Tobacco Mosaic Virus Coat Protein Research Articles

High-density Co-Nx sites anchored on biomass-based carbon nanotubes for efficient removal of plant viruses in water

In this study, we selected rice straw, wheat straw and corn cob as raw materials to successfully prepare cobalt-nitrogen co-doped biomass-based carbon nanotubes composites (BM@CoNCNTs) with Co-Nx sites highly dispersed on porous biochar carrier, which could effectively activate peroxymonosulfate (PMS) to remove tobacco mosaic virus (TMV) in water. Particularly, BM@CoNCNTs-CC, with its confined carbon nanotubes and high-density Co-Nx sites, enhanced the Co2+/Co3+ cycle and direct adsorption of PMS to generate Co(IV)=O and 1O2, playing a dominant role. This led to the effective disruption of TMV coat protein and degradation of its genome within 5 min, achieving up to 6 log10 removal efficiency. Additionally, BM@CoNCNTs-CC/PMS system maintained efficient catalytic removal performance even in complex water matrices (river water and tap water). Overall, our research not only elucidates the production of efficient biomass-based catalysts for environmental remediation, but also highlights the application potential of biomass-based carbon nanotubes composites enriched with Co-Nx sites.

TMV-CP based rational design and discovery of α-Amide phosphate derivatives as anti plant viral agents

The tobacco mosaic virus coat protein (TMV-CP) is indispensable for the virus’s replication, movement and transmission, as well as for the host plant's immune system to recognize it. It constitutes the outermost layer of the virus particle, and serves as an essential component of the virus structure. TMV-CP is essential for initiating and extending viral assembly, playing a crucial role in the self-assembly process of Tobacco Mosaic Virus (TMV). This research employed TMV-CP as a primary target for virtual screening, from which a library of 43,417 compounds was sourced and SH-05 was chosen as the lead compound. Consequently, a series of α-amide phosphate derivatives were designed and synthesized, exhibiting remarkable anti-TMV efficacy. The synthesized compounds were found to be beneficial in treating TMV, with compound 3g displaying a slightly better curative effect than Ningnanmycin (NNM) (EC50 = 304.54 µg/mL) at an EC50 of 291.9 µg/mL. Additionally, 3g exhibited comparable inactivation activity (EC50 = 63.2 µg/mL) to NNM (EC50 = 67.5 µg/mL) and similar protective activity (EC50 = 228.9 µg/mL) to NNM (EC50 = 219.7 µg/mL). Microscale thermal analysis revealed that the binding of 3g (Kd = 4.5 ± 1.9 µM) to TMV-CP showed the same level with NNM (Kd = 5.5 ± 2.6 µM). Results from transmission electron microscopy indicated that 3g could disrupt the structure of TMV virus particles. The toxicity prediction indicated that 3g was low toxicity. Molecular docking showed that 3g interacted with TMV-CP through hydrogen bond, attractive charge interaction and π-Cation interaction. This research provided a novel α-amide phosphate structure target TMV-CP, which may help the discovery of new anti-TMV agents in the future.

Identification of Novel Bisamide-Decorated Benzotriazole Derivatives as Anti-Phytopathogenic Virus Agents: Bioactivity Evaluation and Computational Simulation.

As a notorious phytopathogenic virus, the tobacco mosaic virus (TMV) severely reduced the quality of crops worldwide and caused critical constraints on agricultural production. The development of novel virucides is a persuasive strategy to address this predicament. Herein, a series of novel bisamide-decorated benzotriazole derivatives were elaborately prepared and screened. Biological tests implied that the optimized compound 7d possessed the most brilliant antiviral inactive profile (EC50 = 157.6 μg/mL) and apparently surpassed that of commercial ribavirin (EC50 = 442.1 μg/mL) 2.8-fold. The preliminary antiviral mechanism was elaborately investigated via transmission electron microscopy, microscale thermophoresis (MST) determination, RT-qPCR, and Western blot analysis. The results showed that compound 7d blocked the assembly of TMV by binding with coat protein (Kd = 0.7 μM) and suppressed TMV coat protein gene expression and biosynthesis process. Computational simulations indicated that 7d displayed strong H-bonds and pi interactions with TMV coat protein, affording a lower binding energy (ΔGbind = -17.8 kcal/mol) compared with Ribavirin (ΔGbind = -10.7 kcal/mol). Overall, current results present a valuable perception of bisamide decorated benzotriazole derivatives with appreciably virustatic competence and should be profoundly developed as virucidal candidates in agrochemical.

Advances of Structural Design and Biomedical Applications of Tobacco Mosaic Virus Coat Protein

Recognized as the primary RNA virus to be categorized and extensively studied, the tobacco mosaic virus (TMV) is foundational to advancements in virology, molecular biology, and biomaterials. Over the past few decades, the deep comprehension of the TMV coat protein (TMVcp) molecular structure and assembly principles has stimulated a surge in research on TMVcp structural design using genetic engineering and chemical modification techniques. The unique characteristics of TMVcp, including its nanoscale orderly structure, ease of modification, and considerable drug loading capacity, have enabled significant progress in its biomedical applications. This review summarizes the advanced strategies deployed for TMVcp design and multidimensional assembly and underscores the prototypical applications of TMVcp‐based biomaterials in bioimaging, drug delivery, tissue engineering, and biosensing. Ultimately, the future prospects of TMVcp research in structural design and biomedical applications are explored, which encompass artificial intelligence‐guided structural and functional design, the development of stimulus‐responsive biomaterials, and potential clinical translation.

Antiviral Mechanisms of Anisomycin Produced by Streptomyces albulus SN40 on Potato Virus Y.

Microbial secondary metabolites produced by Streptomyces have diverse application prospects in the control of plant diseases. Herein, the fermentation filtrate of Streptomyces SN40 effectively inhibited the infection of tobacco mosaic virus (TMV) in Nicotiana glutinosa and systemic infection of potato virus Y (PVY) in Nicotiana benthamiana. Additionally, metabolomic analysis indicated that anisomycin (C14H19NO4) and trans-3-indoleacrylic acid (C11H9NO2) were highly abundant in the crude extract and that anisomycin effectively suppressed the infection of TMV as well as PVY. Subsequently, transcriptomic analysis was conducted to elucidate its mechanisms on the induction of host defense responses. Furthermore, the results of molecular docking suggested that anisomycin can potentially bind with the helicase domain (Hel) of TMV replicase, TMV coat protein (CP), and PVY helper component proteinase (HC-Pro). This study demonstrates new functions of anisomycin in virus inhibition and provides important theoretical significance for the development of new biological pesticides to control diverse plant viruses.

Flavonol Derivatives Containing a Quinazolinone Moiety: Design, Synthesis, and Antiviral Activity.

A series of flavonol derivatives containing quinazolinone were designed and synthesized, and their antiviral activities against tobacco mosaic virus (TMV) were evaluated. The results of the half maximal effective concentration (EC50 ) test against TMV showed that the EC50 value of curative activity of K5 was 139.6 μg/mL, which was better than that of the commercial drug ningnanmycin (NNM) 296.0 μg/mL, and the EC50 value of protective activity of K5 was 120.6 μg/mL, which was superior to that of NNM 207.0 μg/mL. The interaction of K5 with TMV coat protein (TMV-CP) was investigated using microscale thermophoresis (MST) and molecular docking and the results showed that K5 can combine with TMV-CP more strongly to TMV-CP than that NNM can. Furthermore, the assay measuring malondialdehyde (MDA) content indicated that K5 had the ability to improve the disease resistance of tobacco. Hence, this study offers strong evidence that flavonol derivatives have potential as novel antiviral agents.

Synthesis of 4H-Pyrazolo[3,4-d]pyrimidin-4-one Hydrazine Derivatives as a Potential Inhibitor for the Self-Assembly of TMV Particles.

Tobacco mosaic virus coat protein (TMV-CP), as a potential target for the development of antiviral agents, can assist in the long-distance movement of viruses and plays an extremely important role in virus replication and propagation. This work focuses on the synthesis and the action mechanism of novel 4H-pyrazolo[3,4-d] pyrimidin-4-one hydrazine derivatives. The synthesized compounds exhibited promising antiviral activity on TMV. Specifically, compound G2 exhibited high inactivating activity (93%) toward TMV, slightly better than commercial reagent NNM (90%). The action of mechanism was further explored by employed molecular docking, molecular dynamics simulation, microscale thermophoresis, qRT-PCR, and transmission electron microscopy. Results indicated that G2 had the capability to interact with amino acid residues such as Trp352, Tyr139, and Asn73 in the active pocket of TMV-CP, creating strong hydrophobic interactions and thus obstructing the virus's self-assembly.

Novel flavonol derivatives containing benzoxazole as potential antiviral agents: design, synthesis, and biological evaluation.

A series of flavonol derivatives containing benzoxazole were designed and synthesized, and the structures of all the target compounds were determined by nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRMS). The structure of X2 was further confirmed by single crystal X-ray diffraction analysis. The results of the bioactivity tests showed that some of the target compounds possessed excellent antiviral activity against tobacco mosaic virus (TMV) in vivo. In particular, the median effective concentration (EC50) values for the curative and protective activities of X17 against TMV were 127.6 and 101.2μg/mL, respectively, which were superior to those of ningnanmycin (320.0 and 234.6μg/mL). The results of preliminary mechanism study indicated that X17 had a strong binding affinity for TMV coat protein (TMV-CP), which might hinder the self-assembly and replication of TMV particles. In addition, X17 was able to effectively inhibit tobacco leaf membrane lipid peroxidation and facilitate the removal of O2- from the body, thereby improving the disease resistance of tobacco plants. Therefore, the design and synthesis of flavonol derivatives containing benzoxazole provides value for the development of new antiviral drugs.

Myricetin derivatives containing the benzoxazinone moiety discovered as potential anti-tobacco mosaic virus agents

A series of myricetin derivatives containing benzoxazinone were designed and synthesized. The structures of all compounds were characterized by NMR and HRMS. The structure of Y4 had been confirmed by single-crystal X-ray diffraction analysis. The test results of EC50 values of tobacco mosaic virus (TMV) suggested that Y8 had the best curative and protective effects, with EC50 values of 236.8, 206.0 μg/mL, respectively, which were higher than that of ningnanmycin (372.4, 360.6 μg/mL). Microscale thermophoresis (MST) experiments demonstrated that Y8 possessed a strong binding affinity for tobacco mosaic virus coat protein (TMV-CP), with a dissociation constant (Kd) value of 0.045 μM, which was superior to the ningnanmycin (0.700 μM). The findings of molecular docking studies revealed that Y8 interacted with multiple amino acid residues of TMV-CP through the formation of non-covalent bonds, which had an effect on the self-assembly of TMV particles. The malondialdehyde (MDA) and superoxide dismutase assay (SOD) content assays also fully verified that Y8 could stimulate the plant immune system and enhance disease resistance by reducing MDA content and increasing SOD content. In summary, myricetin derivatives containing benzoxazinone could be considered to further research and development as novel antiviral agents.

Raman-dielectrophoresis goes viral: towards a rapid and label-free platform for plant virus characterization

An innovative spectroscopic method that allows to chemically and structurally characterize viruses directly in suspension within few minutes was developed. A library of five different plant viruses was obtained combining dielectrophoresis (DEP), performed with a device specifically designed to capture and agglomerate virus particles, and Raman spectroscopy to provide a chemical fingerprint of virions. The tested viruses, purified from infected plants, were chosen for their economic impact on horticultural crops and for their different morphological and structural features. Using the Raman-DEP device, specific profiles for each virus were successfully obtained, relying on chemical differences occurring even with genetically similar viruses belonging to the same taxonomic species and morphologically indiscernible by transmission electron microscopy (TEM). Moreover, we investigated the potentiality of Raman-DEP to follow dynamic changes occurring upon heat treatment of tobacco mosaic virus (TMV) particles. Raman peak deviations linked to TMV coat protein conformation were observed upon treatment at temperatures equal or higher than 85°C, substantiating the rod-to-spherical shape transitions observed by TEM and the concomitant drastic loss of infectivity following plant inoculation. Overall, the Raman-DEP method can be useful for the characterization of virus (nano)particles, setting the basis to create a database suitable for the study of viruses or virus derived-nanoparticles relevant for the agricultural, medical, or biotechnological fields.

Discovery of tryptanthrin analogues bearing F and piperazine moieties as novel phytopathogenic antibacterial and antiviral agents.

Plant bacterial infections and plant viruses seriously affect the yield and quality of crops. Based on the various activities of tryptanthrin, a series of tryptanthrin analogues bearing F and piperazine moieties were designed, synthesized, and evaluated for their biological activities against three plant bacteria and tobacco mosaic virus (TMV). Bioassay results indicated that compounds 6a-6l displayed excellent antibacterial activities in vitro and 6a-6c and 6g exhibited better antiviral activities against TMV than commercial ribavirin. In particular, 6b showed the most effect on Xanthomonas oryzae pv. oryzae (Xoo) with a half-maximal effective concentration (EC50 ) of 1.26 μg mL-1 , compared with the commercial pesticide bismerthiazol (BT; EC50 = 34.3 μg mL-1 ) and thiodiazole copper (TC; EC50 = 73.3 μg mL-1 ). Meanwhile, 6a also had the best antiviral activity at 500 μg mL-1 for curative, protection, and inactivation purposes, compared with ribavirin in vivo. Compound 6b could cause changes in bacterial morphology, induce the accumulation of reactive oxygen species, promote apoptosis of bacterial cells, inhibit the formation of biofilm, and block the growth of Xoo cells. Proteomic analysis revealed major differences in the bacterial secretory system pathways T2SS and T6SS, which inhibited membrane transport. Molecular docking revealed that 6a and 6g could interact with TMV coat protein preventing virus assembly. These results suggest that tryptanthrin analogues bearing F and piperazine moieties could be promising candidate agents for antibacterial and antiviral use in agricultural production. © 2023 Society of Chemical Industry.

Design, synthesis, antibacterial and antiviral evaluation of chalcone derivatives containing benzoxazole

In this study, 24 chalcone derivatives containing benzoxazole were designed and synthesized, and the structures of all target compounds were confirmed by NMR and HRMS. The results of antibacterial activity showed that the median effective concentration (EC50) value of Z2 against Xanthomonas oryzae pv.oryzae (Xoo) was 8.10 μg/mL, which was better than thiodiazole copper (60.62 μg/mL), the antibacterial activity of Z2 was verified by scanning electron microscopy (SEM). Antibacterial mechanism of Z2 on Xoo was preliminarily explored, and the results showed concentrations were 100, 50 mg/L, which had a good cell membrane permeability to Xoo and can inhibit the EPS content and the production of extracellular enzymes, affecting the formation of biofilm, preliminary validation of the antibacterial effect of Z2 on Xoo. At 200 mg/L, Z2 had a better protective activity on harvested potatoes, which was comparable with thiodiazole copper. The activity against tobacco mosaic virus (TMV) was determined with half leaf dry spot method. The EC50 value of curative activity of Z15 was 101.97 μg/mL, which was superior to ningnanmycin (NNM) 294.27 μg/mL, and the EC50 value of protective activity of Z16 was 104.05 μg/mL, which was superior to NNM 185.73 μg/mL. Molecular docking results showed Z15, Z16 had a significantly higher affinity with tobacco mosaic virus coat protein (TMV-CP) than NNM. The above results indicated that chalcone derivatives containing benzoxazole have the potential to become antibacterial and antivirial agents.

Design, Synthesis, and Biological Evaluation of Novel Derivatives of the Marine Natural Product Laurene.

Plant pathogenic fungi and viruses are seriously threatening agricultural production. There is an urgent need to develop novel fungicides and antiviral agents with low toxicity and high efficiency. In this study, we designed and synthesized 32 thiazole-, hydrazone-, and amide-containing derivatives of laurene and systematically evaluated their antiviral activities and fungicidal activities. Structure-simplified compounds 5a-5c, 5i, 5k, 5l, 11a, 11j, and 12c displayed higher antiviral activities than that of ningnanmycin. Compound 11a with a simple chemical structure, convenient synthetic route, and excellent antiviral activity emerged as a secondary lead compound. The docking results show that compounds 5i, 5k, and 11a have strong interactions with the tobacco mosaic virus coat protein (TMV CP). These compounds also exhibited significant fungicidal activities. Compounds 5g, 5k, 11j, and 11l displayed 9.15-17.45 μg/mL EC50 values against Pyricularia grisea, and compounds 5h (EC50: 8.01 μg/mL) and 11i (EC50: 15.23 μg/mL) exhibited a similar level of EC50 values with chlorothalonil (EC50: 7.33 μg/mL) against Physalospora piricola. Preliminary fungicidal mechanism research indicated that compound 5h has a certain destructive effect on the hyphae of P. piricola. This work lays a foundation for the application of laurene derivatives in plant protection.

Protein-Based Model for Energy Transfer between Photosynthetic Light-Harvesting Complexes Is Constructed Using a Direct Protein-Protein Conjugation Strategy.

Photosynthetic organisms utilize dynamic and complex networks of pigments bound within light-harvesting complexes to transfer solar energy from antenna complexes to reaction centers. Understanding the principles underlying the efficiency of these energy transfer processes, and how they may be incorporated into artificial light-harvesting systems, is facilitated by the construction of easily tunable model systems. We describe a protein-based model to mimic directional energy transfer between light-harvesting complexes using a circular permutant of the tobacco mosaic virus coat protein (cpTMV), which self-assembles into a 34-monomer hollow disk. Two populations of cpTMV assemblies, one labeled with donor chromophores and another labeled with acceptor chromophores, were coupled using a direct protein-protein bioconjugation method. Using potassium ferricyanide as an oxidant, assemblies containing o-aminotyrosine were activated toward the addition of assemblies containing p-aminophenylalanine. Both of these noncanonical amino acids were introduced into the cpTMV monomers through amber codon suppression. This coupling strategy has the advantages of directly, irreversibly, and site-selectively coupling donor with acceptor protein assemblies and avoids cross-reactivity with native amino acids and undesired donor-donor or acceptor-acceptor combinations. The coupled donor-acceptor model was shown to transfer energy from an antenna disk containing donor chromophores to a downstream disk containing acceptor chromophores. This model ultimately provides a controllable and modifiable platform for understanding photosynthetic interassembly energy transfer and may lead to the design of more efficient functional light-harvesting materials.

Design, Synthesis, and Bioactivity of Aldisine Derivatives Containing Oxime and Hydrazine Moieties Based on Hydrogen Bonds.

Marine natural products have attracted more and more attention in drug research and development due to their unique structure, diverse biological activities, and novel mode of action. Using antiviral alkaloid aldisine as the lead compound and drawing on the hydrogen bond effect widely used in drug design, derivatives containing oxime and hydrazone moieties were designed and synthesized by introducing functional groups with hydrogen-bond receptors or donors into molecules. The configuration of derivatives was systematically studied through nuclear Overhauser effect (NOE) spectroscopy and single crystal analysis. The antiviral activity test result showed that most derivatives had antiviral activity against tobacco mosaic virus (TMV), and some compounds had better activity than the commercial antiviral drug ribavirin, especially compounds 2 and 24, which had comparable activity to the most effective commercial antiviral drug ningnanmycin. Preliminary mode of action studies showed that compound 2 could affect the assembly of rod-shaped TMVs by promoting the aggregation and fragmentation of TMV coat proteins. Molecular docking experiments demonstrated that the introduction of oxime and hydrazone moieties could indeed increase the hydrogen bond between molecules and target proteins. In addition, we conducted fungicidal and larvicidal activities study of these derivatives. Most of these derivatives had good larvicidal activities against Mythimna separata and Plutella xylostella and showed broad-spectrum fungicidal activities.

Nanoparticle-dsRNA Treatment of Pollen and Root Systems of Diseased Plants Effectively Reduces the Rate of Tobacco Mosaic Virus in Contemporary Seeds.

Most crop viruses are carried and spread by seeds. Virus-infected seeds are seed-borne viral disease infections, and thus, reducing the rate of seed infection is an urgent problem in the seed-production industry. The objective of this study was to use nanoparticles (NPs) to directly deliver dsRNA into plants or pollen to initiate RNA interference (RNAi) to reduce viral carryover in seeds. Chitosan quaternary ammonium salt (HACC), complexed with dsRNAs, was selected for targeting the genes for the tobacco mosaic virus (TMV) coat protein (CP) and TMV RNA-dependent RNA polymerase (RdRP) to form HACC-dsRNA NPs. These NP-based dsRNAs were delivered to the plants using four different methods, including infiltration, spraying, root soaking, and pollen internalization. All four methods were able to reduce the seed-carrying rate of offspring seeds of the TMV-infected plants, with pollen internalization being the most effective in reducing the TMV-carrying rate from 95.1 to 61.1% in the control group. By measuring the plant uptake of fluorescence-labeled NPs and dsRNAs, the transportation of the HACC-dsRNA NPs into the plants was observed, and the uptake of dsRNA in combination with small RNA sequencing was further confirmed, resulting in the silencing of homologous RNA molecules during the topical application. The results demonstrated that the incidence of TMV infection was reduced by various degrees via RNAi induction without the need to develop transgenic plants. These results demonstrate the advantages of NP-based RNAi technology in breeding for disease resistance and developing a new strategy for virus-resistant breeding in plants.

Design, synthesis, antiviral and fungicidal activities of novel polycarpine simplified analogues

Fungal and viral diseases account for 70–80% of agricultural production losses caused by microbial diseases. Synthetic fungicides and antiviral agents have been used to treat plant diseases caused by plant pathogenic fungi and viruses, but their use has been criticized due to their adverse side effects. As alternative strategies, natural fungicides and antiviral agents have attracted many researchers’ interest in recent years. Herein, we designed and synthesized a series of novel polycarpine simplified analogues. Antiviral activity research against tobacco mosaic virus (TMV) revealed that most of the designed compounds have good antiviral activities. The virucidal activities of 4, 6d, 6f, 6h, and 8c are higher than that of polycarpine and similar to that of ningnanmycin. The structure simplified compound 8c was selected for further antiviral mechanism research which showed that compound 8c could inhibit the formation of 20S protein discs by acting on TMV coat protein. These compounds also displayed broad-spectrum fungicidal activities against 7 kinds of plant fungi. This work lays the foundation for the application of polycarpine simplified analogues in crop protection.

Discovery of Barakacin and Its Derivatives as Novel Antiviral and Fungicidal Agents

Pesticides are essential for the development of agriculture. It is urgent to develop green, safe and efficient pesticides. Bisindole alkaloids have unique and concise structures and broad biological activities, which make them an important leading skeleton in the creation of new pesticides. In this work, we synthesized bisindole alkaloid barakacin in a simple seven-step process, and simultaneously designed and synthesized a series of its derivatives. Biological activity research indicated that most of these compounds displayed good antiviral activities against tobacco mosaic virus (TMV). Among them, compound 14b exerted a superior inhibitory effect in comparison to commercially available antiviral agent ribavirin, and could be expected to become a novel antiviral candidate. Molecular biology experiments and molecular docking research found that the potential target of compound 14b was TMV coat protein (CP). These compounds also showed broad-spectrum anti-fungal activities against seven kinds of plant fungi.

Antiviral activity evaluation and action mechanism of myricetin derivatives containing thioether quinoline moiety.

A variety of myricetin derivatives containing thioether quinoline moiety were designed and synthesized. Their structures of title compounds were determined by 1H NMR, 13C NMR, 19F NMR, and HRMS. Single-crystal X-ray diffraction experiments were carried out with B4. Antiviral activity indicated that some of the target compounds exhibited remarkable anti-tobacco mosaic virus (TMV) activity. In particular, compound B6 possessed significant activity. The half maximal effective concentration (EC50) value of the curative activity of compound B6 was 169.0μg/mL, which was superior to the control agent ningnanmycin (227.2μg/mL). Meanwhile, the EC50 value of the protective activity of compound B6 was 86.5μg/mL, which was better than ningnanmycin (179.2μg/mL). Microscale thermophoresis (MST) indicated that compound B6 had a strong binding capability to the tobacco mosaic virus coat protein (TMV-CP) with a dissociation constant (Kd) value of 0.013μmol/L, which was superior to that of myricitrin (61.447μmol/L) and ningnanmycin (3.215μmol/L). And the molecular docking studies were consistent with the experimental results. Therefore, these novel myricetin derivatives containing thioether quinoline moiety could become potential alternative templates for novel antiviral agents.

Plant production of a virus-like particle-based vaccine candidate against porcine reproductive and respiratory syndrome.

Porcine reproductive and respiratory syndrome (PRRS) is a disease leading to spontaneous abortions and stillbirths in sows and lowered life quality and expectancy in growing pigs. PRRS is prevalent worldwide and has significant economic impacts to swine industries around the globe. Co-expression of the two most abundant proteins in the viral envelope, the matrix protein (M) and glycosylated protein 5 (GP5), can produce a neutralizing immune response for the virus providing a potentially effective subunit vaccine against the disease, but these proteins are difficult to express. The goal of this research was to display antigenic portions of the M and GP5 proteins on the surface of tobacco mosaic virus-like particles. A modified tobacco mosaic virus coat protein (TMVc) was transiently expressed in Nicotiana benthamiana leaves and targeted to three subcellular compartments along the secretory pathway to introduce glycosylation patterns important for M-GP5 epitope immunogenicity. We found that accumulation levels in the apoplast were similar to the ER and the vacuole. Because glycans present on plant apoplastic proteins are closest to those present on PRRSV proteins, a TMVc-M-GP5 fusion construct was targeted to the apoplast and accumulated at over 0.5 mg/g of plant fresh weight. TMVc virus-like particles self-assembled in plant cells and surface-displayed the M-GP5 epitope, as visualized by transmission electron microscopy and immunogold localization. These promising findings lay the foundation for immunogenicity and protective-immunity studies in animals to examine the efficacy of this vaccine candidate as a measure to control PRRS.