Tobacco Mosaic Virus Protein Research Articles

In Vivo Delivery of Spherical and Cylindrical In Vitro Reconstituted Virus-like Particles Containing the Same Self-Amplifying mRNA.

The dramatic effectiveness of recent mRNA (mRNA)-based COVID vaccines delivered in lipid nanoparticles has highlighted the promise of mRNA therapeutics in general. In this report, we extend our earlier work on self-amplifying mRNAs delivered in spherical in vitro reconstituted virus-like particles (VLPs), and on drug delivery using cylindrical virus particles. In particular, we carry out separate in vitro assemblies of a self-amplifying mRNA gene in two different virus-like particles: one spherical, formed with the capsid protein of cowpea chlorotic mottle virus (CCMV), and the other cylindrical, formed from the capsid protein of tobacco mosaic virus (TMV). The mRNA gene is rendered self-amplifying by genetically fusing it to the RNA-dependent RNA polymerase (RdRp) of Nodamura virus, and the relative efficacies of cell uptake and downstream protein expression resulting from their CCMV- and TMV-packaged forms are compared directly. This comparison is carried out by their transfections into cells in culture: expressions of two self-amplifying genes, enhanced yellow fluorescent protein (EYFP) and Renilla luciferase (Luc), packaged alternately in CCMV and TMV VLPs, are quantified by fluorescence and chemiluminescence levels, respectively, and relative numbers of the delivered mRNAs are measured by quantitative real-time PCR. The cellular uptake of both forms of these VLPs is further confirmed by confocal microscopy of transfected cells. Finally, VLP-mediated delivery of the self-amplifying-mRNA in mice following footpad injection is shown by in vivo fluorescence imaging to result in robust expression of EYFP in the draining lymph nodes, suggesting the potential of these plant virus-like particles as a promising mRNA gene and vaccine delivery modality. These results establish that both CCMV and TMV VLPs can deliver their in vitro packaged mRNA genes to immune cells and that their self-amplifying forms significantly enhance in situ expression. Choice of one VLP (CCMV or TMV) over the other will depend on which geometry of nucleocapsid is self-assembled more efficiently for a given length and sequence of RNA, and suggests that these plant VLP gene delivery systems will prove useful in a wide variety of medical applications, both preventive and therapeutic.

Analysis of Codon Usage Bias of Six Genes of Replicase/Coat Protein of Tobacco Mosaic Virus

Background: Tobacco mosaic virus (TMV) stands as a highly studied virus and consequently, its features and composition are extensively understood. It has been found to induce diverse infections in numerous plant species, with tobacco leaves being notably affected, showing mottled browning. Presently, the sole available method to control its spread is by removing infected plants. Understanding codon use bias is crucial as it could play a pivotal role in molecular interventions aimed at halting the virus’s replication and multiplication, thereby helping to contain its propagation. Methods: Currently, the research focuses on assessing codon bias within six genes related to the replicase/coat protein of TMV, namely TMVgp1, TMVgp2, TMVgp3, TMVgp4, TMVgp5 and TMVgp6. To conduct this analysis, various methods such as relative dinucleotide abundance, relative synonymous codon usage (RSCU), neutrality plot and parity rule 2 (PR2) plot were employed. Result: All of the identified genes had a modest codon bias, according to the study on codon usage, as well as the function of mutation pressure in gene TMVgp3 and natural selection in genes TMVgp1, TMVgp2, TMVgp4, TMVgp5 and TMVgp6. The Research into codon use bias showed that the TMV virus’s chosen genes are subjected to naturally occurring selection as well as mutational pressure.

First Report of Tobacco Mosaic Virus Infecting Anisomeles indica (L.) Kuntze in China.

Anisomeles indica (L.) Kuntze is a perennial erect herb that belongs to the genus Epimeredi, family Labiatae (Hsieh et al., 2008). This herb is distributed in several southern provinces such as Yunnan, Sichuan and Guizhou in China, and it is also exported to Southeast Asian countries such as Singapore and Malaysia (Li., 2010; Yao et al., 2019). Due to its market potential and broad development prospects, the herb has been cultivated in Yunnan. In August 2021, virus-like symptoms on leaves, including shrinking, mosaic, and yellow mottling(Fig S1. A) appeared on approximately 80% of A. indica in the experimental fields of the Kunming Institute of Botany, Chinese Academy of Science, in Kunming, Yunnan. To unveil the possible viral agents associated with the disease symptoms, leaf samples were collected from 5 plants for transmission electron microscopy (TEM) analysis using negative staining (Zhang et al., 2016). Rhabditiform-shaped particles around 300 × 18 nm (Fig S1. C) were observed, which resemble those of tobamoviruses. To identify the exact virus, total RNA was extracted from the 20 leaf samples using the RNA-easy Isolation Reagent (Vazyme, Nanjing, China), followed by reverse transcription (RT)-PCR with a degenerate tobamovirus primer pair (Li et al., 2014). A 480-bp amplicon was obtained from each sample and cloned into the pMD18-T vector for Sanger sequencing (Takara, Dalian, China). BLASTn-analysis revealed that the 20 amplicons were identical and shared 100% nucleotide sequence identity with tobacco mosaic virus (TMV) isolate Bei Cang Zhu from Atractylodes lancea (acc. no. KU198186) One sequence was deposited in the GenBank under the accession number OK489807. ELISA testing with TMV-specific antibody (Agdia, USA) produced positive results for all of the 20 leaf samples. In order to understand the difference between TMV isolates from A. indica and those form other host plants, the sequences of movement protein (MP, 807 bp) and RNA-dependent RNA polymerase (RdRp, 3351 bp) of TMV were also obtained from one of the TMV infected samples using the target gene special primers (Tab. S1), and submitted to GenBank under the accession number OM3662406 (MP) and OM366242 (RdRp). BLASTn-analysis revealed that the amplicon of MP shared 97.75% nucleotide sequence identity with TMV isolate Henan 9-2-2017 from sweet potato (MN186255.1) and RdRp shared 97.43% nucleotide sequence identity with TMV isolate SXFQ from Solanum lycopersicum (JX993906.1). Phylogenetic analysis indicated that the isolate of A. indica grouped with several TMV isolates (e.g., tomato, AF103779.1 and tobacco, HE818449.1) from Northern China. The virus was successfully transmitted onto healthy A.indica plants (n = 5) upon mechanical inoculation as the plants not only developed foliar distortion symptoms but also tested positive for TMV by RT-PCR with the CP-specific primers (Tab. S1). Taken together, our results demonstrated that the diseased A. indica plants were infected with TMV. To our knowledge, this is the first report of TMV infected A. indica (L.) Kuntze in China. Symptomatic phenotype-based field surveys on some plantations in Yunnan Province indicated that the disease incidence ranged from 70% to 90%, resulting in significant loss of production of A. indica. It is necessary to monitor the viruses in the fields and find effective methods to protect TMV in the A. indica (L.) Kuntze industry.

The coat protein of tobacco mosaic virus as an anti-tobacco mosaic virus: a molecular dynamics simulation

The Coat Protein (CP) of the Tobacco Mosaic Virus (TMV) executes an important duty in the protection of virus RNA. The interaction between the virus CP and host plant proteins induces infection in the host and creates dark and light green mosaics on crops, which disturb the growth and function of the plant. The interaction between the virus CP and the modified CP, expressed in transgenic plants, causes Coat Protein-Mediated Resistance (CP-MR), which reduces virus infection in transgenic plants. In this study, a model is suggested for resistance as “stop assembly of CP” in the virus. It is based on the fact that the CP, when mutated, acts as a dead-end in virus assembly. For evaluation of the model, we investigated the effect of four mutants including CBT28I, ABT42W, ABD77R, and ABT89W complexes on plant resistance against TMV infection by molecular dynamics simulation. Previous studies had shown the influence of such mutations on the CP-MR. The MD results of in the present study further confirmed the mentioned effect and demonstrated how the mutations could be the cause of CP-MR. The results are calculated by the RMSD, Rg, H-bond, and g-MMPBSA scripts. The change in binding energy between two chains is consistent with CP-MR such that with increase in binding energy, the affinity between two chains was reduced and the CP-MR increased. Based on this model, it is possible to design mutants with a high level of efficiency.Communicated by Ramaswamy H. Sarma

Inhibition of Cell-Free Translation and Replication of Tobacco Mosaic Virus RNA by Exogenously Added 5'-Proximal Fragments of the Genomic RNA.

Replication proteins of tobacco mosaic virus (TMV), a positive-sense RNA virus, co-translationally bind to a 5′-proximal ~70-nucleotide (nt) region of the genomic RNA, referred to as the nuclease-resistant (NR) region for replication template selection. Therefore, disruption of the interaction between the viral replication proteins and viral genomic RNA is expected to inhibit the replication of TMV. In this study, we demonstrate that the addition of small RNA fragments (18–33 nts in length) derived from different regions within the NR region inhibit the binding of TMV replication proteins to viral RNA and TMV RNA replication in a cell-free system. Intriguingly, some of the small RNA fragments also inhibited the translation of mRNA in a sequence-nonspecific manner. These results highlight the pleiotropic roles of the 5′-proximal region of the TMV genome.

The coat protein p25 from maize chlorotic mottle virus involved in symptom development and systemic movement of tobacco mosaic virus hybrids.

Viral coat protein (CP) has numerous critical functions in plant infection, but little is known about p25, the CP of maize chlorotic mottle virus (MCMV; Machlomovirus), which causes severe yield losses in maize worldwide. Here, we investigated the roles of p25 in pathogenicity and systemic movement, as well as potential interactions with host plants, using a hybrid tobacco mosaic virus (TMV)-based expression system. Highly conserved protein p25 is predicted to contain a membrane-anchored nuclear localization signal (NLS) sequence and an extracellular sequence. In transgenic Nicotiana benthamiana plants containing the movement protein (MP) of TMV (TMV-MP), p25 induced severe symptoms, including dwarf and foliar necrosis, and was detected in inoculated and non-inoculated leaves. After the deletion of NLS from nuclear-located p25, the protein was found throughout the host cell, and plant stunting and starch granule deformity were reduced. Systemic movement and pathogenicity were significantly impaired when the C-terminal regions of p25 were absent. Using virus-induced gene silencing (VIGS), the transcript level of heat shock protein HSP90 was distinctly lower in host plants in association with the absence of leaf necrosis induced by TMV-p25. Our results revealed crucial roles for MCMV p25 in viral pathogenicity, long-distance movement, and interactions with N. benthamiana.

Palmitoylation Is Indispensable for Remorin to Restrict Tobacco Mosaic Virus Cell-to-Cell Movement in Nicotiana benthamiana.

Remorin (REM) is a plant-specific plasma membrane-associated protein regulating plasmodesmata plasticity and restricting viral cell-to-cell movement. Here, we show that palmitoylation is broadly present in group 1 remorin proteins in Nicotiana benthamiana and is crucial for plasma membrane localization and accumulation. By screening the four members of N. benthamiana group 1 remorin proteins, we found that only NbREM1.5 could significantly hamper tobacco mosaic virus (TMV) cell-to-cell movement. We further showed that NbREM1.5 interacts with the movement protein of TMV in vivo and interferes with its function of expanding the plasmodesmata size exclusion limit. We also demonstrated that palmitoylation is indispensable for NbREM1.5 to hamper plasmodesmata permeability and inhibit TMV cell-to-cell movement.

Negative modulation of SA signaling components by the capsid protein of tobacco mosaic virus is required for viral long-distance movement.

An important aspect of plant-virus interaction is the way viruses dynamically move over long distances and how plant immunity modulates viral systemic movement. Salicylic acid (SA), a well-characterized hormone responsible for immune responses against virus, is activated through different transcription factors including TGA and WRKY. In tobamoviruses, evidence suggests that capsid protein (CP) is required for long-distance movement, although its precise role has not been fully characterized yet. Previously, we showed that the CP of Tobacco Mosaic Virus (TMV)-Cg negatively modulates the SA-mediated defense. In this study, we analyzed the impact of SA-defense mechanism on the long-distance transport of a truncated version of TMV (TMV ∆CP virus) that cannot move to systemic tissues. The study showed that the negative modulation of NPR1 and TGA10 factors allows the long-distance transport of TMV ∆CP virus. Moreover, we observed that the stabilization of DELLA proteins promotes TMV ∆CP systemic movement. We also characterized a group of genes, part of a network modulated by CP, involved in TMV ∆CP long-distance transport. Altogether, our results indicate that CP-mediated downregulation of SA signaling pathway is required for the virus systemic movement, and this role of CP may be linked to its ability to stabilize DELLA proteins.

How tobacco mosaic virus goes the distance.

Human viruses are the current focus of many virologists worldwide. However, the first virus that was discovered was a plant virus. In the nineteenth century, several researchers were investigating a disease in tobacco plants that caused mottled browning of leaves (Rifkind and Freeman, 2005). The Russian microbiologist Dmitry Ivanovsky found that this disease was caused by something so small that it was able to pass through a filter with pores that could hold back bacteria. Later, the Dutch botanist Willem Beijerinck showed that the disease-causing agent was ‘alive’ and self-replicating rather than a chemical toxin. The virus, now called tobacco mosaic virus (TMV), is still of major concern because—despite its name—it causes disease in a wide range of plants, including tomato (Solanum lycopersicum), cucumber (Cucumis sativus), and several ornamental flowers. The infectious virus particle—or virion—of TMV is composed of a single strand of RNA covered by a protein shell (capsid) that assembles into a rod-shaped structure. The capsid is entirely made up of subunits of a single protein, called coat protein. Without it, the virus is not able to form a virion. Virion assembly was long thought to play important roles in other aspects, such as the ability to move systemically through the phloem of the plant to reach distal tissues. Indeed, previous research showed that the virus is not able to move over long distances without the coat protein (Saito et al., 1990). However, more recent findings indicate the coat protein also has a function in the systemic movement of the virus that is not linked with the formation of virions (Bendahmane et al., 2007). In this issue, researchers from the Instituto de Agrobiotecnología y Biología Molecular, Argentina investigated how the coat protein of TMV facilitates long-distance movement (Venturuzzi et al., 2021). For this purpose, they used a mutant virus that lacks the gene encoding the coat protein and therefore cannot move systemically. When inoculated with the mutant virus, only a low number of plants accumulated the virus in tissues distal to the infection site. But when the authors co-inoculated the mutant virus with a vector encoding the coat protein (to complement the mutation), the virus was able to move systemically in most plants. Furthermore, when they inoculated one leaf with the virus and another, distal leaf with the coat protein-encoding vector, they observed long-distance movement as well. This indicates that the coat protein allows long-distance movement independent from virion formation. The salicylic acid (SA)-mediated defense response is a key pathway involved in the restriction of systemic infections in plants. Therefore, the authors investigated the role of this defense pathway in the long-distance movement of TMV by performing experiments with several SA-pathway mutants. This drew their attention towards NON-EXPRESSER OF PR GENES 1 (NPR1) and the TGACG-binding transcription factor TGA10. When they infected plant mutants of these genes with wild-type virus, they noticed that the virus accumulated at a higher level than in wild-type plants. Moreover, comparisons of gene expression levels of infected and non-infected plants showed that several genes that are downregulated by the coat protein in wild-type plants show a different expression pattern in npr1/tga10 mutant plants. This indicates that NPR1 and TGA10 might be involved in repressing viral movement upon recognition of the capsid protein. To test this hypothesis, the authors performed infection experiments in NPR1- and TGA10-silenced plants with a mutant virus that lacks the coat protein. In most of these plants, systemic movement of the mutant virus was observed. Previous studies from the same research group showed that the coat protein can stabilize DELLA transcription factors. As TGA10 is known to interact with DELLAs, the authors decided to investigate if these transcription factors are also involved in systemic movement of the virus. They found that overexpression of GIBBERELLIC ACID INSENSITIVE (GAI) allowed the systemic movement of the coat protein-deficient mutant. The same was true for plants in which DELLAs were stabilized through the application of the GA biosynthesis inhibitor Paclobutrazol. This indicates that DELLAs facilitate systemic movement, potentially through stabilization by the coat protein. The authors constructed a model to explain the roles of NPR1, TGA transcription factors, and DELLA proteins together with several other defense-related proteins that are involved in the systemic movement of TMV (Figure ). The corresponding author Sebastian Asurmendi explains that in the future, he aims to study these different players in more detail to establish their molecular functions. He is especially interested in understanding how the coat protein stabilizes DELLAs. DELLA proteins are involved in many physiological and developmental plant processes, and therefore a target for plant breeders. That they also play a role in viral movement implies that genetic manipulations of DELLAs might also affect plant immunity. For now, good advice for breeders targeting DELLAs: test how the plants respond to viral infection.

Identification of Proteins of Tobacco Mosaic Virus by Using a Method of Feature Extraction.

Tobacco mosaic virus, TMV for short, is widely distributed in the global tobacco industry and has a significant impact on tobacco production. It can reduce the amount of tobacco grown by 50–70%. In this research of study, we aimed to identify tobacco mosaic virus proteins and healthy tobacco leaf proteins by using machine learning approaches. The experiment's results showed that the support vector machine algorithm achieved high accuracy in different feature extraction methods. And 188-dimensions feature extraction method improved the classification accuracy. In that the support vector machine algorithm and 188-dimensions feature extraction method were finally selected as the final experimental methods. In the 10-fold cross-validation processes, the SVM combined with 188-dimensions achieved 93.5% accuracy on the training set and 92.7% accuracy on the independent validation set. Besides, the evaluation index of the results of experiments indicate that the method developed by us is valid and robust.

Transglutaminase-mediated assembly of multi-enzyme pathway onto TMV brush surfaces for synthesis of bacterial autoinducer-2

Bioelectronic microdevices, with spatially arranged biosynthetic machinery, can be programmed to convert raw materials to high-value products in a controlled manner. Generic methods for biofunctionalization that enable precise control over biocomponent assembly at the nano and meso scales are necessary to diversify the range and capabilities of these systems. Here, we used tobacco mosaic virus (TMV) derived virus like particles (VLPs) as 3D interfacial scaffolds for the assembly of biosynthetic enzymes onto gold electrodes. The TMV capsids are aligned in a vertical brush configuration by cysteine modifications to the capsid protein and by taking advantage of the well-known gold/cysteine affinity. This alignment enables high surface density and biosynthetic enzyme-enzyme proximity. Enzymes are covalently tethered to the capsid protein of TMV by the N- and C-terminal addition of lysine-rich assembly domains which react with surface exposed glutamine residues on the capsid surfaces; the lysine/glutamine linkages are mediated by a microbial transglutaminase (mTG). We demonstrate flexible mTG-mediated assembly of a three-enzyme biosynthetic pathway that converts S-adenosylmethionine (SAM) to autoinducer-2 (AI-2), a bacterial signal molecule that mediates quorum sensing behavior. We propose that our VLP and mTG based fabrication approach will help in the modular assembly of biological components onto microelectronic devices and that these will find utility in many applications including sensing and lab on chip devices.

Programming Dynamic Assembly of Viral Proteins with DNA Origami.

Biomolecular assembly in biological systems is typically a complex dynamic process regulated by the exchange of molecular information between biomolecules such as proteins and nucleic acids. Here, we demonstrate a nucleic-acid-based system that can program the dynamic assembly process of viral proteins. Tobacco mosaic virus (TMV) genome-mimicking RNA is anchored on DNA origami nanostructures via hybridization with a series of DNA strands which also function as locks that prevent the packaging of RNA by the TMV proteins. The selective, sequential releasing of the RNA via toehold-mediated strand displacement allows us to program the availability of RNA and subsequently the TMV growth in situ. Furthermore, the programmable dynamic assembly of TMV on DNA templates also enables the production of new DNA-protein hybrid nanostructures, which are not attainable by using previous assembly methods.

Synthesis, Biological Activity and Action Mechanism Study of Novel Chalcone Derivatives Containing Malonate

A series of novel chalcone malonate derivatives were synthesized and their antibacterial and antiviral activities were evaluated. All target compounds were characterized by spectral data. The results of antimicrobial bioassay showed that one compound (diethyl [3-(naphthalen-2-yl)-1-(3-nitrophenyl)-3-oxopropyl]propanedioate) showed excellent antibacterial activity against Xanthomonas oryzae pv. oryzae (Xoo), with an EC50 value of 10.2 μg/mL, which is significantly superior to bismerthiazol (71.7 μg/mL) and thiodiazole copper (97.8 μg/mL). At the same time, the mechanism of two compounds was confirmed by scanning electron microscopy. In addition, another compound (diethyl [3-(naphthalen-2-yl)-1-(4-nitrophenyl)-3-oxopropyl]propanedioate) showed significant curative activity to tobacco mosaic virus, with a value of 74.3 %, which was superior to 53.3 % of ningnanmycin. The results of microscale thermophoresis also showed that the Kd value of the combination of two compounds with the coat protein of tobacco mosaic virus was 0.211 and 0.166 μmol/L, which was better than 0.596 μmol/L of ningnanmycin. At the same time, the molecular docking of two compounds with tobacco mosaic virus-coat protein shows that the compound is well embedded in the pocket between the two subunits of tobacco mosaic virus-coat protein. These results show that chalcone derivatives containing malonate group can be considered as activators in the design of antibacterial and antiviral agents.

Complexes Formed via Bioconjugation of Genetically Modified TMV Particles with Conserved Influenza Antigen: Synthesis and Characterization.

Recently we obtained complexes between genetically modified Tobacco Mosaic Virus (TMV) particles and proteins carrying conserved influenza antigen such as M2e epitope. Viral vector TMV-N-lys based on TMV-U1 genome was constructed by insertion of chemically active lysine into the exposed N-terminal part of the coat protein. Nicotiana benthamiana plants were agroinjected and TMV-N-lys virions were purified from non-inoculated leaves. Preparation was analyzed by SDS-PAGE/Coomassie staining; main protein with electrophoretic mobility of 21 kDa was detected. Electron microscopy confirmed the stability of modified particles. Chemical conjugation of TMV-N-lys virions and target influenza antigen M2e expressed in E. coli was performed using 5 mM 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and 1 mM N-hydroxysuccinimide. The efficiency of chemical conjugation was confirmed by Western blotting. For additional characterization we used conventional electron microscopy. The diameter of the complexes did not differ significantly from the initial TMV-N-lys virions, but complexes formed highly organized and extensive network with dense "grains" on the surface. Dynamic light scattering demonstrated that the single peaks, reflecting the complexes TMV-N-lys/DHFR-M2e were significantly shifted relative to the control TMV-N-lys virions. The indirect enzyme-linked immunosorbent assay with TMV- and DHFR-M2e-specific antibodies showed that the complexes retain stability during overnight adsorption. Thus, the results allow using these complexes for immunization of animals with the subsequent preparation of a candidate universal vaccine against the influenza virus.

Synthesis and bioactivity evaluation of penta-1,4-diene-3-one oxime ether derivatives

A series of penta-1,4-diene-3-one oxime ether derivatives were synthesized, and their antiviral and antifungal activities were evaluated. Bioactivity evaluations showed that most target compounds had significant antiviral effects against tobacco mosaic virus (TMV). Among them, (1E,3Z,4E)-1-(4-(benzyloxy)phenyl)-5-(furan-2-yl)penta-1,4-dien-3-one O-(3-fluorobenzyl) oxime (5e) was found to have good curative activity against TMV, with an inhibition rate of 64.6%, which was better than that of ribavirin (45.2%). (1E,3Z,4E)-1-(4-(benzyloxy) phenyl)-5-(furan-2-yl)penta-1,4-dien-3-one O-((6-chloropyridin-3-yl)methyl) oxime (5d) had a remarkable protective effect against TMV, with an inhibitory rate of 66.9%, which was better than that of ribavirin (61.8%). The inhibitory rate of (1E,3Z,4E)-1-(2-(benzyloxy)phenyl)-5-(furan-2-yl)penta-1,4-dien-3-one O-(4-chlorobenzyl) oxime(5m) in inactivation activity against TMV was 87.0%, which was better than that of ribavirin (77.9%). Further molecular docking studies indicated that compound 5m shows strong binding affinities toward the coat protein of tobacco mosaic virus. This result indicates that penta-1,4-diene-3-one oxime ether derivatives can play a significant role in discovering new antiviral agents.

Functionalized Tobacco Mosaic Virus Coat Protein Monomers and Oligomers as Nanocarriers for Anti-Cancer Peptides.

Components with self-assembly properties derived from plant viruses provide the opportunity to design biological nanoscaffolds for the ordered display of agents of diverse nature and with complementing functions. With the aim of designing a functionalized nanoscaffold to target cancer, the coat protein (CP) of Tobacco mosaic virus (TMV) was tested as nanocarrier for an insoluble, highly hydrophobic peptide that targets the transmembrane domain of the Neuropilin-1 (NRP1) receptor in cancer cells. The resulting construct CPL-K (CP-linker-“Kill”) binds to NRP1 in cancer cells and disrupts NRP1 complex formation with PlexA1 as well as downstream Akt survival signaling. The application of CPL-K also inhibits angiogenesis and cell migration. CP was also fused to a peptide that targets the extracellular domain of NRP1 and this fusion protein (CPL-F, CP-Linker-“Find”) is shown to bind to cultured cancer cells and to inhibit NRP1-dependent angiogenesis as well. CPL-K and CPL-F maintain their anti-angiogenic properties upon co-assembly to oligomers/nanoparticles together with CPL. The observations show that the CP of TMV can be employed to generate a functionalized nanoparticle with biological activity. Remarkably, fusion to CPL allowed us to solubilize the highly insoluble transmembrane NRP1 peptide and to retain its anti-angiogenic effect.

Elementary Steps in Plant‐Pathogen Recognition Revealed by Enhancing the Stability and Structure of Domains of Plant Innate Immune Receptors

Pathogens elicit an immune response in plants through the recognition of the avirulent effectors by multi‐domain intracellular disease resistance receptors. The nature and specificity of plant‐pathogen interactions and the molecular mechanism of eliciting an immune response is being explored using the N protein from Nicotianaglutinosa. The N protein is an archetype of the nucleotide binding (NB), leucine rich repeat (LRR) family of innate immune receptors (NLRs), and the interaction with its avirulent effector, the helicase domain (HEL) of the replicase protein of tobacco mosaic virus (TMV) triggers a classic hypersensitive cell death response in plants. An initial step in activating cell death pathways upon pathogen recognition is a structure change that enables NLR assembly through their N‐terminal Toll‐Interlukin1 Receptor‐like (TIR) domains. Stabilized domains with increased tendency to self‐associate have been constructed by focused mutagenesis guided by computational modeling of a 3.0 Å crystal structure of the N‐TIR domain. The energetics of TIR dimerization is being correlated with the severity of the hypersensitive response by measuring salicylic acid using LC‐MS to examine the hypothesis that receptor assembly is correlated with NLR signaling. Yeast surface display is being used to probe the manner whereby host NLRs recognize pathogenic determinants, and reveals that the interaction of N with its pathogen effector, the HEL domain of TMV, is mediated via its LRR domain. Binding analyses show that a stabilized LRR domain binds HEL about 250‐fold more strongly than a non‐virulent homologue that does not display a hypersensitive response. Variants of the full‐length N protein are being rebuilt from the individually stabilized TIR and LRR domains optimized for functional analyses to enable biochemical and structural investigations of plant NLR receptors.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Molecular Mechanics Simulations and Improved Tight-Binding Hamiltonians for Artificial Light Harvesting Systems: Predicting Geometric Distributions, Disorder, and Spectroscopy of Chromophores in a Protein Environment.

We present molecular mechanics and spectroscopic calculations on prototype artificial light harvesting systems consisting of chromophores attached to a tobacco mosaic virus (TMV) protein scaffold. These systems have been synthesized and characterized spectroscopically, but information about the microscopic configurations and geometry of these TMV-templated chromophore assemblies is largely unknown. We use a Monte Carlo conformational search algorithm to determine the preferred positions and orientations of two chromophores, Coumarin 343 together with its linker and Oregon Green 488, when these are attached at two different sites (104 and 123) on the TMV protein. The resulting geometric information shows that the extent of disorder and aggregation properties and therefore the optical properties of the TMV-templated chromophore assembly are highly dependent on both the choice of chromophores and the protein site to which they are bound. We use the results of the conformational search as geometric parameters together with an improved tight-binding Hamiltonian to simulate the linear absorption spectra and compare with experimental spectral measurements. The ideal dipole approximation to the Hamiltonian is not valid because the distance between chromophores can be very small. We found that using the geometries from the conformational search is necessary to reproduce the features of the experimental spectral peaks.

Tobacco Mosaic Virus with Peroxidase-Like Activity for Cancer Cell Detection through Colorimetric Assay.

Cell-based ELISA (CELLISA) has been widely used in disease diagnosis due to its simplicity and low cost. Recently, peroxidase-like nanomaterials have emerged as promising systems for CELLISA applications. In this work, tobacco mosaic virus (TMV) was simultaneously tailored with peroxidase-like inorganic nanoparticles (platinum nanoparticles) and cancer cell target groups (folic acid, FA) to obtain TMV-FA-Pt nanoparticles for cancer cell detection. Induced by the uniformly distributed reactive groups and well-defined structure of the TMV particle, platinum nanoparticles could be grown in situ on the exterior surface of TMV with excellent monodispersity and uniform spatial distribution. Meanwhile, FA with a PEG1000 linker was successfully conjugated to the coat proteins of TMV through the Cu(I)-catalyzed alkyne-azide cycloaddition reaction, an efficient "click" chemistry. Our study demonstrated that the resultant TMV-FA-Pt had specific affinity to cancer cells and was successfully used to detect cancer cells through CELLISA. Less than 1.0 × 104 cells/mL of cancer cells could be readily detected.

Binding studies between cytosinpeptidemycin and the superfamily 1 helicase protein of tobacco mosaic virus.

Tobacco mosaic virus (TMV) helicases play important roles in viral multiplication and interactions with host organisms. They can also be targeted by antiviral agents. Cytosinpeptidemycin has a good control effect against TMV. However, the mechanism of action is unclear. In this study, we expressed and purified TMV superfamily 1 helicase (TMV-Hel) and analyzed its three-dimensional structure. Furthermore, the binding interactions of TMV-Hel and cytosinpeptidemycin were studied. Microscale thermophoresis and isothermal titration calorimetry experiments showed that cytosinpeptidemycin bound to TMV-Hel with a dissociation constant of 0.24–0.44 μM. Docking studies provided further insights into the interaction of cytosinpeptidemycin with the His375 of TMV-Hel. Mutational and Microscale thermophoresis analyses showed that cytosinpeptidemycin bound to a TMV-Hel mutant (H375A) with a dissociation constant of 14.5 μM. Thus, His375 may be the important binding site for cytosinpeptidemycin. The data are important for designing and synthesizing new effective antiphytoviral agents.