Assembly Of Tobacco Mosaic Virus Research Articles

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.

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.

Discovery of novel phenothiazine derivatives as new agrochemical alternatives for treating plant viral diseases.

Plant viral diseases, namely 'plant cancer', are extremely difficult to control. Even worse, few antiviral agents can effectively control and totally block viral infection. There is an urgent need to explore and discover novel agrochemicals with high activity and a unique mode of action to manage these refractory diseases. Forty-one new phenothiazine derivatives were prepared and their inhibitory activity against tobacco mosaic virus (TMV) was assessed. Compound A8 had the highest protective activity against TMV, with a half-maximal effective concentration (EC50 ) of 115.67 μg/mL, which was significantly better than that of the positive controls ningnanmycin (271.28 μg/mL) and ribavirin (557.47 μg/mL). Biochemical assays demonstrated that compound A8 could inhibit TMV replication by disrupting TMV self-assembly, but also enabled the tobacco plant to enhance its defense potency by increasing the activities of various defense enzymes. In this study, novel phenothiazine derivatives were elaborately fabricated and showed remarkable anti-TMV behavior that possessed the dual-action mechanisms of inhibiting TMV assembly and invoking the defense responses of tobacco plants. Moreover, new agrochemical alternatives based on phenothiazine were assessed for their antiviral activities and showed extended agricultural application. © 2023 Society of Chemical Industry.

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.

Elucidation of the viral disassembly switch of tobacco mosaic virus.

Stable capsid structures of viruses protect viral RNA while they also require controlled disassembly for releasing the viral genome in the host cell. A detailed understanding of viral disassembly processes and the involved structural switches is still lacking. This process has been extensively studied using tobacco mosaic virus (TMV), and carboxylate interactions are assumed to play a critical part in this process. Here, we present two cryo‐EM structures of the helical TMV assembly at 2.0 and 1.9 Å resolution in conditions of high Ca2+ concentration at low pH and in water. Based on our atomic models, we identify the conformational details of the disassembly switch mechanism: In high Ca2+/acidic pH environment, the virion is stabilized between neighboring subunits through carboxyl groups E95 and E97 in close proximity to a Ca2+ binding site that is shared between two subunits. Upon increase in pH and lower Ca2+ levels, mutual repulsion of the E95/E97 pair and Ca2+ removal destabilize the network of interactions between adjacent subunits at lower radius and release the switch for viral disassembly.

Phthalocyanine-Virus Nanofibers as Heterogeneous Catalysts for Continuous-Flow Photo-Oxidation Processes.

The generation of highly reactive oxygen species (ROS) at room temperature for application in organic synthesis and wastewater treatment represents a great challenge of the current chemical industry. In fact, the development of biodegradable scaffolds to support ROS-generating active sites is an important prerequisite for the production of environmentally benign catalysts. Herein, the electrostatic cocrystallization of a cationic phthalocyanine (Pc) and negatively charged tobacco mosaic virus (TMV) is described, together with the capacity of the resulting crystals to photogenerate ROS. To this end, a novel peripherally crowded zinc Pc (1) is synthesized. With 16 positive charges, this photosensitizer shows no aqueous aggregation, and is able to act as a molecular glue in the unidimensional assembly of TMV. A step-wise decrease of ionic strength in mixtures of both components results in exceptionally long fibers, constituted by hexagonally bundled viruses thoroughly characterized by electron and confocal microscopy. The fibers are able to produce ROS in a proof-of-concept microfluidic device, where they are immobilized and irradiated in several cycles, showing a resilient performance. The bottom-up approach also enables the light-triggered disassembly of fibers after use. This work represents an important example of a biohybrid material with projected application in light-mediated heterogeneous catalysis.

Natural Products for Drug Discovery: Discovery of Gramines as Novel Agents against a Plant Virus.

Plant viral diseases seriously affect crop yield and quality. The natural product gramine (1) and its simple structural analogues 2-35 were synthesized from indoles, amines, and aldehydes in one step. The antiviral effects of these alkaloids were evaluated systematically. Most of these compounds were found to have higher antiviral effects than commercial ribavirin for the first time. Especially compounds 22, 30, and 31 exhibited significantly higher effects than ningnanmycin, thereby emerging as novel antiviral leads for further optimization. The preliminary implementation indicated that these compounds likely inhibit the assembly of tobacco mosaic virus (TMV) by cross-linking TMV capsid protein. Gramine analogues were also found to have broad-spectrum fungicidal effects. Although gramine has been reported to have influence on germination and development of Erysiphe graminis, these compounds displayed no fungicidal effects against Blumeria graminis f. sp. tritici on wheat in our test. Some of these compounds also exhibited certain insecticidal activities.

Synthesis, bioactivity and mode of action of 5A 5B 6C tricyclic spirolactones as novel antiviral lead compounds.

Plant viral diseases cause tremendous decreases in yield and quality. Natural polycyclic compounds such as those containing carbocycles are often very important lead compounds for drug and pesticide development. Tricyclic spiranoid lactones with 5A 5B 6C -ring fusion topologies possess various bioactivities. In this study, 33 new 5A 5B 6C tricyclic spirolactones were rationally designed, synthesized, characterized and evaluated for antiviral activities. These compounds showed no apparent toxicity against Italian honeybees up to 2.73 µg bee-1 . Spirolactones 14, 16, 19, 23 and 28 at a concentration of 100 µg mL-1 inactivated 90% of tobacco mosaic virus (TMV) infection, making these compounds much more potent than the positive controls. Significantly, compound 19 displayed the best inactivation activity causing inhibition of up to 98%. The results of the bioassays and QSAR studies indicated that the carbon-containing cyclic moiety was the antiviral pharmacophore, and derivative 19, which showed the best inactivation activity, could emerge as a potential antiviral agent against TMV. In vitro capsid protein (CP) assembly and TMV assembly inhibition determinations indicated that these compounds induced crosslinking in the TMV and prevented its uncoating, which was a putative new mode of action for TMV inactivation. © 2018 Society of Chemical Industry.

Selective in Situ Assembly of Viral Protein onto DNA Origami.

Engineering hybrid protein-DNA assemblies in a controlled manner has attracted particular attention, for their potential applications in biomedicine and nanotechnology due to their intricate folding properties and important physiological roles. Although DNA origami has served as a powerful platform for spatially arranging functional molecules, in situ assembly of proteins onto DNA origami is still challenging, especially in a precisely controlled and facile manner. Here, we demonstrate in situ assembly of tobacco mosaic virus (TMV) coat proteins onto DNA origami to generate programmable assembly of hybrid DNA origami-protein nanoarchitectures. The protein nanotubes of controlled length are precisely anchored on the DNA origami at selected locations using TMV genome-mimicking RNA strands. This study opens a new route to the organization of protein and DNA into sophisticated protein-DNA nanoarchitectures by harnessing the viral encapsidation mechanism and the programmability of DNA origami.

RNA-Directed Assembly of Tobacco Mosaic Virus (TMV)-Like Carriers with Tunable Fractions of Differently Addressable Coat Proteins.

Taking advantage of the ability for in vitro assembly of the plant-infecting virus tobacco mosaic virus (TMV), rod-shaped nanoscale scaffolds presenting different addressable groups can be obtained. We have established procedures resulting in virus-like particles with randomly distributed functional groups, with different groups arranged in striped but randomized structures, and even with distinct groups clustered in adjacent, better-defined domains. The TMV coat protein (CP) variants combined in these approaches can either originate all from TMV mutants propagated in planta, or be mixed with CP expressed in E. coli (CPEc). Protocols for expression and purification of a CPEc-His6 mutant in E. coli as well as the different methods for in vitro assembly and the visualization by decoration of one CP type are explained in detail.

Integrated Methods to Manufacture Hydrogel Microparticles with High Protein Conjugation Capacity and Binding Kinetics via Viral Nanotemplate Display.

Genetically modified tobacco mosaic virus (TMV) can serve as a potent nanotemplate for high capacity protein conjugation through covalent coupling to its coat proteins with precise nanoscale spacing. TMV's own genomic RNA can also be exploited for orientationally controlled assembly onto various platforms with sequence and spatial selectivity via nucleic acid hybridization. Here we describe detailed methods for fabrication of hydrogel microparticles with capture DNA sequences, chemical activation and programming of TMV templates, TMV assembly with the microparticles and protein conjugation via bio-orthogonal click reactions.

Dynamic DNA-controlled "stop-and-go" assembly of well-defined protein domains on RNA-scaffolded TMV-like nanotubes.

A DNA-based approach allows external control over the self-assembly process of tobacco mosaic virus (TMV)-like ribonucleoprotein nanotubes: their growth from viral coat protein (CP) subunits on five distinct RNA scaffolds containing the TMV origin of assembly (OAs) could be temporarily blocked by a stopper DNA oligomer hybridized downstream (3') of the OAs. At two upstream (5') sites tested, simple hybridization was not sufficient for stable stalling, which correlates with previous findings on a non-symmetric assembly of TMV. The growth of DNA-arrested particles could be restarted efficiently by displacement of the stopper via its toehold by using a release DNA oligomer, even after storage for twelve days. This novel strategy for growing proteinaceous tubes under tight kinetic and spatial control combines RNA guidance and its site-specific but reversible interruption by DNA blocking elements. As three of the RNA scaffolds contained long heterologous non-TMV sequence portions that included the stopping sites, this method is applicable to all RNAs amenable to TMV CP encapsidation, albeit with variable efficiency most likely depending on the scaffolds' secondary structures. The use of two distinct, selectively addressable CP variants during the serial assembly stages finally enabled an externally configured fabrication of nanotubes with highly defined subdomains. The "stop-and-go" strategy thus might pave the way towards production routines of TMV-like particles with variable aspect ratios from a single RNA scaffold, and of nanotubes with two or even more adjacent protein domains of tightly pre-defined lengths.

Nanomechanical characterization of rod-like superlattice assembled from tobacco mosaic viruses

Tobacco mosaic virus (TMV) and TMV-derived materials have demonstrated their great potential in biomedical applications, where the mechanical properties are determining factors for their proper functionalities and structural integrity. Recently, it has been found that a superlattice structure can be formed by two-dimensional hexagonal packing TMV self-assembly in Barium ions solution. In parallel to the exploration of possible applications of TMV superlattice, the mechanical properties were characterized by the atomic force microscopy based nanoindentation. The elastic modulus of 2.14 GPa was obtained by application of the extended Johnson-Kendall-Roberts (JKR) model with the force vs sample deformation data. The adhesion force was taken into consideration, and an easy-to-implement approach of using the extended JKR model was proposed by processing both the theoretical model and the experimental data. Finite element analysis was conducted to evaluate the reinforcing effect of the like-charge forces between the TMVs and the mechanical properties of the TMV superlattice. Using the Halpin-Tsai model, the transverse elastic modulus of the superlattice sample varied within 2.00–4.38 GPa, depending on the indentation locations. Attraction-repulsion equilibrium was found to maintain the packing of TMVs. This provides useful information to address the sources of the attraction and repulsion forces to control the TMV assembly.

Slam-Freezing as a Cryopreservation Tool for X-Ray Scattering Study on Membrane Samples

Reducing radiation damage is critical in X-ray scattering experiments for biological systems. A common technique involves rapidly freezing the sample and preserving it at cryogenic temperatures. However, this approach is only applicable to small samples, whose sizes are usually less than several tens of microns. To study 2D structures on a large, flat surface, we adopted the slam-freezing technique by using a modified cryostat and a computer-controlled linear motor. A special sample cell with an 80 µm-thick, double side polished silicon wafer was designed, with the aqueous solution having a contact area with silicon substrate of about 1 cm2. The influence of Polyethylene glycol (PEG) as a cryoprotectant was investigated. The aqueous solution could reach vitreous state at a PEG concentration of about 50% (v/v), verified by both in-situ grazing small angle X-ray scattering (GISAXS) and wide angle X-ray diffraction (WAXD) experiments. 2D assembly of tobacco mosaic virus (TMV) on lipid mono-layer grown on Octadecyltrichlorosilane-coated (OTS-coated) silicon surface was tested under both native state (in solution) and vitreous state using slam-freezing technique. It was shown that current protocol could significantly increase the dose tolerance of the sample in X-ray scattering experiments.

Fabrication of Chitosan-Poly(ethylene glycol) Hybrid Hydrogel Microparticles via Replica Molding and Its Application toward Facile Conjugation of Biomolecules

We demonstrate a facile scheme to fabricate nonspherical chitosan-poly(ethylene glycol) (PEG) microparticle platforms for conjugation of biomolecules with high surface density. Specifically, we show that PEG microparticles containing short chitosan oligomers are readily fabricated via replica molding (RM). Fluorescence and FTIR microscopy results illustrate that these chitosan moieties are incorporated with PEG networks in a stable manner while retaining chemical reactivity toward amine-reactive chemistries. The chitosan-PEG particles are then conjugated with single-stranded (ss) DNAs via Cu-free click chemistry. Fluorescence and confocal microscopy results show facile conjugation of biomolecules with the chitosan-PEG particles under mild conditions with high selectivity. These ssDNA-conjugated chitosan-PEG particles are then enlisted to assemble tobacco mosaic virus (TMV) via nucleic acid hybridization as an example of orientationally controlled conjugation of supramolecular targets. Results clearly show controllable TMV assembly with high surface density, indicating high surface DNA density on the particles. Combined, these results demonstrate a facile fabrication-conjugation scheme for robust biomolecular conjugation or assembly platforms. We expect that our approach can be enlisted in a wide array of biomolecular targets and applications.

Antofine Analogues Can Inhibit Tobacco Mosaic Virus Assembly through Small‐Molecule–RNA Interactions

The extra unpaired base(s) or bulged structures of nucleic acids are capable either of forming complexes with nucleic-acid-binding proteins or of acting as binding sites for small molecules. We are interested in developing bulge-specific agents as potential drugs or chemical tools in biological research. Antofine can selectively bind with DNA and RNA bulged structures (Xi et al., Bioorg. Med. Chem. Lett. 2006, 16, 4300-4304). Furthermore, a series of antofine analogues suitable for selective binding with TMV RNA rather than with TMV coat protein (CP) were found. Biochemical studies indicated that antofine and its analogues disrupt in vitro virus assembly through small-molecule-RNA interactions. A structural model to illustrate these effects has been proposed. It is suggested that antofine analogues bind selectively with RNA bulged structures and therefore disrupt interaction between TMV RNA and TMV CP.

A Kinetic Zipper Model and the Assembly of Tobacco Mosaic Virus

We put forward a modified Zipper model inspired by the statics and dynamics of the spontaneous reconstitution of rodlike tobacco mosaic virus particles in solutions containing the coat protein and the single-stranded RNA of the virus. An important ingredient of our model is an allosteric switch associated with the binding of the first protein unit to the origin-of-assembly domain of the viral RNA. The subsequent addition and conformational switching of coat proteins to the growing capsid we believe is catalyzed by the presence of the helical arrangement of bound proteins to the RNA. The model explains why the formation of complete viruses is favored over incomplete ones, even though the process is quasi-one-dimensional in character. We numerically solve the relevant kinetic equations and show that time evolution is different for the assembly and disassembly of the virus, the former exhibiting a time lag even if all forward rate constants are equal. We find the late-stage assembly kinetics in the presence of excess protein to be governed by a single-exponential relaxation, which agrees with available experimental data on TMV reconstruction.

From Virus Structure to Chromatin: X-ray Diffraction to Three-Dimensional Electron Microscopy

Early influences led me first to medical school with a view to microbiology, but I felt the lack of a deeper foundation and changed to chemistry, which in turn led me to physics and mathematics. I moved to the University of Cape Town to work on the X-ray crystallography of some small organic compounds. I developed a new method of using molecular structure factors to solve the crystal structure, which won me a research studentship to Trinity College Cambridge and the Cavendish Laboratory. There I worked on the austenite-pearlite transition in steel. This is governed by the dissipation of latent heat, and I ended up numerically solving partial differential equations. I used the idea of nucleation and growth during the phase change, which had its echo when I later tackled the assembly of Tobacco mosaic virus (TMV) from its constituent RNA and protein subunits. I wanted to move on to X-ray structure analysis of large biological molecules and obtained a Nuffield Fellowship to work in J.D. Bernal's department at Birkbeck College, London. There, I met Rosalind Franklin, who had taken up the study of TMV. I was able to interpret some of Franklin's beautiful X-ray diffraction patterns of the virus particle. From then on, my fate was sealed. After Franklin's untimely death in 1958, I moved in 1962 to the newly built MRC Laboratory of Molecular Biology in Cambridge, which, under Max Perutz, housed the original MRC unit from the Cavendish Laboratory. I was thus privileged to join the Laboratory at an early stage in its expansion and consequently able to take advantage of, and to help build up, its then unique environment of intellectual and technological sophistication. There I have remained ever since.

Self-Assembly of Tobacco Mosaic Virus at Oil/Water Interfaces

The oil/water interfacial assembly of tobacco mosaic virus (TMV) has been studied in situ by tensiometry and small-angle X-ray and neutron scattering (SAXS and SANS). TMV showed different orientations at the perfluorodecalin/water interface, depending on the initial TMV concentration in the aqueous phase. At low TMV concentration, the rods oriented parallel to the interface, mediating the interfacial interactions at the greatest extent per particle. At high TMV concentrations, the rods were oriented normal to the interface, mediating the interfacial interactions and also neutralizing inter-rod electrostatic repulsion. We found that the inter-rod repulsive forces between TMVs dominated the in-plane packing, which was strongly affected by the ionic strength and the bulk solution but not by the pH in the range of pH = 6-8.