Getting Hold of the Tobamovirus Particle-Why and How? Purification Routes over Time and a New Customizable Approach.

This article develops a multi-perspective view on motivations and methods for tobamovirus purification through the ages and presents a novel, efficient, easy-to-use approach that can be well-adapted to different species of native and functionalized virions. We survey the various driving forces prompting researchers to enrich tobamoviruses, from the search for the causative agents of mosaic diseases in plants to their increasing recognition as versatile nanocarriers in biomedical and engineering applications. The best practices and rarely applied options for the serial processing steps required for successful isolation of tobamoviruses are then reviewed.

Soft Sub-Structured Multi-Material Biosensor Hydrogels with Enzymes Retained by Plant Viral Scaffolds.

An all-soft multi-material combination consisting of a hydrogel based on poly(ethylene glycol) (PEG) coated with spatially defined spots of gelatin methacryloyl (GM) containing selectively addressable viral nanorods is presented, and its basic application as a qualitative biosensor with reporter enzymes displayed on the tobacco mosaic virus (TMV) bioscaffolds within the GM is demonstrated. Biologically inert PEG supports are equipped with GM spots serving as biological matrix for enzymes clustered on TMV particles preventing diffusion out of the gel. For this multi-material combination, i) the PEG-based hydrogel surface is modified to achieve a clear boundary between coated and non-coated regions by introducing either isothiouronium or thiol groups.

Facile Purification and Use of Tobamoviral Nanocarriers for Antibody-Mediated Display of a Two-Enzyme System.

Immunosorbent turnip vein clearing virus (TVCV) particles displaying the IgG-binding domains D and E of protein A (PA) on every coat protein (CP) subunit (TVCV) were purified from plants via optimized and new protocols. The latter used polyethylene glycol (PEG) raw precipitates, from which virions were selectively re-solubilized in reverse PEG concentration gradients. This procedure improved the integrity of both TVCV and the wild-type subgroup 3 tobamovirus.

Differential Effects of RNA-Dependent RNA Polymerase 6 (RDR6) Silencing on New and Old World Begomoviruses in .

RNA-dependent RNA polymerases (RDRs) are key players in the antiviral defence mediated by RNA silencing in plants. RDR6 is one of the major components of the process, regulating the infection of certain RNA viruses. To better clarify its function against DNA viruses, we analyzed the effect of RDR6 inactivation (RDR6i) in plants on two phloem-limited begomoviruses, the bipartite Abutilon mosaic virus (AbMV) and the monopartite tomato yellow leaf curl Sardinia virus (TYLCSV).

Convenient site-selective protein coupling from bacterial raw lysates to coenzyme A-modified tobacco mosaic virus (TMV) by Bacillus subtilis Sfp phosphopantetheinyl transferase.

A facile enzyme-mediated strategy enables site-specific covalent one-step coupling of genetically tagged luciferase molecules to coenzyme A-modified tobacco mosaic virus (TMV-CoA) both in solution and on solid supports. Bacillus subtilis surfactin phosphopantetheinyl transferase Sfp produced in E. coli mediated the conjugation of firefly luciferase N-terminally extended by eleven amino acids forming a 'ybbR tag' as Sfp-selective substrate, which even worked in bacterial raw lysates.

Towards Multi-Analyte Detection with Field-Effect Capacitors Modified with Bioparticles as Enzyme Nanocarriers.

Utilizing an appropriate enzyme immobilization strategy is crucial for designing enzyme-based biosensors. Plant virus-like particles represent ideal nanoscaffolds for an extremely dense and precise immobilization of enzymes, due to their regular shape, high surface-to-volume ratio and high density of surface binding sites. In the present work, (TMV) particles were applied for the co-immobilization of penicillinase and urease onto the gate surface of a field-effect electrolyte-insulator-semiconductor capacitor (EISCAP) with a p-Si-SiO-TaO layer structure for the sequential detection of penicillin and urea.

Enzymatic activity of individual bioelectrocatalytic viral nanoparticles: dependence of catalysis on the viral scaffold and its length.

The enzymatic activity of tobacco mosaic virus (TMV) nanorod particles decorated with an integrated electro-catalytic system, comprising the quinoprotein glucose-dehydrogenase (PQQ-GDH) enzyme and ferrocenylated PEG chains as redox mediators, is probed at the individual virion scale by atomic force microscopy-scanning electrochemical atomic force microscopy (AFM-SECM). A marked dependence of the catalytic activity on the particle length is observed. This finding can be explained by electron propagation along the viral backbone, resulting from electron exchange between ferrocene moieties, coupled with enzymatic catalysis.

The Effect of Pooling on the Detection of the Nucleocapsid Protein of SARS-CoV-2 with Rapid Antigen Tests.

The COVID-19 pandemic puts significant stress on the viral testing capabilities of many countries. Rapid point-of-care (PoC) antigen tests are valuable tools but implementing frequent large scale testing is costly. We have developed an inexpensive device for pooling swabs, extracting specimens, and detecting viral antigens with a commercial lateral flow test for the nucleocapsid protein of SARS-CoV-2 as antigen.

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 SiO-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.

Light-Addressable Actuator-Sensor Platform for Monitoring and Manipulation of pH Gradients in Microfluidics: A Case Study with the Enzyme Penicillinase.

The feasibility of light-addressed detection and manipulation of pH gradients inside an electrochemical microfluidic cell was studied. Local pH changes, induced by a light-addressable electrode (LAE), were detected using a light-addressable potentiometric sensor (LAPS) with different measurement modes representing an actuator-sensor system. Biosensor functionality was examined depending on locally induced pH gradients with the help of the model enzyme penicillinase, which had been immobilized in the microfluidic channel.

Capacitive Field-Effect Biosensor Studying Adsorption of Particles.

Plant virus-like particles, and in particular, (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, TaO-gate field-effect capacitive sensors have been applied for the label-free electrical detection of TMV adsorption.

Field-Effect Sensors for Virus Detection: From Ebola to SARS-CoV-2 and Plant Viral Enhancers.

Coronavirus disease 2019 (COVID-19) is a novel human infectious disease provoked by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Currently, no specific vaccines or drugs against COVID-19 are available. Therefore, early diagnosis and treatment are essential in order to slow the virus spread and to contain the disease outbreak.

Phosphorylations of the Abutilon Mosaic Virus Movement Protein Affect Its Self-Interaction, Symptom Development, Viral DNA Accumulation, and Host Range.

The genome of bipartite geminiviruses in the genus comprises two circular DNAs: DNA-A and DNA-B. The DNA-B component encodes a nuclear shuttle protein (NSP) and a movement protein (MP), which cooperate for systemic spread of infectious nucleic acids within host plants and affect pathogenicity. MP mediates multiple functions during intra- and intercellular trafficking, such as binding of viral nucleoprotein complexes, targeting to and modification of plasmodesmata, and release of the cargo after cell-to-cell transfer.

From stars to stripes: RNA-directed shaping of plant viral protein templates-structural synthetic virology for smart biohybrid nanostructures.

The self-assembly of viral building blocks bears exciting prospects for fabricating new types of bionanoparticles with multivalent protein shells. These enable a spatially controlled immobilization of functionalities at highest surface densities-an increasing demand worldwide for applications from vaccination to tissue engineering, biocatalysis, and sensing. Certain plant viruses hold particular promise because they are sustainably available, biodegradable, nonpathogenic for mammals, and amenable to in vitro self-organization of virus-like particles.

[Mortality after high-risk surgery in Jehovah's Witness patients].

Background: Jehovah's Witness (JW) patients strictly refuse allogeneic blood transfusion for religious reasons. Nevertheless, JW also wish to benefit from modern therapeutic concepts including major surgical procedures without facing an excessive risk of death. The Northwest Hospital in Frankfurt am Main Germany is a confidential clinic of JW and performs approximately 100 surgical interventions per year on this patient group.

Hydrophobization of Tobacco Mosaic Virus to Control the Mineralization of Organic Templates.

The robust, anisotropic tobacco mosaic virus (TMV) provides a monodisperse particle size and defined surface chemistry. Owing to these properties, it became an excellent bio-template for the synthesis of diverse nanostructured organic/inorganic functional materials. For selective mineralization of the bio-template, specific functional groups were introduced by means of different genetically encoded amino acids or peptide sequences into the polar virus surface.

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 Particles: The Journey From Fundamental Studies to Bionanotechnology Applications.

Ever since its initial characterization in the 19th century, tobacco mosaic virus (TMV) has played a prominent role in the development of modern virology and molecular biology. In particular, research on the three-dimensional structure of the virus particles and the mechanism by which these assemble from their constituent protein and RNA components has made TMV a paradigm for our current view of the morphogenesis of self-assembling structures, including viral particles. More recently, this knowledge has been applied to the development of novel reagents and structures for applications in biomedicine and bionanotechnology.

Plant virus-based materials for biomedical applications: Trends and prospects.

Nanomaterials composed of plant viral components are finding their way into medical technology and health care, as they offer singular properties. Precisely shaped, tailored virus nanoparticles (VNPs) with multivalent protein surfaces are efficiently loaded with functional compounds such as contrast agents and drugs, and serve as carrier templates and targeting vehicles displaying e.g.

TMV-Based Adapter Templates for Enhanced Enzyme Loading in Biosensor Applications.

Nanotubular tobacco mosaic virus (TMV) particles and RNA-free lower-order coat protein (CP) aggregates have been employed as enzyme carriers in different diagnostic layouts and compared for their influence on biosensor performance. In the following, we describe a label-free electrochemical biosensor for improved glucose detection by use of TMV adapters and the enzyme glucose oxidase (GOD). A specific and efficient immobilization of streptavidin-conjugated GOD ([SA]-GOD) complexes on biotinylated TMV nanotubes or CP aggregates was achieved via bioaffinity binding.

Dual Functionalization of Rod-Shaped Viruses on Single Coat Protein Subunits.

Plant viruses are emerging as versatile tools for nanotechnology applications since it is possible to modify their multivalent protein surfaces and thereby introduce and display new functionalities. In this chapter, we describe a tobacco mosaic virus (TMV) variant that exposes two selectively addressable amino acid moieties on each of its 2130 coat protein (CP) subunits. A lysine as well as a cysteine introduced at accessible sites of every CP can be modified with amino- and/or thiol-reactive chemistry such as N-hydroxysuccinimide esters (NHS ester) and maleimide containing reagents alone or simultaneously.

Bioinspired Silica Mineralization on Viral Templates.

Plant virus capsids are attractive entities for nanotechnological applications because of their variation in shape and natural assembly ability. This chapter describes the production and modification of three differently shaped plant virus capsids for silica mineralization purposes. The chosen plant viruses exhibit either an icosahedral (cowpea mosaic virus, CPMV), or a flexuous rod-like structure (potato virus X, PVX), or a rigid rod-like shape (tobacco mosaic virus, TMV), and are well-known and frequently used plant viruses for biotechnological applications.

Bottom-Up Assembly of TMV-Based Nucleoprotein Architectures on Solid Supports.

RNA-guided self-assembly of tobacco mosaic virus (TMV)-like nucleoprotein nanotubes is possible using 3'-terminally surface-linked scaffold RNAs containing the viral origin of assembly (OAS). In combination with TMV coat protein (CP) preparations, these scaffold RNAs can direct the growth of selectively addressable multivalent carrier particles directly at sites of interest on demand. Serving as adapter templates for the installation of functional molecules, they may promote an integration of active units into miniaturized technical devices, or enable their presentation on soft-matter nanotube systems at high surface densities advantageous for, for example, biodetection or purification applications.

Electrochemically-Driven Insertion of Biological Nanodiscs into Solid State Membrane Pores as a Basis for "Pore-In-Pore" Membranes.

Nanoporous membranes are of increasing interest for many applications, such as molecular filters, biosensors, nanofluidic logic and energy conversion devices. To meet high-quality standards, e.g.

Field-effect biosensor using virus particles as scaffolds for enzyme immobilization.

A field-effect biosensor employing tobacco mosaic virus (TMV) particles as scaffolds for enzyme immobilization is presented. Nanotubular TMV scaffolds allow a dense immobilization of precisely positioned enzymes with retained activity. To demonstrate feasibility of this new strategy, a penicillin sensor has been developed by coupling a penicillinase with virus particles as a model system.