant cells to treat peanut allergy has opened the door for the development of affordable oral drugs that can be manufactured and distributed in remote areas without cold storage infrastructure and that eliminate the need for expensive purification steps and sterile delivery by injection., (© 2021 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Infectious diseases, also known as transmissible or communicable diseases, are caused by pathogens or parasites that spread in communities by direct contact with infected individuals or contaminated materials, through droplets and aerosols, or via vectors such as insects. Such diseases cause ˜17% of all human deaths and their management and control places an immense burden on healthcare systems worldwide. Traditional approaches for the prevention and control of infectious diseases include vaccination programmes, hygiene measures and drugs that suppress the pathogen, treat the disease symptoms or attenuate aggressive reactions of the host immune system. The provision of vaccines and biologic drugs such as antibodies is hampered by the high cost and limited scalability of traditional manufacturing platforms based on microbial and animal cells, particularly in developing countries where infectious diseases are prevalent and poorly controlled. Molecular farming, which uses plants for protein expression, is a promising strategy to address the drawbacks of current manufacturing platforms. In this review article, we consider the potential of molecular farming to address healthcare demands for the most prevalent and important epidemic and pandemic diseases, focussing on recent outbreaks of high-mortality coronavirus infections and diseases that disproportionately affect the developing world., (© 2021 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Bluetongue (BT) is a severe and economically important disease of ruminants that is widely distributed around the world, caused by the bluetongue virus (BTV). More than 28 different BTV serotypes have been identified in serum neutralisation tests (SNT), which, along with geographic variants (topotypes) within each serotype, reflect differences in BTV outer-capsid protein VP2. VP2 is the primary target for neutralising antibodies, although the basis for cross-reactions and serological variations between and within BTV serotypes is poorly understood. Recombinant BTV VP2 proteins (rVP2) were expressed in Nicotiana benthamiana , based on sequence data for isolates of thirteen BTV serotypes (primarily from Europe), including three 'novel' serotypes (BTV-25, -26 and -27) and alternative topotypes of four serotypes. Cross-reactions within and between these viruses were explored using rabbit anti-rVP2 sera and post BTV-infection sheep reference-antisera, in I-ELISA (with rVP2 target antigens) and SNT (with reference strains of BTV-1 to -24, -26 and -27). Strong reactions were generally detected with homologous rVP2 proteins or virus strains/serotypes. The sheep antisera were largely serotype-specific in SNT, but more cross-reactive by ELISA. Rabbit antisera were more cross-reactive in SNT, and showed widespread, high titre cross-reactions against homologous and heterologous rVP2 proteins in ELISA. Results were analysed and visualised by antigenic cartography, showing closer relationships in some, but not all cases, between VP2 topotypes within the same serotype, and between serotypes belonging to the same 'VP2 nucleotype'.

The past 30 years have seen the growth of plant molecular farming as an approach to the production of recombinant proteins for pharmaceutical and biotechnological uses. Much of this effort has focused on producing vaccine candidates against viral diseases, including those caused by enveloped viruses. These represent a particular challenge given the difficulties associated with expressing and purifying membrane-bound proteins and achieving correct assembly. Despite this, there have been notable successes both from a biochemical and a clinical perspective, with a number of clinical trials showing great promise. This review will explore the history and current status of plant-produced vaccine candidates against enveloped viruses to date, with a particular focus on virus-like particles (VLPs), which mimic authentic virus structures but do not contain infectious genetic material.

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.

The production of plant helical virus-like particles (VLPs) via plant-based expression has been problematic with previous studies suggesting that an RNA scaffold may be necessary for their efficient production. To examine this, we compared the accumulation of VLPs from two potexviruses, papaya mosaic virus and alternanthera mosaic virus (AltMV), when the coat proteins were expressed from a replicating potato virus X- based vector (pEff) and a non-replicating vector (pEAQ- HT ). Significantly greater quantities of VLPs could be purified when pEff was used. The pEff system was also very efficient at producing VLPs of helical viruses from different virus families. Examination of the RNA content of AltMV and tobacco mosaic virus VLPs produced from pEff revealed the presence of vector-derived RNA sequences, suggesting that the replicating RNA acts as a scaffold for VLP assembly. Cryo-EM analysis of the AltMV VLPs showed they had a structure very similar to that of authentic potexvirus particles. Thus, we conclude that vectors generating replicating forms of RNA, such as pEff, are very efficient for producing helical VLPs.

Dengue virus (DENV) is an emerging threat causing an estimated 390 million infections per year. Dengvaxia, the only licensed vaccine, may not be adequately safe in young and seronegative patients; hence, development of a safer, more effective vaccine is of great public health interest. Virus-like particles (VLPs) are a safe and very efficient vaccine strategy, and DENV VLPs have been produced in various expression systems. Here, we describe the production of DENV VLPs in Nicotiana benthamiana using transient expression. The co-expression of DENV structural proteins (SP) and a truncated version of the non-structural proteins (NSPs), lacking NS5 that contains the RNA-dependent RNA polymerase, led to the assembly of DENV VLPs in plants. These VLPs were comparable in appearance and size to VLPs produced in mammalian cells. Contrary to data from other expression systems, expression of the protein complex prM-E was not successful, and strategies used in other expression systems to improve the VLP yield did not result in increased yields in plants but, rather, increased purification difficulties. Immunogenicity assays in BALB/c mice revealed that plant-made DENV1-SP + NSP VLPs led to a higher antibody response in mice compared with DENV-E domain III displayed inside bluetongue virus core-like particles and a DENV-E domain III subunit. These results are consistent with the idea that VLPs could be the optimal approach to creating candidate vaccines against enveloped viruses., (© 2020 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

The side effects of chemotherapy can be reduced by targeting tumor cells with an enzyme (or the corresponding gene) that converts a nontoxic prodrug into a toxic drug inside the tumor cells, also killing the surrounding tumor cells via the bystander effect. Viruses are the most efficient gene delivery vehicles because they have evolved to transfer their own nucleic acids into cells, but their efficiency must be balanced against the risks of infection, the immunogenicity of nucleic acids, and the potential for genomic integration. We therefore tested the effectiveness of genome-free virus-like particles (VLPs) for the delivery of Herpes simplex virus 1 thymidine kinase (HSV1-TK), the most common enzyme used in prodrug conversion therapy. HSV1-TK is typically delivered as a gene, but in the context of VLPs, it must be delivered as a protein. We constructed VLPs and smaller core-like particles (CLPs) based on Bluetongue virus, with HSV1-TK fused to the inner capsid protein VP3. TK-CLPs and TK-VLPs could be produced in large quantities in plants. The TK-VLPs killed human glioblastoma cells efficiently in the presence of ganciclovir, with an IC 50 value of 14.8 μM. Conversely, CLPs were ineffective because they remained trapped in the endosomal compartment, in common with many synthetic nanoparticles. VLPs are advantageous because they can escape from endosomes and therefore allow HSV1-TK to access the cytosolic adenosine triphosphate (ATP) required for the phosphorylation of ganciclovir. The VLP delivery strategy of TK protein therefore offers a promising new modality for the treatment of cancer with systemic prodrugs such as ganciclovir.

Geminivirus particles, consisting of a pair of twinned isometric structures, have one of the most distinctive capsids in the virological world. Until recently, there was little information as to how these structures are generated. To address this, we developed a system to produce capsid structures following the delivery of geminivirus coat protein and replicating circular single-stranded DNA (cssDNA) by the infiltration of gene constructs into plant leaves. The transencapsidation of cssDNA of the Begomovirus genus by coat protein of different geminivirus genera was shown to occur with full-length but not half-length molecules. Double capsid structures, distinct from geminate capsid structures, were also generated in this expression system. By increasing the length of the encapsidated cssDNA, triple geminate capsid structures, consisting of straight, bent and condensed forms were generated. The straight geminate triple structures generated were similar in morphology to those recorded for a potato-infecting virus from Peru. These finding demonstrate that the length of encapsidated DNA controls both the size and stability of geminivirus particles.

Virus-like particles (VLPs) can be used as nano-carriers and antigen-display systems in vaccine development and therapeutic applications. Conjugation of peptides or whole proteins to VLPs can be achieved using different methods such as the SpyTag/SpyCatcher system. Here we investigate the conjugation of tandem Hepatitis B core (tHBcAg) VLPs and the model antigen GFP in vivo in Nicotiana benthamiana. We show that tHBcAg VLPs could be successfully conjugated with GFP in the cytosol and ER without altering VLP formation or GFP fluorescence. Conjugation in the cytosol was more efficient when SpyCatcher was displayed on tHBcAg VLPs instead of being fused to GFP. This effect was even more obvious in the ER, showing that it is optimal to display SpyCatcher on the tHBcAg VLPs and SpyTag on the binding partner. To test transferability of the GFP results to other antigens, we successfully conjugated tHBcAg VLPs to the HIV capsid protein P24 in the cytosol. This work presents an efficient strategy which can lead to time and cost saving post-translational, covalent conjugation of recombinant proteins in plants.

The harsh conditions of the gastro-intestinal (GI) milieu pose a major barrier to the oral delivery of protein nanocages. Here we studied the stability of Nudaurelia capensis omega virus (NωV) virus-like particles (VLPs) in simulated GI fluids. NωV VLPs capsids and procapsids were transiently expressed in plants, the VLPs were incubated in various simulated GI fluids and their stability was determined by gel electrophoresis, density gradient ultracentrifugation and transmission electron microscopy (TEM). The results showed that the capsids were highly resistant to simulated gastric fluids at pH ≥ 3. Even under the harshest conditions, which consisted of a pepsin solution at pH 1.2, NωV capsids remained assembled as VLPs, though some digestion of the coat protein occurred. Moreover, 80.8% (±10.2%) stability was measured for NωV capsids upon 4 h incubation in simulated intestinal fluids. The high resistance of this protein cage to digestion and denaturation can be attributed to its distinctively compact structure. The more porous form of the VLPs, the procapsid, was less stable under all conditions. Our results suggest that NωV VLPs capsids are likely to endure transit through the GI tract, designating them as promising candidate protein nanocages for oral drug delivery., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: G.P.L. declares that he is a named inventor on granted patent WO 29,087,391 A1 that describes the CPMV-HT system used for transient expression in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Plant molecular farming (PMF) is rapidly gaining traction as a viable alternative to the currently accepted paradigm of producing biologics. While the platform is potentially cheaper and more scalable than conventional manufacturing systems, expression yields and appropriate post-translational modifications along the plant secretory pathway remain a challenge for certain proteins. Viral fusion glycoproteins in particular are often expressed at low yields in plants and, in some cases, may not be appropriately processed. Recently, however, transiently or stably engineering the host plant has shown promise as a strategy for producing heterologous proteins with more complex maturation requirements. In this study we investigated the co-expression of a suite of human chaperones to improve the production of a human immunodeficiency virus (HIV) type 1 soluble gp140 vaccine candidate in Nicotiana benthamiana plants. The co-expression of calreticulin (CRT) resulted in a dramatic increase in Env expression and ameliorated the endoplasmic reticulum (ER) stress response - as evidenced by lower transcript abundance of representative stress-responsive genes. The co-expression of CRT similarly improved accumulation of glycoproteins from Epstein-Barr virus (EBV), Rift Valley fever virus (RVFV) and chikungunya virus (CHIKV), suggesting that the endogenous chaperone machinery may impose a bottleneck for their production. We subsequently successfully combined the co-expression of human CRT with the transient expression of human furin, to enable the production of an appropriately cleaved HIV gp140 antigen. These transient plant host engineering strategies are a promising approach for the production of high yields of appropriately processed and cleaved viral glycoproteins., (© 2020 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

The Hepatitis E virus (HEV) is a causative agent of acute hepatitis, mainly transmitted by the fecal-oral route or zoonotic. Open reading frame (ORF) 2 encodes the viral capsid protein, which is essential for virion assembly, host interaction, and inducing neutralizing antibodies. In this study, we investigated whether full-length and N- and C-terminally modified versions of the capsid protein transiently expressed in N. benthamiana plants could assemble into highly-immunogenic, virus-like particles (VLPs). We also assessed whether such VLPs can act as a carrier of foreign immunogenic epitopes, such as the highly-conserved M2e peptide from the Influenza virus. Plant codon-optimized HEV ORF2 capsid genes were constructed in which the nucleotides coding the N-terminal, the C-terminal, or both parts of the protein were deleted. The M2e peptide was inserted into the P2 loop after the residue Gly556 of HEV ORF2 protein by gene fusion, and three different chimeric constructs were designed. Plants expressed all versions of the HEV capsid protein up to 10% of total soluble protein (TSP), including the chimeras, but only the capsid protein consisting of aa residues 110 to 610 (HEV 110-610) and chimeric M2 HEV 110-610 spontaneously assembled in higher order structures. The chimeric VLPs assembled into particles with 22-36 nm in diameter and specifically reacted with the anti-M2e antibody., Competing Interests: The authors declare no conflict of interest

The Luteoviridae are pathogenic plant viruses responsible for significant crop losses worldwide. They infect a wide range of food crops, including cereals, legumes, cucurbits, sugar beet, sugarcane, and potato and, as such, are a major threat to global food security. Viral replication is strictly limited to the plant vasculature, and this phloem limitation, coupled with the need for aphid transmission of virus particles, has made it difficult to generate virus in the quantities needed for high-resolution structural studies. Here, we exploit recent advances in heterologous expression in plants to produce sufficient quantities of virus-like particles for structural studies. We have determined their structures to high resolution by cryoelectron microscopy, providing the molecular-level insight required to rationally interrogate luteovirid capsid formation and aphid transmission, thereby providing a platform for the development of preventive agrochemicals for this important family of plant viruses., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: The growing field of plant molecular farming relies on expression vectors that allow high yields of recombinant proteins to be produced through transient gene expression. While numerous expression vectors currently exist for this purpose, there are very few examples of systematic efforts to improve upon these. Moreover, the current generation of expression systems makes use of naturally-occurring regulatory elements, typically selected from plant viruses, to maximise yields. This study aims to use rational design to generate synthetic sequences that can rival existing ones., Results: In this work, we present the rational design of novel synthetic 5' and 3' untranslated regions (UTRs) which can be used in various combinations to modulate accumulation levels of transiently-expressed recombinant proteins. Using the pEAQ- HT expression vector as a point of comparison, we show that pre-existing expression systems can be improved by the deployment of rationally designed synthetic UTRs. Notably, we show that a suite of short, synthetic 5'UTRs behave as expression enhancers that outperform the HT 5'UTR present in the CPMV- HT expression system. Furthermore, we confirm the critical role played by the 3'UTR of cowpea mosaic virus RNA-2 in the performance of the CPMV- HT system. Finally, we use the knowledge obtained from these results to develop novel expression vectors (named pHRE and pHREAC) that equal or outperform pEAQ- HT in terms of recombinant protein yield. These new vectors are also domesticated for the use of certain Type IIS restriction enzymes, which allows for quicker cloning and straightforward assessment of different combinations of UTRs., Conclusions: We have shown that it is possible to rationally design a suite of expression modulators in the form of synthetic UTRs. We have created novel expression vectors that allow very high levels of recombinant protein expression in a transient expression context. This will have important consequences for future efforts to develop ever-better plant transient overexpression vectors for research or industrial applications., Competing Interests: Competing interestsGP.L. declares that he is a named inventor on granted patent WO 29087391 A1 that describes the CPMV-HT system used for transient expression in this paper.

The icosahedral capsid of cowpea mosaic virus is formed by 60 copies of the large (L) and small (S) coat protein subunits. The 24-amino-acid C-terminal peptide of the S coat protein can undergo proteolytic cleavage without affecting particle stability or infectivity. Mutagenic studies have shown that this sequence is involved in particle assembly, virus movement, RNA encapsidation and suppression of gene silencing. However, it is unclear how these processes are related, and which part(s) of the sequence are involved in each process. Here, we have analysed the effect of mutations in the C-terminal region of the S protein on the assembly of empty virus-like particles and on the systemic movement of infectious virus. The results confirmed the importance of positively charged amino acids adjacent to the cleavage site for particle assembly and revealed that the C-terminal 11 amino acids are important for efficient systemic movement of the virus.

Bluetongue is a severe, economically important disease of ruminants that is widely distributed in tropical and temperate regions around the world. It is associated with major production losses, restrictions of animal movements and trade, as well as costs associated with developing and implementing effective surveillance and control measures. Mammalian hosts infected with bluetongue virus (BTV) generate a protective neutralising antibody response targeting the major BTV outer-capsid protein and serotype-specific antigen, VP2. BTV VP2 proteins that have been expressed in plants are soluble, with a native conformation displaying neutralising epitopes and can assemble with other BTV structural proteins to form virus-like particles (VLPs). His-tagged VP2 proteins of BTV serotypes 4 and 8 were transiently expressed in Nicotiana benthamiana then purified by immobilised metal affinity chromatography (IMAC). Antisera from IFNAR -/- mice prime/boost vaccinated with the purified proteins, were shown to contain VP2-specific antibodies by Indirect ELISA (I-ELISA), western blotting and serum neutralisation tests (SNT). Vaccinated mice, subsequently challenged with either the homologous or heterologous BTV serotype, developed viraemia by day 3 post-infection. However, no clinical signs were observed in mice challenged with the homologous serotype (either prime-boost or single-shot vaccinated), all of which survived for the duration of the study. In contrast, all of the vaccinated mice challenged with a heterologous serotype, died, showing no evidence of cross-protection or suppression of viraemia, as detected by real-time RT-qPCR or virus isolation. The induction of protective, serotype-specific neutralising antibodies in IFNAR -/- mice, indicates potential for the use of plant-expressed BTV VP2s as subunit vaccine components, or as a basis for serotype-specific serological assays.

Plant viruses can cause devastating losses to agriculture and are therefore a major threat to food security. The rapid identification of virally-infected crops allowing containment is essential to limit such threats, but plant viral diseases can be extremely challenging to diagnose. An ideal method for plant virus diagnosis would be a device which can be implemented easily in the field. Such devices require a binding reagent that is specific for the virus of interest. We chose to investigate the use of Affimer reagents, artificial binding proteins and a model plant virus Cowpea Mosaic virus (CPMV) empty virus like particles (eVLPs). CPMV-eVLP mimic the morphology of wild-type (WT) CPMV but lack any infectious genomic material and so do not have biocontainment issues. We have produced and purified an Affimer reagent selected for its ability to bind to CPMV-eVLP and have shown that the selected Affimer also specifically binds to WT CPMV. We have produced a 3.4 Å structure of WT CPMV bound to the Affimer using cryo-electron microscopy. Finally, we have shown that this Affimer is capable of reliably detecting the virus in crude extracts of CPMV-infected leaves and can therefore form the basis for the future development of diagnostic tests.

Satellite tobacco necrosis virus 1 (STNV-1) is a model system for in vitro RNA encapsidation studies (N. Patel, E. C. Dykeman, R. H. A. Coutts, G. P. Lomonossoff, et al., Proc Natl Acad Sci U S A 112:2227-2232, 2015, https://doi.org/10.1073/pnas.1420812112; N. Patel, E. Wroblewski, G. Leonov, S. E. V. Phillips, et al., Proc Natl Acad Sci U S A 114:12255-12260, 2017, https://doi.org/10.1073/pnas.1706951114), leading to the identification of degenerate packaging signals (PSs) proposed to be involved in the recognition of its genome by the capsid protein (CP). The aim of the present work was to investigate whether these putative PSs can confer selective packaging of STNV-1 RNA in vivo and to assess the prospects of using decoy RNAs in antiviral therapy. We have developed an in planta packaging assay based on the transient expression of STNV-1 CP and have assessed the ability of the resulting virus-like particles (VLPs) to encapsidate mutant STNV-1 RNAs expected to have different encapsidation potential based on in vitro studies. The results revealed that >90% of the encapsidated RNAs are host derived, although there is some selectivity of packaging for STNV-1 RNA and certain host RNAs. Comparison of the packaging efficiencies of mutant STNV-1 RNAs showed that they are encapsidated mainly according to their abundance within the cells, rather than the presence or absence of the putative PSs previously identified from in vitro studies. In contrast, subsequent infection experiments demonstrated that host RNAs represent only <1% of virion content. Although selective encapsidation of certain host RNAs was noted, no direct correlation could be made between this preference and the presence of potential PSs in the host RNA sequences. Overall, the data illustrate that the differences in RNA packaging efficiency identified through in vitro studies are insufficient to explain the specific packaging of STNV-1 RNA. IMPORTANCE Viruses preferentially encapsidate their own genomic RNA, sometimes as a result of the presence of clearly defined packaging signals (PSs) in their genome sequence. Recently, a novel form of short degenerate PSs has been proposed (N. Patel, E. C. Dykeman, R. H. A. Coutts, G. P. Lomonossoff, et al., Proc Natl Acad Sci U S A 112:2227-2232, 2015, https://doi.org/10.1073/pnas.1420812112; N. Patel, E. Wroblewski, G. Leonov, S. E. V. Phillips, et al., Proc Natl Acad Sci U S A 114:12255-12260, 2017, https://doi.org/10.1073/pnas.1706951114) using satellite tobacco necrosis virus 1 (STNV-1) as a model system for in vitro studies. It has been suggested that competing with these putative PSs may constitute a novel therapeutic approach against pathogenic single-stranded RNA viruses. Our work demonstrates that the previously identified PSs have no discernible significance for the selective packaging of STNV-1 in vivo in the presence and absence of competition or replication: viral sequences are encapsidated mostly on the basis of their abundance within the cell, while encapsidation of host RNAs also occurs. Nevertheless, the putative PSs identified in STNV-1 RNA may still have applications in bionanotechnology, such as the in vitro selective packaging of RNA molecules., (Copyright © 2019 Kotta-Loizou et al.)

Dengue fever is currently ranked as the top emerging tropical disease, driven by increased global travel, urbanization, and poor hygiene conditions as well as global warming effects which facilitate the spread of Aedes mosquitoes beyond their current distribution. Today, more than 100 countries are affected most of which are tropical Asian and Latin American nations with limited access to medical care. Hence, the development of a dengue vaccine that is dually cost-effective and able to confer a comprehensive protection is ultimately needed. In this study, a consensus sequence of the antigenic dengue viral glycoprotein domain III (cEDIII) was used aiming to provide comprehensive coverage against all four circulating dengue viral serotypes and potential clade replacement event. Utilizing hepatitis B tandem core technology, the cEDIII sequence was inserted into the immunodominant c/e1 loop region so that it could be displayed on the spike structures of assembled particles. The tandem core particles displaying cEDIII epitopes (tHBcAg-cEDIII) were successfully produced in Nicotiana benthamiana via Agrobacterium -mediated transient expression strategy to give a protein of ∼54 kDa, detected in both soluble and insoluble fractions of plant extracts. The assembled tHBcAg-cEDIII virus-like particles (VLPs) were also visualized from transmission electron microscopy. These VLPs had diameters that range from 32 to 35 nm, presenting an apparent size increment as compared to tHBcAg control particles without cEDIII display (namely tEL). Mice immunized with tHBcAg-cEDIII VLPs showed a positive seroconversion to cEDIII antigen, thereby signifying that the assembled tHBcAg-cEDIII VLPs have successfully displayed cEDIII antigen to the immune system. If it is proven to be successful, tHBcAg-cEDIII has the potential to be developed as a cost-effective vaccine candidate that confers a simultaneous protection against all four infecting dengue viral serotypes.

Protein-corona formation in body fluids and/or entrapment of nanoparticles in protein matrices (e.g. food and mucus) can hinder the delivery of nanoparticles, irrespective of the route of administration. Here we demonstrate that certain viral nanoparticles (VNPs) can evade the adhesion of a broad panel of macromolecules from several biological milieus. We also show that the permeability of VNPs through mucin gels is far superior to that of synthetic nanoparticles. The non-sticky nature of VNPs implies that they will be able to readily cross most non-specific protein and glycoprotein barriers encountered, ubiquitously, upon administration through mucosal, and non-mucosal routes.

To elucidate the linkage between replication and encapsidation in Picornavirales , we have taken advantage of the bipartite nature of a plant-infecting member of this order, cowpea mosaic virus (CPMV), to decouple the two processes. RNA-free virus-like particles (empty virus-like particles [eVLPs]) can be generated by transiently coexpressing the RNA-2-encoded coat protein precursor (VP60) with the RNA-1-encoded 24,000-molecular-weight (24K) protease, in the absence of the replication machinery (K. Saunders, F. Sainsbury, and G. P. Lomonossoff, Virology 393:329-337, 2009, https://doi.org/10.1016/j.virol.2009.08.023). We have made use of the ability to produce assembled capsids of CPMV in the absence of replication to examine the putative linkage between RNA replication and packaging in the Picornavirales We have created a series of mutant RNA-1 and RNA-2 molecules and have assessed the effects of the mutations on both the replication and packaging of the viral RNAs. We demonstrate that mutations that affect replication have a concomitant impact on encapsidation and that RNA-1-mediated replication is required for encapsidation of both RNA-1 and RNA-2. This close coupling between replication and encapsidation provides a means for the specific packaging of viral RNAs. Moreover, we demonstrate that this feature of CPMV can be used to specifically encapsidate custom RNA by placing a sequence of choice between the RNA-2 sequences required for replication. IMPORTANCE The mechanism whereby members of the order Picornavirales specifically package their genomic RNAs is poorly understood. Research with monopartite members of the order, such as poliovirus, indicated that packaging is linked to replication, although the presence of "packaging signals" along the length of the viral RNA has also been suggested. Thanks to the bipartite nature of the CPMV genome, which allows the manipulation of RNA-1 without modifying RNA-2, we show here that this specificity is due to a functional link between the two processes of viral replication and encapsidation. This has important implications for our understanding of the fundamental molecular biology of Picornavirales and opens the door to novel research and therapeutic applications in the field of custom RNA packaging and delivery technologies., (Copyright © 2019 Kruse et al.)

Geminiviruses are major plant pathogens that threaten food security globally. They have a unique architecture built from two incomplete icosahedral particles, fused to form a geminate capsid. However, despite their importance to agricultural economies and fundamental biological interest, the details of how this is realized in 3D remain unknown. Here we report the structure of Ageratum yellow vein virus at 3.3 Å resolution, using single-particle cryo-electron microscopy, together with an atomic model that shows that the N-terminus of the single capsid protein (CP) adopts three different conformations essential for building the interface between geminate halves. Our map also contains density for ~7 bases of single-stranded DNA bound to each CP, and we show that the interactions between the genome and CPs are different at the interface than in the rest of the capsid. With additional mutagenesis data, this suggests a central role for DNA binding-induced conformational change in directing the assembly of geminate capsids.

Cowpea mosaic virus (CPMV) is a plant virus which is being extensively investigated as a drug delivery and vaccine nanocarrier for parenteral administration. However, to date little is known about the suitability of plant-based nanocarriers for oral delivery. In this study, the colloidal (i.e. aggregation), physical (i.e. denaturation) and chemical (i.e. digestion of the polypeptides) stability of CPMV and its empty virus-like particles (eVLPs) in conditions resembling the gastrointestinal fluids were evaluated. The nanoparticles were incubated in various simulated gastric and intestinal fluids and in pig gastric and intestinal fluids. CPMV and eVLPs had similar stabilities. In simulated gastric media, they were stable at pH ≥ 2.5. At lower pH destabilisation of the particle structure occurred, which, in turn, rendered the polypeptides extremely sensitive to pepsin digestion. However, both CPMV and eVLPs were stable in simulated intestinal fluids, in pig gastric fluids and in pig intestinal fluids. Thus CPMV, despite being a protein-based nanoparticle, was much more resistant to the harsh GI conditions than soluble proteins. Remarkably, both CPMV and eVLPs incubated in pig gastric and intestinal fluids were not subject to protein adsorption, with no formation of a detectable protein corona. The lack of a protein corona on CPMV and eVLP surfaces in GI fluids would imply that, if orally administered, these nanoparticles could maintain their native surface characteristics; thus, their biological interactions would remain predictable and unchanged. In summary, CPMV and eVLPs can be considered promising nanocarriers for applications requiring oral delivery, given their chemical, physical and colloidal stability and lack of protein adsorption from the environment in most of the tested conditions.

When the coat protein reading frame of turnip crinkle virus (TCV) is transiently expressed in leaves, virus-like particles (VLPs) are readily formed. However, after introducing genetic modifications to the full-length coat protein sequence, such as the introduction of an epitope-specific sequence within the coat protein sequence or the in-frame carboxyl terminal fusion of GFP, the formation of such modified VLPs is poor. However, by coexpression of one of these modified forms with wild-type TCV coat protein by the coinfiltration of appropriate Agrobacterium suspensions, VLP generation is enhanced through the formation of "mosaics," that is, individual VLPs consisting of both modified and wild-type subunits (also known as phenotypically mixed VLPs). Here we describe methods for the introduction of genetic modifications into the TCV coat protein sequence, the production of mosaic TCV VLPs and their characterization.

The discovery of a new class of pathogen, viruses, in the late 19th century, ushered in a period of study of the biochemical and structural properties of these entities in which plant viruses played a prominent role. This was, in large part, due to the relative ease with which sufficient quantities of material could be produced for such analyses. As analytical techniques became increasingly sensitive, similar studies could be performed on the viruses from other organisms. However, plant viruses continued to play an important role in the development of molecular biology, including the demonstration that RNA can be infectious, the determination of the genetic code, the mechanism by which viral RNAs are translated, and some of the early studies on gene silencing. Thus, the study of plant viruses should not be considered a "niche" subject but rather part of the mainstream of virology and molecular biology., (© 2018 Elsevier Inc. All rights reserved.)

This chapter provides a practical guide to the in planta transient production of bluetongue virus-like particles containing a fluorescent cargo protein. Bluetongue virus (BTV) particles are icosahedral, multishelled entities of a relatively large size. Heterologous expression of the four main structural proteins of BTV results in the assembly of empty virus-like particles which resemble the native virus externally, but are devoid of nucleic acid. The space within the particles is sufficient to allow incorporation of relatively large cargo proteins, such as green fluorescent protein (GFP), by genetic fusion to the structural protein VP3. The method described utilizes the pEAQ vectors for high-level transient expression of such particles in Nicotiana benthamiana.

Ever since its initial characterization in the 19th century, tobacco mosaic virus (TMV) has played a prominent role in the development of modern virology and molecular biology. In particular, research on the three-dimensional structure of the virus particles and the mechanism by which these assemble from their constituent protein and RNA components has made TMV a paradigm for our current view of the morphogenesis of self-assembling structures, including viral particles. More recently, this knowledge has been applied to the development of novel reagents and structures for applications in biomedicine and bionanotechnology. In this article, we review how fundamental science has led to TMV being at the vanguard of these new technologies., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Particles of cowpea mosaic virus (CPMV) have enjoyed considerable success as nanoparticles. The development of a system for producing empty virus-like particles (eVLPs) of the virus, which are non-infectious and have the potential to be loaded with heterologous material, has increased the number of possible applications for CPMV-based particles. However, for this potential to be realised, it was essential to demonstrate that eVLPs were accurate surrogates for natural virus particles, and this information was provided by high-resolution cryo-EM studies of eVLPs. This demonstration has enabled the approaches developed for the production of modified particles developed with natural CPMV particles to be applied to eVLPs. Furthermore, a combination of cryo-EM and mutagenic studies allowed the development of particles which are permeable but which could still assemble efficiently. These particles were shown to be loadable with cobalt, indicating that they can, indeed, be used as nano-containers., (© 2017 The Author(s).)

We have utilized plant-based transient expression to produce tobacco mosaic virus (TMV)-based nano-rods of predetermined lengths. This is achieved by expressing RNAs containing the TMV origin of assembly sequence (OAS) and the sequence of the TMV coat protein either on the same RNA molecule or on two separate constructs. We show that the length of the resulting nano-rods is dependent upon the length of the RNA that possesses the OAS element. By expressing a version of the TMV coat protein that incorporates a metal-binding peptide at its C-terminus in the presence of RNA containing the OAS we have been able to produce nano-rods of predetermined length that are coated with cobalt-platinum. These nano-rods have the properties of defined-length nano-wires that make them ideal for many developing bionanotechnological processes.

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.

Nanotechnology is a rapidly expanding field seeking to utilize nano-scale structures for a wide range of applications. Biologically derived nanostructures, such as viruses and virus-like particles (VLPs), provide excellent platforms for functionalization due to their physical and chemical properties. Plant viruses, and VLPs derived from them, have been used extensively in biotechnology. They have been characterized in detail over several decades and have desirable properties including high yields, robustness, and ease of purification. Through modifications to viral surfaces, either interior or exterior, plant-virus-derived nanoparticles have been shown to support a range of functions of potential interest to medicine and nano-technology. In this review we highlight recent and influential achievements in the use of plant virus particles as vehicles for diverse functions: from delivery of anticancer compounds, to targeted bioimaging, vaccine production to nanowire formation. WIREs Nanomed Nanobiotechnol 2017, 9:e1447. doi: 10.1002/wnan.1447 For further resources related to this article, please visit the WIREs website., (© 2017 John Innes Centre. Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology Published by Wiley Periodicals, Inc.)