Tobacco Mosaic Virus Particles Research Articles

Pathological Changes in Sweet Basil (Ocimum basilicum L.) Caused by Mixed Infection of Tobacco Mosaic Virus and Phytoplasma

Ocimum basilicum L. (a medicinal plant known as sweet basil) was investigated and showed disease symptoms, including mosaic mottling, leaf distortion, blistering, witches’ broom, and phyllody. The plants were found to be infected by both tobacco mosaic virus (TMV) and phytoplasma. Positive TMV was indicated by serological identification using direct antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). Phytoplasma infection was identified molecularly using the phytoplasma P1/P7 primer pair and the nested primer, R16F2n/R16R2, which successfully generated a DNA band at ~1200 bp. The virus was successfully transmitted mechanically to Nicotiana tabacum, Datura metal, Datura stramonium, and Phaseolus vulgaris. Light and transmission electron microscopy showed pleomorphic phytoplasmas and rod-shaped particles (300 nm) of TMV, respectively. Histopathological and cytopathological studies revealed changes in the cell behavior of the infected leaves of sweet basil due to virus and phytoplasma infection. Hypoplasia and hyperplasia were observed in the mosaic areas of the mesophyll. Alterations in the vascular tissues and degeneration of the organelles were observed in ultrathin sections of the infected mesophyll. The presence of phytoplasma cells and crystalline bodies were indicative of the mixed infection. To our knowledge, this is the first report of a mixed infection of TMV and phytoplasmas in sweet basil.

Analysis of Engineered Tobacco Mosaic Virus and Potato Virus X Nanoparticles as Carriers for Biocatalysts.

Plant virus nanoparticles are promising candidates for the development of novel materials, including nanocomposites and scaffolds/carriers for functional molecules such as enzymes. Their advantages for enzyme immobilization include a modular organization, a robust and programmable structure, and a simple, cost-effective production. However, the activity of many enzymes relies on posttranslational modification and most plant viruses replicate in the cytoplasm, so functional enzymes cannot be displayed on the virus surface by direct coat protein fusions. An alternative display system to present the Trichoderma reesei endoglucanase Cel12A on potato virus X (PVX) using SpyTag/SpyCatcher (ST/SC) technology was recently developed by the authors, which allows the carrier and enzyme to be produced separately before isopeptide conjugation. Although kinetic analysis clearly indicated efficient biocatalyst activity, the PVX carrier interfered with substrate binding. To overcome this, the suitability of tobacco mosaic virus (TMV) was tested, which can also accommodate a larger number of ST peptides. We produced TMV particles displaying ST as a new platform for the immobilization of enzymes such as Cel12A, and compared its performance to the established PVX-ST platform in terms of catalytic efficiency. Although more enzyme molecules were immobilized on the TMV-ST particles, we found that the rigid scaffold and helical spacing significantly affected enzyme activity.

Identification of anti-TMV active flavonoid glycosides and their mode of action on virus particles from Clematis lasiandra Maxim.

Tobacco mosaic virus (TMV) is a disreputable plant pathogen that causes a decline in the quality and yield of various economic crops. Natural products are important potential sources of biopesticides to control TMV. This study focuses on the discovery of anti-TMV active flavonoid glycosides and their mode of action on TMV particles from Clematis lasiandra Maxim. A new benzoyl acylated flavonoid glycoside, kaempferol 3-O-(2''-benzoyl)-β-d-glucopyranosyl-7-O-α-l-rhamnopyranoside (1), and nine known flavonoids (2-10) were identified first from C. lasiandra. The hydroxyl group at C-7, E-p-coumarate at C-6'' in the Glc of C-6, and the glucuronic acid at C-3 were functional groups for the antiviral flavonoid glycosides. Flavonoids 2, 5, and 6 showed higher inactivation efficacies of 64.62% to 82.54% compared with ningnanmycin at 500 μg ml-1 . The protective and curative efficacies for 2 and 5 were 57.44-59.00% and 41.17-43.92% at 500 μg ml-1 , respectively. Compound 5 showed higher TMV systemic resistance with control efficacies of 41.64%, 36.56% and 27.62% at concentrations of 500, 250 and 125 μg ml-1 compared with ningnanmycin in K326 tobaccos, respectively. Compound 5 can directly fracture TMV particles into small fragments combining with the fusion phenomena, and TMV-CP was an important target for 5 to break TMV particles. Flavonoid glycosides from C. lasiandra showed potent antiviral activities against TMV with multiple modes of action including inactivation, protective and curative effects, and inducing systemic resistance. TMV-CP was an important target for active flavonoid glycosides to fracture TMV particles. The results provided evidence that flavonoid glycosides from C. lasiandra have the potential to control TMV.

Detection of plant virus particles with a capacitive field-effect sensor

Plant viruses are major contributors to crop losses and induce high economic costs worldwide. For reliable, on-site and early detection of plant viral diseases, portable biosensors are of great interest. In this study, a field-effect SiO2-gate electrolyte-insulator-semiconductor (EIS) sensor was utilized for the label-free electrostatic detection of tobacco mosaic virus (TMV) particles as a model plant pathogen. The capacitive EIS sensor has been characterized regarding its TMV sensitivity by means of constant-capacitance method. The EIS sensor was able to detect biotinylated TMV particles from a solution with a TMV concentration as low as 0.025 nM. A good correlation between the registered EIS sensor signal and the density of adsorbed TMV particles assessed from scanning electron microscopy images of the SiO2-gate chip surface was observed. Additionally, the isoelectric point of the biotinylated TMV particles was determined via zeta potential measurements and the influence of ionic strength of the measurement solution on the TMV-modified EIS sensor signal has been studied.

(IMCS Second Place Best Paper Award) High-Density Immobilization of Tobacco Mosaic Virus Nanotubes As Enzyme Nanocarriers Onto Field-Effect Biosensor Structures

Introduction Viruses are not only infectious agents, they are also known as promising functional building blocks for application in nano- and biotechnologies. The tobacco mosaic virus (TMV) was the first studied plant virus and is widely distributed; it infects vegetables, like tomato, bell pepper, beans and other members of the family Solanaceae, while it is totally harmless for mammals [1]. It is one of the most studied plant viruses and its genome is completely sequenced, whereby genetical and chemical modification is easy. TMV has a nanotube-like shape with a length of 300 nm, an outer diameter of 18 nm and an inner diameter of 4 nm. Since it possesses a high chemical and physical robustness, it can be integrated with different electronic transducers for bio- and chemical sensing applications [2].Recently, we presented a TMV-based amperometric glucose biosensor [3] and a potentiometric penicillin biosensor [4]. TMV was used as enzyme nanocarrier for the enzyme glucose oxidase and penicillinase, respectively [3,4]. The sensitivity and detection limit of these biosensors, among others, depend on the density of TMVs on the sensor surface. The surface density of the immobilized TMVs is strongly influenced by the electrostatic interactions between the charged TMVs and sensor surface as well as by the inter-TMV-nanotubes repulsion, which could be changed by varying the pH value and the ionic strength of the TMV solution. In this study, we investigated an impact of the pH value and ionic strength of the TMV solution on the surface density of TMV nanotubes immobilized onto Ta2O5-gate capacitive field-effect electrolyte-insulator-semiconductor (EIS) sensors. Materials and Methods TMV particles modified with biotin-linker molecules (TMVBio), which serve as binding sites for streptavidin-conjugated enzymes, have been immobilized onto capacitive field-effect EIS sensors with Ta2O5 as transducer layer, as shown in Fig. 1. Immobilization was performed from TMVBiosolutions with different values of pH between pH 3.0 and pH 9.5 and ionic strength between 0.1 mM and 750 mM. The sensors have been electrochemically characterized before and after TMVBio immobilization by capacitance-voltage- and constant-capacitance methods, respectively. In addition, the density of the immobilized TMVs and morphology of the sensor surface has been investigated by means of scanning electron microscopy (SEM). Results and Conclusions The TMVBio density on the Ta2O5 sensor surface was influenced by varying the pH value (as exemplarily shown in Fig. 1) and ionic strength (not shown) of the TMVBio solution. The amplitude of the field-effect sensor signal correlates well with the density of the immobilized TMVBionanotubes. Thus, optimized conditions for the high-density immobilization of TMVBio nanotubes onto the Ta2O5 surface and thereby enhanced biosensing have been found. Details of the experiments and the obtained results will be presented and discussed. Acknowledgements The authors like to thank Dr. Claudia Koch and Rebecca Hummel, Stuttgart, for scientific and technical support.

Near-Infrared Chiral Plasmonic Microwires through Precision Assembly of Gold Nanorods on Soft Biotemplates

Directing the assembly of plasmonic nanoparticles into chiral superstructures has diverse applications including, chiroptical sensing, nonlinear optics, and biomedicine. Though soft template-mediated assemblies of both spherical and nonspherical gold nanoparticles have made significant progress, most approaches require sophisticated chemical synthesis or advanced methodologies. Besides, reports of structurally precise chiral plasmonic assemblies beyond nanoscale are limited. Here, we propose an efficient yet simple strategy to grow such precision assemblies up to mesoscale, which is beneficial for a broader community. Briefly, cationic gold nanorods (AuNRs) are allowed to systematically assemble along atomically precise, chiral, rodlike tobacco mosaic virus (TMV) particles via electrostatic attraction under ambient condition. This leads to spontaneous formation of helical hybrid microwires with high structural precision, as evidenced by cryogenic transmission electron microscopy and tomography. Resulting composite superstructures show a strong circular dichroism response at the plasmon wavelength of the AuNRs, which is supported by simulations using discrete dipole approximation. Further, chirality of the system is investigated at a single-microwire level using polarized dark-field scattering microscopy. An alternative chiral template, negatively charged colloidal cellulose nanocrystals, also arrange AuNRs into similar chiral microstructures. Thus, our report proposes a generic methodology to obtain chiral plasmonic response at the NIR region using inexpensive templates that will encourage the exploration of a wider range of nanoscale templates for creating hybrid mesostructures with emerging optoelectronic properties.

In vivo antiviral activity and disassembly mechanism of novel 1-phenyl-5-amine-4-pyrazole thioether derivatives against Tobacco mosaic virus

A series of novel 1-phenyl-5-amine-4-pyrazole thioether derivatives containing a 1,3,4-oxadiazole moiety was designed and synthesised. In vivo antiviral bioassay results showed that most of the target compounds exhibited excellent inactivation activity against Tobacco mosaic virus (TMV). The EC50 values of the inactivation activities for T2, T7, T9, T24, T25 and T27 were 15.7, 15.7, 15.5, 11.9, 12.5 and 16.5 μg/mL, respectively, which were remarkably superior over that of the commercialised antiviral agent ningnanmycin (40.3 μg/mL). Morphological study using AFM and TEM of TMV treated with T24 showed that T24 could significantly shorten the polymerization length of TMV particles and formed a distinct break on the rod-shaped TMV. Investigations for virus infection efficiency on tobacco leaves demonstrated that infectivity of virion had been reduced obviously upon T24 treatment. Subsequently, a strong interaction between T24 and TMV-CP (Kd = 3.8 μM, score 6.11) was observed through MST experiments. Molecular docking study further revealed that target compounds interact with amino acid residue Glu50 in TMV CP, causing disassembly of virion, shorting the length of the virion and reducing the infectivity of virion, and resulting in high inactivating activity of target compounds. This study provides a new insight for discovery of antiviral compounds through a new action mechanism with a new binding site.

Capacitive Field-Effect Biosensor Studying Adsorption of Tobacco Mosaic Virus Particles.

Plant virus-like particles, and in particular, tobacco mosaic virus (TMV) particles, are increasingly being used in nano- and biotechnology as well as for biochemical sensing purposes as nanoscaffolds for the high-density immobilization of receptor molecules. The sensitive parameters of TMV-assisted biosensors depend, among others, on the density of adsorbed TMV particles on the sensor surface, which is affected by both the adsorption conditions and surface properties of the sensor. In this work, Ta2O5-gate field-effect capacitive sensors have been applied for the label-free electrical detection of TMV adsorption. The impact of the TMV concentration on both the sensor signal and the density of TMV particles adsorbed onto the Ta2O5-gate surface has been studied systematically by means of field-effect and scanning electron microscopy methods. In addition, the surface density of TMV particles loaded under different incubation times has been investigated. Finally, the field-effect sensor also demonstrates the label-free detection of penicillinase immobilization as model bioreceptor on TMV particles.

Reticine A, a new potent natural elicitor: isolation from the fruit peel of Citrus reticulate and induction of systemic resistance against tobacco mosaic virus and other plant fungal diseases.

Systemic acquired resistance (SAR) induced by elicitors is a highly satisfying form of resistance that protects plants against invading pathogens. Exploration and development of new elicitors is a promising alternative to conventional biocides in resistant pest management. In our previous broad screening, fruit peel extract of Citrus reticulata Blanco exhibited the ability to induce SAR in tobacco. A new potent elicitor reticine A was isolated from the fruit peel extract of industrial crop C. reticulate and its structure was well elucidated. In vivo assays showed that reticine A had considerable control efficacies at 100 and 500 μg mL-1 , being superior to commercial elicitor benzothiadiazole (BTH) (100 μg mL-1 ). Reticine A had no significant impact on the virulence of tobacco mosaic virus (TMV) particles under in vitro conditions. Application of reticine A induced a local hypersensitive reaction (HR), systemic accumulation of H2 O2 and salicylic acid (SA), systemic increase in defensive enzyme activities and systemic upregulated expression of pathogenesis-related (PR) proteins, suggesting its induction of SAR in tobacco. The expression of NPR1 and SA biosynthesis genes ICS and PAL were systemically upregulated. SAR induced by reticine A against TMV in tobacco was demonstrated and the mechanism might be attributed to activating the expression of several defensive genes mediated by an SA signal. This study highlights the potential of reticine A which is recommended to be applied directly or as an active ingredient in the crude extract formulation ahead of time in the field, as well as being a potential lead compound for further optimization.

High-Density Immobilization of Tobacco Mosaic Virus Nanotubes As Enzyme Nanocarriers Onto Field-Effect Biosensor Structures

Introduction Viruses are not only infectious agents, they are also known as promising functional building blocks for application in nano- and biotechnologies. The tobacco mosaic virus (TMV) was the first studied plant virus and is widely distributed; it infects vegetables, like tomato, bell pepper, beans and other members of the family Solanaceae, while it is totally harmless for mammals [1]. It is one of the most studied plant viruses and its genome is completely sequenced, whereby genetical and chemical modification is easy. TMV has a nanotube-like shape with a length of 300 nm, an outer diameter of 18 nm and an inner diameter of 4 nm. Since it possesses a high chemical and physical robustness, it can be integrated with different electronic transducers for bio- and chemical sensing applications [2].Recently, we presented a TMV-based amperometric glucose biosensor [3] and a potentiometric penicillin biosensor [4]. TMV was used as enzyme nanocarrier for the enzyme glucose oxidase and penicillinase, respectively [3,4]. The sensitivity and detection limit of these biosensors, among others, depend on the density of TMVs on the sensor surface. The surface density of the immobilized TMVs is strongly influenced by the electrostatic interactions between the charged TMVs and sensor surface as well as by the inter-TMV-nanotubes repulsion, which could be changed by varying the pH value and the ionic strength of the TMV solution. In this study, we investigated an impact of the pH value and ionic strength of the TMV solution on the surface density of TMV nanotubes immobilized onto Ta2O5-gate capacitive field-effect electrolyte-insulator-semiconductor (EIS) sensors. Materials and Methods TMV particles modified with biotin-linker molecules (TMVBio), which serve as binding sites for streptavidin-conjugated enzymes, have been immobilized onto capacitive field-effect EIS sensors with Ta2O5 as transducer layer, as shown in Fig. 1. Immobilization was performed from TMVBiosolutions with different values of pH between pH 3.0 and pH 9.5 and ionic strength between 0.1 mM and 750 mM. The sensors have been electrochemically characterized before and after TMVBio immobilization by capacitance-voltage- and constant-capacitance methods, respectively. In addition, the density of the immobilized TMVs and morphology of the sensor surface has been investigated by means of scanning electron microscopy (SEM). Results and Conclusions The TMVBio density on the Ta2O5 sensor surface was influenced by varying the pH value (as exemplarily shown in Fig. 1) and ionic strength (not shown) of the TMVBio solution. The amplitude of the field-effect sensor signal correlates well with the density of the immobilized TMVBionanotubes. Thus, optimized conditions for the high-density immobilization of TMVBio nanotubes onto the Ta2O5 surface and thereby enhanced biosensing have been found. Details of the experiments and the obtained results will be presented and discussed.Figure 1: Measurement set-up with schematic layer structure of the capacitive EIS sensor modified with negatively charged TMVBio particles (a). SEM images of the Ta2O5-sensor surface modified with TMVBio nanotubes at pH 4.5 (b) and pH 7.0 (c), respectively. Acknowledgements The authors like to thank Dr. Claudia Koch and Rebecca Hummel, Stuttgart, for scientific and technical support.

Discovery of novel indole derivatives containing dithioacetal as potential antiviral agents for plants

Thirty unreported indole derivatives containing dithioacetal moiety were synthesized and evaluated for anti-plant viral activity. Bioassay results displayed that some of the target compounds showed better activities against tobacco mosaic virus (TMV) than the commercial Ribavirin in vivo. In particular, anti-TMV curative, protective and inactivating activity of 4p were 55.1, 57.2, and 80.3%, respectively, and EC50 value for inactivating activity was 88.5 μg/mL. The observation of transmission electron microscope showed that 4p may have a certain destructive effect on TMV particles. To further study, microscale thermophoresis analysis result also demonstrated that 4p powerfully interacted with TMV coat protein in vitro. Hence, this study provides a strong evidence suporting that indole derivatives might be applied as new 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.

Optimization of a Plasmodium falciparum circumsporozoite protein repeat vaccine using the tobacco mosaic virus platform

Plasmodium falciparum vaccine RTS,S/AS01 is based on the major NPNA repeat and the C-terminal region of the circumsporozoite protein (CSP). RTS,S-induced NPNA-specific antibody titer and avidity have been associated with high-level protection in naïve subjects, but efficacy and longevity in target populations is relatively low. In an effort to improve upon RTS,S, a minimal repeat-only, epitope-focused, protective, malaria vaccine was designed. Repeat antigen copy number and flexibility was optimized using the tobacco mosaic virus (TMV) display platform. Comparing antigenicity of TMV displaying 3 to 20 copies of NPNA revealed that low copy number can reduce the abundance of low-affinity monoclonal antibody (mAb) epitopes while retaining high-affinity mAb epitopes. TMV presentation improved titer and avidity of repeat-specific Abs compared to a nearly full-length protein vaccine (FL-CSP). NPNAx5 antigen displayed as a loop on the TMV particle was found to be most optimal and its efficacy could be further augmented by combination with a human-use adjuvant ALFQ that contains immune-stimulators. These data were confirmed in rhesus macaques where a low dose of TMV-NPNAx5 elicited Abs that persisted at functional levels for up to 11 mo. We show here a complex association between NPNA copy number, flexibility, antigenicity, immunogenicity, and efficacy of CSP-based vaccines. We hypothesize that designing minimal epitope CSP vaccines could confer better and more durable protection against malaria. Preclinical data presented here supports the evaluation of TMV-NPNAx5/ALFQ in human trials.

Design, synthesis, anti-TMV activity, and preliminary mechanism of cinnamic acid derivatives containing dithioacetal moiety

A series of cinnamic acid derivatives, which contained dithioacetal moiety, were designed and synthesized, and their anti-plant virus activity against Tobacco mosaic virus (TMV) were evaluated. Most target compounds exhibited good anti-plant virus activities. Compound 2y, especially at 500 mg/L concentration, had an excellent activity against TMV, and its curative, protective, and inactivating activities were 62.5%, 61.8%, and 83.5%, respectively. These activity values were significantly superior to those of ribavirin (45.9%, 39.8%, and 70.3%) and xiangcaoliusuobingmi (44.7%, 48.3%, and 71.7%) and comparable to those of ningnanmycin (61.9%, 53.3%, and 85.2%). Compound 2y presented an EC50 value of 50.7 mg/L for inactivating activity against TMV, which was superior to those of ningnanmycin (51.5 mg/L), ribavirin (160.4 mg/L), and xiangcaoliusuobingmi (83.0 mg/L). Through transmission electron microscopy, we found that compound 2y caused a certain degree of damage to TMV particles, which caused them to break and bend. Four conventional hydrogen bonds were formed with amino acid residues GLN34, THR37, ARG90, and ARG46 of TMV coat protein (CP) through molecular docking. Microscale thermophoresis test results showed that compound 2y with TMV CP had a strong binding force, and the dissociation constant (Kd) was 1.6 μM. In summary, the cinnamic acid derivatives containing dithioacetal moiety provide a foundation for further research on antiviral agents.

New chalcone derivatives: synthesis, antiviral activity and mechanism of action.

In this work, twenty-eight chalcone derivatives containing a purine (sulfur) ether moiety were synthesized and their antiviral activities were evaluated. Biological results showed that compound 5d exhibited outstanding inactive activity against tobacco mosaic virus (TMV) in vivo (EC50 = 65.8 μg mL−1), which is significantly superior to that of ribavirin (EC50 = 154.3 μg mL−1). Transmission electron microscopy indicated that compound 5d can break the integrity of TMV particles. The results of microscale thermophoresis, fluorescence titration and molecular docking showed that compound 5d had stronger combining affinity (Ka = 1.02 ×105 L mol−1, Kd = 13.4 μmol L−1) with TMV coat protein (TMV-CP), which is due to the formation of five hydrogen bonds between compound 5d and the amino-acid residues of TMV-CP. These findings revealed that compound 5d can effectively inhibit the infective ability of TMV. This work provides inspiration and reference for the discovery of new antiviral agents.

Inhibitory Effect of Osthole from Cnidium monnieri on Tobacco Mosaic Virus (TMV) Infection in Nicotiana glutinosa.

The coumarin compound of osthole was extracted from Cnidium monnieri and identified by LC-MS and 1H- and 13C-NMR. Osthole was tested for anti-virus activity against tobacco mosaic virus (TMV) using the half-leaf method. The results showed that stronger antiviral activity on TMV infection appeared in Nicotiana glutinosa than that of eugenol and ningnanmycin, with inhibitory, protective, and curative effects of 72.57%, 70.26%, and 61.97%, respectively. Through observation of the TMV particles, we found that osthole could directly affect the viral particles. Correspondingly, the level of coat protein detected by Western blot was significantly reduced when the concentrations of osthole increased in tested plants compared to that of the control. These results suggest that osthole has anti-TMV activity and may be used as a biological reagent to control the plant virus in the half-leaf method.

Synthesis of Anthranilic Diamide Derivatives Containing Moieties of Trifluoromethylpyridine and Hydrazone as Potential Anti-Viral Agents for Plants.

A series of novel anthranilic diamide derivatives (5a-5ab) containing moieties of trifluoromethylpyridine and hydrazone was designed and synthesized. The synthesized compounds were evaluated in vivo for their activities against tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV). Most of the synthesized compounds displayed good to excellent antiviral activities. The compounds 5i, 5k, 5s, 5w, 5x, and 5z had the curative activity over 65% against TMV at the concentration of 500 μg/mL, which were significantly higher than those of ningnanmycin (55.0%) and ribavirin (37.9%). Notably, the curative activity of compound 5i was up to 79.5%, with the EC50 value of 75.9 μg/mL, whereas the EC50 value of ningnanmycin was 362.4 μg/mL. The pot experiments also further demonstrated the significantly curative effect of 5i. Meanwhile, compounds 5h, 5p and 5x displayed more protective activities on TMV than that of ningnanmycin. Moreover, compounds 5a, 5e, 5f, and 5i showed inactivation activity similar to ningnanmycin at 500 μg/mL, and the EC50 value of 5e (41.5 μg/mL) was lower than ningnanmycin (50.0 μg/mL). The findings of transmission electron microscopic (TEM) indicated that the synthesized compounds exhibited strong and significant binding affinity to TMV coat protein (CP) and could obstruct the self-assembly and increment of TMV particles. Microscale thermophoresis (MST) studies on TMV-CP and CMV CP revealed that some of the active compounds, particularly 5i, exhibited a strong binding capability to TMV-CP or CMV-CP. This study revealed that anthranilic diamide derivatives containing moieties of trifluoromethylpyridine and hydrazone could be used as novel antiviral agents for controlling the plant viruses.

A single-molecule atomic force microscopy study reveals the antiviral mechanism of tannin and its derivatives.

Antiviral agents work by stopping or intervening the virus replication. Virus replication is a fast and multi-step process while effective antiviral intervention requires agents to interact with the protein coat, genetic RNA/DNA or both during virus replication. Thus, quantifying these interactions at the molecular level, although it is quite challenging, is very important for an understanding of the underlying molecular mechanism of antiviral intervention. Here, at the single molecule level, we employ single molecule force spectroscopy (SMFS) in combination with AFM imaging and choose tobacco mosaic virus (TMV)/tannin as a model system of tubular virus to directly study how the inhibitor influences the interactions of RNA and coat protein. We illustrated the antiviral mechanism of tannin during the three main stages of TMV infection, i.e., before the entry of cells, the disassembly of genetic RNA and reassembly of genetic RNA, respectively. Our SMFS results show that tannin and its derivatives can stabilize the TMV complex by enhancing the interactions between RNA and coat protein via weak interactions, such as hydrogen bonding and hydrophobic interactions. In addition, the stabilization effect showed molecular weight dependence, i.e., for higher molecular weight tannin the stabilization occurs after genetic RNA gets partially disassembled from the protein coat, while the lower molecular weight tannin hydrolyte starts experiencing the stabilization effect before the RNA disassembly. Furthermore, the cycling stretching-relaxation experiments in the presence/absence of tannin proved that tannin can prevent the assembling of RNA and coat protein. In addition, the AFM imaging results demonstrate that tannin can cause the aggregation of TMV particles in a concentration-dependent manner; a higher concentration of tannin will cause more severe aggregations. These results deepen our understanding of the antiviral mechanism of tannin and its derivatives, which facilitate the rational design of efficient agents for antiviral therapy.

Precise Assembly of Genetically Functionalized Magnetosomes and Tobacco Mosaic Virus Particles Generates a Magnetic Biocomposite

Magnetosomes represent magnetic nanoparticles with unprecedented characteristics. Both their crystal morphology and the composition of the enveloping membrane can be manipulated by genetic means, allowing the display of functional moieties on the particle surface. In this study, we explore the generation of a new biomaterial assembly by coupling magnetosomes with tobacco mosaic virus (TMV) particles, both functionalized with complementary recognition sites. TMV consists of single-stranded RNA encapsidated by more than 2100 coat proteins, which enable chemical modification via functional groups. Incubation of EmGFP- or biotin-decorated TMV particles with magnetosomes genetically functionalized with GFP-binding nanobodies or streptavidin, respectively, results in the formation of magnetic, mesoscopic, strand-like biocomposites. TMV facilitates the agglomeration of magnetosomes by providing a scaffold. The size of the TMV-magnetosome mesostrands can be adjusted by varying the TMV-magnetosome particle ratios. The versatility of this novel material combination is furthermore demonstrated by coupling magnetosomes and terminal, 5'-functionalized TMV particles with high molecular precision, which results in "drumstick"-like TMV-magnetosome complexes. In summary, our approaches provide promising strategies for the generation of new biomaterial assemblies that could be used as scaffold for the introduction of further functionalities, and we foresee a broad application potential in the biomedical and biotechnological field.

Magnetron sputtering deposition of ultra-thin metal coatings for the visualization of protein-containing objects of nanometer size by electron microscopy

Electron microscopy is widely used in biological and medical research, enabling to obtain images of the investigated objects with nanometer resolution. Exposure of biological objects under electron beam during the visualization can, however, cause their damage. Destructive effect of the electron beam can be significantly decreased by formation of metal coating on the surface of the objects to be studied. In the present study, an approach for the formation of ultra-thin metal coatings on the surface of protein-containing structures is proposed. Magnetron sputtering in argon plasma in DC mode was employed to form 8 Å-thick tungsten films on the surface of model protein-containing objects— tobacco mosaic virus particles — for their subsequent visualization by electron microscopy.