Movement Proteins (BC1 and BV1) of Abutilon Mosaic Geminivirus Are Cotransported in and between Cells of Sink but Not of Source Leaves as Detected by Green Fluorescent Protein Tagging

AbstractTwo movement proteins (BV1 and BC1) facilitate the intra- and intercellular transport of begomoviruses in plants. In contrast to other geminiviruses the movement protein BC1 of Abutilon mosaic virus (AbMV) remained in the supernatant after centrifuging plant extracts at 20,000 g. To test whether this unusual behavior results from a distinct intracellular distribution of the protein, the BC1 gene has been fused to the gene of green fluorescent protein (GFP). The resulting plasmids were delivered into nonhost plants (Allium cepa) as well as into mature and immature cells of host plants (Nicotiana tabacum, N. benthamiana) by biolistic bombardment for transient expression in planta. BC1 directed GFP to two different cellular sites. In the majority of nonhost cells as well as in mature cells of host leaves, BC1 was mainly localized in small punctate flecks at the cell periphery or, to a lesser extent, around the nucleus. In sink leaves of host plants, GFP:BC1 additionally developed disc-like structures in the cell periphery. Cobombardment of GFP:BC1 with its cognate infectious DNA A and B did not change their subcellular distribution patterns in source leaves but led to the formation of peculiar needle-like structures in sink leaves. The nuclear shuttle protein (BV1) of AbMV accumulated mainly inside the nuclei as shown by immunohistochemical staining and GFP tagging. In sink cells of host plants it was mobilized to the plasma membrane and to the nucleus of the neighboring cell by coexpressed BC1, GFP:BC1, BC1:GFP, or after cobombardment with the cognate viral DNA. Only under these conditions were GFP:BC1 and BC1:GFP also found in the recipient cell

Apertureless scanning near-field optical microscopy of sparsely labeled tobacco mosaic viruses and the intermediate filament desmin

Harder A, Dieding M, Walhorn V, et al. Apertureless scanning near-field optical microscopy of sparsely labeled tobacco mosaic viruses and the intermediate filament desmin. Beilstein Journal of Nanotechnology. 2013;4:510-516.Both fluorescence imaging and atomic force microscopy (AFM) are highly versatile and extensively used in applications ranging from nanotechnology to life sciences. In fluorescence microscopy luminescent dyes serve as position markers. Moreover, they can be used as active reporters of their local vicinity. The dipolar coupling of the tip with the incident light and the fluorophore give rise to a local field and fluorescence enhancement. AFM topographic imaging allows for resolutions down to the atomic scale. It can be operated in vacuum, under ambient conditions and in liquids. This makes it ideal for the investigation of a wide range of different samples. Furthermore an illuminated AFM cantilever tip apex exposes strongly confined non-propagating electromagnetic fields that can serve as a coupling agent for single dye molecules. Thus, combining both techniques by means of apertureless scanning near-field optical microscopy (aSNOM) enables concurrent high resolution topography and fluorescence imaging. Commonly, among the various (apertureless) SNOM approaches metallic or metallized probes are used. Here, we report on our custom-built aSNOM setup, which uses commercially available monolithic silicon AFM cantilevers. The field enhancement confined to the tip apex facilitates an optical resolution down to 20 nm. Furthermore, the use of standard mass-produced AFM cantilevers spares elaborate probe production or modification processes. We investigated tobacco mosaic viruses and the intermediate filament protein desmin. Both are mixed complexes of building blocks, which are fluorescently labeled to a low degree. The simultaneous recording of topography and fluorescence data allows for the exact localization of distinct building blocks within the superordinate structures

Virus-Templated Near-Amorphous Iron Oxide Nanotubes

© 2016 American Chemical Society. We present a simple synthesis of iron oxide nanotubes, grown under very mild conditions from a solution containing Fe(II) and Fe(III), on rod-shaped tobacco mosaic virus templates. Their well-defined shape and surface chemistry suggest that these robust bionanoparticles are a versatile platform for synthesis of small, thin mineral tubes, which was achieved efficiently. Various characterization tools were used to explore the iron oxide in detail: Electron microscopy (SEM, TEM), magnetometry (SQUID-VSM), diffraction (XRD, TEM-SAED), electron spectroscopies (EELS, EDX, XPS), and X-ray absorption (XANES with EXAFS analysis). They allowed determination of the structure, crystallinity, magnetic properties, and composition of the tubes. The protein surface of the viral templates was crucial to nucleate iron oxide, exhibiting analogies to biomineralization in natural compartments such as ferritin cages

Novel roles for well-known players: from tobacco mosaic virus pests to enzymatically active assemblies

The rod-shaped nanoparticles of the widespread plant pathogen tobacco mosaic virus (TMV) have been a matter of intense debates and cutting-edge research for more than a hundred years. During the late 19th century, their behavior in filtration tests applied to the agent causing the \u27plant mosaic disease\u27 eventually led to the discrimination of viruses from bacteria. Thereafter, they promoted the development of biophysical cornerstone techniques such as electron microscopy and ultracentrifugation. Since the 1950s, the robust, helically arranged nucleoprotein complexes consisting of a single RNA and more than 2100 identical coat protein subunits have enabled molecular studies which have pioneered the understanding of viral replication and self-assembly, and elucidated major aspects of virus–host interplay, which can lead to agronomically relevant diseases. However, during the last decades, TMV has acquired a new reputation as a well-defined high-yield nanotemplate with multivalent protein surfaces, allowing for an ordered high-density presentation of multiple active molecules or synthetic compounds. Amino acid side chains exposed on the viral coat may be tailored genetically or biochemically to meet the demands for selective conjugation reactions, or to directly engineer novel functionality on TMV-derived nanosticks. The natural TMV size (length: 300 nm) in combination with functional ligands such as peptides, enzymes, dyes, drugs or inorganic materials is advantageous for applications ranging from biomedical imaging and therapy approaches over surface enlargement of battery electrodes to the immobilization of enzymes. TMV building blocks are also amenable to external control of in vitro assembly and re-organization into technically expedient new shapes or arrays, which bears a unique potential for the development of \u27smart\u27 functional 3D structures. Among those, materials designed for enzyme-based biodetection layouts, which are routinely applied, e.g., for monitoring blood sugar concentrations, might profit particularly from the presence of TMV rods: Their surfaces were recently shown to stabilize enzymatic activities upon repeated consecutive uses and over several weeks. This review gives the reader a ride through strikingly diverse achievements obtained with TMV-based particles, compares them to the progress with related viruses, and focuses on latest results revealing special advantages for enzyme-based biosensing formats, which might be of high interest for diagnostics employing \u27systems-on-a-chip\u27

Immune Checkpoint Profiling in Humanized Breast Cancer Mice Revealed Cell-Specific LAG-3/PD-1/TIM-3 Co-Expression and Elevated PD-1/TIM-3 Secretion

Checkpoint blockade is particularly based on PD-1/PD-L1-inhibiting antibodies. However, an efficient immunological tumor defense can be blocked not only by PD-(L)1 but also by the presence of additional immune checkpoint molecules. Here, we investigated the co-expression of several immune checkpoint proteins and the soluble forms thereof (e.g., PD-1, TIM-3, LAG-3, PD-L1, PD-L2 and others) in humanized tumor mice (HTM) simultaneously harboring cell line-derived (JIMT-1, MDA-MB-231, MCF-7) or patient-derived breast cancer and a functional human immune system. We identified tumor-infiltrating T cells with a triple-positive PD-1, LAG-3 and TIM-3 phenotype. While PD-1 expression was increased in both the CD4 and CD8 T cells, TIM-3 was found to be upregulated particularly in the cytotoxic T cells in the MDA-MB-231-based HTM model. High levels of soluble TIM-3 and galectin-9 (a TIM-3 ligand) were detected in the serum. Surprisingly, soluble PD-L2, but only low levels of sPD-L1, were found in mice harboring PD-L1-positive tumors. Analysis of a dataset containing 3039 primary breast cancer samples on the R2 Genomics Analysis Platform revealed increased TIM-3, galectin-9 and LAG-3 expression, not only in triple-negative breast cancer but also in the HER2+ and hormone receptor-positive breast cancer subtypes. These data indicate that LAG-3 and TIM-3 represent additional key molecules within the breast cancer anti-immunity landscape

Peptide-equipped tobacco mosaic virus templates for selective and controllable biomineral deposition

The coating of regular-shaped, readily available nanorod biotemplates with inorganic compounds has attracted increasing interest during recent years. The goal is an effective, bioinspired fabrication of fiber-reinforced composites and robust, miniaturized technical devices. Major challenges in the synthesis of applicable mineralized nanorods lie in selectivity and adjustability of the inorganic material deposited on the biological, rod-shaped backbones, with respect to thickness and surface profile of the resulting coating, as well as the avoidance of aggregation into extended superstructures. Nanotubular tobacco mosaic virus (TMV) templates have proved particularly suitable towards this goal: Their multivalent protein coating can be modified by high-surface-density conjugation of peptides, inducing and governing silica deposition from precursor solutions in vitro. In this study, TMV has been equipped with mineralization-directing peptides designed to yield silica coatings in a reliable and predictable manner via precipitation from tetraethoxysilane (TEOS) precursors. Three peptide groups were compared regarding their influence on silica polymerization: (i) two peptide variants with alternating basic and acidic residues, i.e. lysine–aspartic acid (KD)$_{χ}$ motifs expected to act as charge-relay systems promoting TEOS hydrolysis and silica polymerization; (ii) a tetrahistidine-exposing polypeptide (CA$_{4}$H$_{4}$) known to induce silicification due to the positive charge of its clustered imidazole side chains; and (iii) two peptides with high ZnO binding affinity. Differential effects on the mineralization of the TMV surface were demonstrated, where a (KD)$_{χ}$ charge-relay peptide (designed in this study) led to the most reproducible and selective silica deposition. A homogenous coating of the biotemplate and tight control of shell thickness were achieved

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. We describe the production (including genetic or chemical modification) and purification of the plant viruses or of empty virus-like particles in the case of CPMV, as well as the characterization of these harvested templates. The mineralization procedures and differences in the protocols specific to the distinct viruses are described, and the analyses of the mineralization results are explained

Interaction of DNA with the Movement Proteins of Geminiviruses Revisited

Geminiviruses manage the transport of their DNA within plants with the help of three proteins, the coat protein (CP), the nuclear shuttle protein (NSP), and the movement protein (MP). The DNA-binding capabilities of CP, NSP, and MP of Abutilon mosaic virus (AbMV; family Geminiviridae; genus Begomovirus) were scrutinized using gel mobility shift assays and electron microscopy. CP and NSP revealed a sequence-independent affinity for both double-stranded and single-stranded DNA, as has been previously reported for other begomoviruses. MP interacted selectively with dimeric supercoiled plasmid DNA in the electrophoretic assay. Further apparent size- and form-selective binding capacities of MP have been previously reported for another geminivirus (Bean dwarf mosaic virus), but in the case of AbMV, they have been identified as the result of electrophoretic interference rather than of complex formation. Without these complications, electron microscopy confirmed the assembly of double-stranded supercoiled DNA with NSP and MP into conspicuous structures and provided the first direct evidence for cooperative interaction of MP, NSP, and DNA. Based on these results and previous ones, a transport model of geminiviruses is discussed in which NSP packages DNA and MP anchors this complex to the protoplasmic leaflets of plasma membranes and microsomes for cell-to-cell movement

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. Adaptations for distinct particle species, pitfalls, and ‘forgotten’ or underrepresented technologies are considered as well. The article is topped off with our own development of a method for virion preparation, rooted in historical protocols. It combines selective re-solubilization of polyethylene glycol (PEG) virion raw precipitates with density step gradient centrifugation in biocompatible iodixanol formulations, yielding ready-to-use particle suspensions. This newly established protocol and some considerations for perhaps worthwhile further developments could serve as putative stepping stones towards preparation procedures appropriate for routine practical uses of these multivalent soft-matter nanorods

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