Ultrastructural Characterization of Turnip Mosaic Virus-Induced Cellular Rearrangements Reveals Membrane-Bound Viral Particles Accumulating in Vacuoles.

UNLABELLED: Positive-strand RNA [(+) RNA] viruses remodel cellular membranes to facilitate virus replication and assembly. In the case of turnip mosaic virus (TuMV), the viral membrane protein 6K2 plays an essential role in endomembrane alterations. Although 6K2-induced membrane dynamics have been widely studied by confocal microscopy, the ultrastructure of this remodeling has not been extensively examined. In this study, we investigated the formation of TuMV-induced membrane changes by chemical fixation and high-pressure freezing/freeze substitution (HPF/FS) for transmission electron microscopy at different times of infection. We observed the formation of convoluted membranes connected to rough endoplasmic reticulum (rER) early in the infection process, followed by the production of single-membrane vesicle-like (SMVL) structures at the midstage of infection. Both SMVL and double-membrane vesicle-like structures with electron-dense cores, as well as electron-dense bodies, were found late in the infection process. Immunogold labeling results showed that the vesicle-like structures were 6K2 tagged and suggested that only the SMVL structures were viral RNA replication sites. Electron tomography (ET) was used to regenerate a three-dimensional model of these vesicle-like structures, which showed that they were, in fact, tubules. Late in infection, we observed filamentous particle bundles associated with electron-dense bodies, which suggests that these are sites for viral particle assembly. In addition, TuMV particles were observed to accumulate in the central vacuole as membrane-associated linear arrays. Our work thus unravels the sequential appearance of distinct TuMV-induced membrane structures for viral RNA replication, viral particle assembly, and accumulation. IMPORTANCE: Positive-strand RNA viruses remodel cellular membranes for different stages of the infection process, such as protein translation and processing, viral RNA synthesis, particle assembly, and virus transmission. The ultrastructure of turnip mosaic virus (TuMV)-induced membrane remodeling was investigated over several days of infection. The first change that was observed involved endoplasmic reticulum-connected convoluted membrane accumulation. This was followed by the formation of single-membrane tubules, which were shown to be viral RNA replication sites. Later in the infection process, double-membrane tubular structures were observed and were associated with viral particle bundles. In addition, TuMV particles were observed to accumulate in the central vacuole as membrane-associated linear arrays. This work thus unravels the sequential appearance of distinct TuMV-induced membrane structures for viral RNA replication, viral particle assembly, and accumulation.

A synthetic nanomaterial for virus recognition produced by surface imprinting.

Major stumbling blocks in the production of fully synthetic materials designed to feature virus recognition properties are that the target is large and its self-assembled architecture is fragile. Here we describe a synthetic strategy to produce organic/inorganic nanoparticulate hybrids that recognize non-enveloped icosahedral viruses in water at concentrations down to the picomolar range. We demonstrate that these systems bind a virus that, in turn, acts as a template during the nanomaterial synthesis. These virus imprinted particles then display remarkable selectivity and affinity. The reported method, which is based on surface imprinting using silica nanoparticles that act as a carrier material and organosilanes serving as biomimetic building blocks, goes beyond simple shape imprinting. We demonstrate the formation of a chemical imprint, comparable to the formation of biosilica, due to the template effect of the virion surface on the synthesis of the recognition material.

Crystallization, structural diversity and anisotropy effects in 2D arrays of icosahedral viruses.

We investigate two-dimensional (2D) assembly of the icosahedral turnip yellow mosaic virus (TYMV) under cationic lipid monolayers at the aqueous solution­vapor interface. The 2D crystallization of TYMV has been achieved by enhancing electrostatically induced interfacial adsorption, an approach recently demonstrated for another virus. In situ X-ray scattering reveals two close-packed 2D crystalline phases of TYMV that are distinct from the previously reported hexagonal and centered square (√2 × âˆš2) arrays of TYMV. One of the newly observed phases arises from either a dimeric double-square (2 × 1) or tetrameric square (2 × 2) unit cell. The other is a rhombic crystal with a lattice angle of 80°. The two observed crystal phases are substantially less dense (by over 10%) than a 2D lattice of TYMV could be according to its known size and shape, indicating that local anisotropic interparticle interactions play a key role in stabilizing these crystals. TYMV's anisotropy attributes and numerical analysis of 2D arrays of virus-shaped particles are used to derive a model for the rhombic crystal in which the particle orientation is consistent with the electrostatic lipid­TYMV attraction and the interparticle contacts exhibit steric complementarity. The interplay between particle anisotropy and packing is contrasted between the rhombic crystal model and the square (√2 × âˆš2) crystal. This study highlights how the high symmetry and subtle asphericity of icosahedral particles enrich the variety and complexity of ordered 2D structures that can be generated through self-assembly.

Polyvalent display of RGD motifs on turnip yellow mosaic virus for enhanced stem cell adhesion and spreading.

Turnip yellow mosaic virus (TYMV) is a stable 28 nm icosahedral plant virus that can be isolated in gram quantities. In order to study the polyvalent effect of Arg-Gly-Asp (RGD) clustering on the response of bone marrow stem cells (BMSCs), an RGD motif was genetically displayed on the coat protein of the TYMV capsid. Composite films composed of either wild-type TYMV or TYMV-RGD44, in combination with poly(allylamine hydrochloride) (PAH), were fabricated by a layer-by-layer adsorption of virus and PAH. The deposition process was studied by quartz crystal microbalance, UV-visible spectroscopy and atomic force microscopy. BMSC adhesion assays showed enhanced cell adhesion and spreading on TYMV-RGD44 coated substrates compared to native TYMV. These results demonstrate the potential of TYMV as a viable scaffold for bioactive peptide display and cell culturing studies.

Interfacial assembly of turnip yellow mosaic virus nanoparticles.

An extensive study of the factors that affect the interfacial assembly of bionanoparticles at the oil/water (O/W) interface is reported. Bionanoparticles, such as viruses, have distinctive structural properties due to the unique arrangement of their protein structures. The assembly process of such bionanoparticles at interfaces is governed by factors including the ionic strength and pH of the aqueous layer, concentration of the particles, and nature of the oil phase. This study highlights the impact of these factors on the interfacial assembly of bionanoparticles at the O/W interface using native turnip yellow mosaic virus (TYMV) as the prototype. Robust monolayer assemblies of TYMV were produced by self-assembly at the O/W interface using emulsions and planar interfaces. TYMV maintained its structure and integrity under different assembly conditions. For the emulsion droplets, they were fully covered with TYMV as evidenced by transmission electron microscopy (TEM) and scanning force microscopy (SFM). Tensiometry and small-angle neutron scattering (SANS) further supported this finding. Although the emulsions offered a complete coverage by TYMV particles, they lacked long-range ordering due to rapid exchange at the interface. By altering the assembly process, highly ordered, hexagonal arrays of TYMV were obtained at planar O/W interfaces. The pH, ionic strength, and viscosity of the solution played a crucial role in enhancing the lateral ordering of TYMV assembled at the planar O/W interface. This interfacial ordering of TYMV particles was further stabilized by introduction of a positively charged dehydroabietyl amine (DHAA) in the organic phase which held the assembly together by electrostatic interactions. The long-range array formation was observed using TEM and SFM. The results presented here illustrate that the interfacial assembly at the O/W interface is a versatile approach to achieve highly stable self-assembled structures.

Complete nucleotide sequence and experimental host range of Okra mosaic virus.

Okra mosaic virus (OkMV) is a tymovirus infecting members of the family Malvaceae. Early infections in okra (Abelmoschus esculentus) lead to yield losses of 12-19.5%. Besides intensive biological characterizations of OkMV only minor molecular data were available. Therefore, we determined the complete nucleotide sequence of a Nigerian isolate of OkMV. The complete genomic RNA (gRNA) comprises 6,223 nt and its genome organization showed three major ORFs coding for a putative movement protein (MP) of M r 73.1 kDa, a large replication-associated protein (RP) of M r 202.4 kDa and a coat protein (CP) of M r 19.6 kDa. Prediction of secondary RNA structures showed three hairpin structures with internal loops in the 5'-untranslated region (UTR) and a 3'-terminal tRNA-like structure (TLS) which comprises the anticodon for valine, typical for a member of the genus Tymovirus. Phylogenetic comparisons based on the RP, MP and CP amino acid sequences showed the close relationship of OkMV not only to other completely sequenced tymoviruses like Kennedya yellow mosaic virus (KYMV), Turnip yellow mosaic virus (TYMV) and Erysimum latent virus (ErLV), but also to Calopogonium yellow vein virus (CalYVV), Clitoria yellow vein virus (CYVV) and Desmodium yellow mottle virus (DYMoV). This is the first report of a complete OkMV genome sequence from one of the various OkMV isolates originating from West Africa described so far. Additionally, the experimental host range of OkMV including several Nicotiana species was determined.

Formation of two-dimensional crystals of icosahedral RNA viruses.

UNLABELLED: The formation of 2D arrays of three small icosahedral RNA viruses with known 3D structures (tomato bushy stunt virus, turnip yellow mosaic virus and bromegrass mosaic virus) has been investigated to determine the role of each component of a negative staining solution containing ammonium molybdate and polyethylene glycol. Virion association was monitored by dynamic light scattering (DLS) and virus array formation was visualised by conventional transmission electron microscopy and cryo-electron microscopy after negative staining. The structural properties of viral arrays prepared in vitro were compared to those of microcrystals found in the leaves of infected plants. A novel form of macroscopic 3D crystals of turnip yellow mosaic virus has been grown in the negative staining solution. On the basis of the experimental results, the hypothesis is advanced that microscopic arrays might be planar crystallisation nuclei. The formation of 2D crystals and the enhancing effect of polyethylene glycol on the self-organisation of virions at the air/water interface are discussed. SYNOPSIS: The formation of 2D arrays of icosahedral viruses was investigated by spectroscopic and transmission electron microscopic methods.

Chimeric tymovirus-like particles displaying foot-and-mouth disease virus non-structural protein epitopes and its use for detection of FMDV-NSP antibodies.

Expression of Physalis mottle tymovirus (PhMV) coat protein (CP) in Escherichia coli (E. coli) was earlier shown to self-assemble into empty capsids that are nearly identical to the capsids formed in vivo. Aminoacid substitutions were made at the N-terminus of wild-type PhMV CP with single or tandem repeats of infection related B-cell epitopes of foot-and-mouth disease virus (FMDV) non-structural proteins (NSPs) 3B1, 3B2, 3AB, 3D and 3ABD of lengths 48, 66, 49, 51 and 55, respectively to produce chimeras pR-Ph-3B1, pR-Ph-3B2, pR-Ph- 3AB, pR-Ph-3D and pR-Ph-3ABD. Expression of these constructs in E. coli resulted in chimeric proteins which self-assembled into chimeric tymovirus-like particles (TVLPs), Ph-3B1, Ph-3B2, Ph-3AB, Ph-3D and Ph-3ABD as determined by ultracentrifugation and electron microscopy. Ph-3B1, Ph-3B2, Ph-3AB and Ph-3ABD reacted with polyclonal anti-3AB antibodies in ELISA and electroblot immunoassay, while wild-type PhMV TVLP and Ph-3D antigens did not react. An indirect ELISA (I-ELISA) was developed using Ph-3AB to detect FMDV-NSP antibodies in sera of animals that showed clinical signs of FMD. Field serum samples from cattle, buffalos, sheep, goats and pigs were examined by using these chimeric TVLPs for the differentiation of FMDV infected animals from vaccinated animals (DIVA). The assay was demonstrated to be highly specific (100%) and reproducible with sensitivity levels (94%) comparable to the Ceditest kit (P>0.05).

Atomic force microscopy investigation of Turnip Yellow Mosaic Virus capsid disruption and RNA extrusion.

Turnip Yellow Mosaic Virus (TYMV) was subjected to a variety of procedures which disrupted the protein capsids and produced exposure of the ssRNA genome. The results of the treatments were visualized by atomic force microscopy (AFM). Both in situ and ex situ freeze-thawing produced RNA emission, though at low efficiency. The RNA lost from such particles was evident, in some cases in the process of exiting the virions. More severe disruption of TYMV and extrusion of intact RNA onto the substrate were produced by drying the virus and rehydrating with neutral buffer. Similar products were also obtained by heating TYMV to 70 -75 degrees C and by exposure to alkaline pH. Experiments showed the nucleic acid to have an elaborate secondary structure distributed linearly along its length.

Molecular characterization of isolates of anagyris vein yellowing virus, plantago mottle virus and scrophularia mottle virus -- comparison of various approaches for tymovirus classification.

The complete nucleotide sequences were determined for the genomic RNAs of three tymoviruses, i.e. isolates of anagyris vein yellowing virus (AVYV), plantago mottle virus (PlMoV) and scrophularia mottle virus (SrMV) which are all serologically closely related to ononis yellow mosaic virus (ibid) and to Nemesia ring necrosis virus (NeRNV), a recently described recombinant virus which is widely spread in commercially grown ornamental plant species belonging to the Scrophulariaceae. Total nucleotide and coat protein amino acid sequence identities revealed similar groupings in the genus tymovirus as serological studies did. The latter, however, tended to suggest much closer relationships than the molecular data and may fail to recognise the distinctiveness of new tymovirus species. The usefulness of various species demarcation criteria for the classification of tymoviruses is discussed.

The RNA of turnip yellow mosaic virus exhibits icosahedral order.

Difference electron density maps, based on structure factor amplitudes and experimental phases from crystals of wild-type turnip yellow mosaic virus and those of empty capsids prepared by freeze-thawing, show a large portion of the encapsidated RNA to have an icosahedral distribution. Four unique segments of base-paired, double-helical RNA, one to two turns in length, lie between 33-A and 101-A radius and are organized about either 2-fold or 5-fold icosahedral axes. In addition, single-stranded loops of RNA invade the pentameric and hexameric capsomeres where they contact the interior capsid surface. The remaining RNA, not seen in electron density maps, must serve as connecting links between these secondary structural elements and is likely icosahedrally disordered. The distribution of RNA observed crystallographically appears to be in agreement with models based on biochemical data and secondary structural analyses.

Atomic force microscopy analysis of icosahedral virus RNA.

Single-stranded genomic RNAs from four icosahedral viruses (poliovirus, turnip yellow mosaic virus (TYMV), brome mosaic virus (BMV), and satellite tobacco mosaic virus (STMV)) along with the RNA from the helical tobacco mosaic virus (TMV) were extracted using phenol/chloroform. The RNAs were imaged using atomic force microscopy (AFM) under dynamic conditions in which the RNA was observed to unfold. RNAs from the four icosahedral viruses initially exhibited highly condensed, uniform spherical shapes with diameters consistent with those expected from the interiors of their respective capsids. Upon incubation at 26 degrees C, poliovirus RNA gradually transformed into chains of globular domains having the appearance of thick, irregularly segmented fibers. These ultimately unwound further to reveal segmented portions of the fibers connected by single strands of RNA of 0.5-1 nm thickness. Virtually the same transformations were shown by TYMV and BMV RNA, and with heating, the RNA from STMV. Upon cooling, the chains of domains of poliovirus RNA and STMV RNA condensed and re-formed their original spherical shapes. TMV RNAs initially appeared as single-stranded threads of 0.5-1.0 nm diameter but took on the structure of the multidomain chains upon further incubation at room temperature. These ultimately condensed into short, thick chains of larger domains. Our observations suggest that classical extraction of RNA from icosahedral virions produces little effect on overall conformation. As tertiary structure is lost however, it is evident that secondary structural elements are arranged in a sequential, linear fashion along the polynucleotide chain. At least in the case of poliovirus and STMV, the process of tertiary structure re-formation from the linear chain of secondary structural domains proceeds in the absence of protein. RNA base sequence, therefore, may be sufficient to encode the conformation of the encapsidated RNA even in the absence of coat proteins.

The formation of empty shells upon pressure induced decapsidation of turnip yellow mosaic virus.

The stability of turnip yellow mosaic virus (TYMV) was investigated under pressure, using solution neutron small angle scattering. Dissociation products were characterized by analytical ultracentrifugation and electron microscopy. At pH 6.0, TYMV remained unaffected by pressure, up to 260 Megapascals (MPa), the highest pressure reached in these experiments. At pH 8.0, TYMV remained unaffected by pressure up to 160 MPa, but decapsidated irreversibly above 200 MPa, giving rise to more and more empty shells upon increasing pressure. The organization of these empty shells was similar to that of the capsid of native virions, apart from the presence of a hole corresponding to the loss of a group of 5-8 coat protein subunits, through which the RNA may have escaped. At variance with other small isometric viruses, the capsid of TYMV never dissociated under pressure into subunits or small aggregates of subunits. This exceptional behavior of TYMV is probably due to the importance of van der Waals contacts and hydrogen bonds in the stability of its capsid.

An approach to an objective background subtraction for elemental mapping with core-edges down to 50 eV: description, evaluation and application.

To image the distribution of a specific element in a specimen with an energy filtering TEM, the element-unspecific background under the core-edge has to be subtracted. The most commonly used procedure is the three-window power-law method leading to considerable systematic errors for low-energy core-edges. Here a new method is described which can be considered as a generalized difference method. Characteristic examples for element detection in biological specimens using this method are shown. The background under the core-edge can be described by one or two pre-edge windows as a polynome of third order. This function can be deduced from specimen areas that are not known to contain the element or from a second specimen used as a standard. Control experiments showed that background subtraction for on-overlapping core-edges in the low-loss region (50-200 eV) needs two pre-edge images, whereas at higher-energy losses (> 300 eV) only one pre-edge image is necessary. With the method described, objective elemental mapping becomes possible even for edges at 50-100 eV. This was proven for the M2,3-edge of iron at 60 eV. The detection of phosphorous was possible with a signal-to-noise ratio five times higher than when using the three-window method. Preliminary data showed that it should be possible to detect calcium with only one image before the edge.

Surface processes in the crystallization of turnip yellow mosaic virus visualized by atomic force microscopy.

In situ atomic force microscopy (AFM) was used to investigate surface evolution during the growth of single crystals of turnip yellow mosaic virus (TYMV). Growth of the (101) face of TYMV crystals proceeded by two-dimensional nucleation. The molecular structure of the step edges and adsorption of individual virus particles and their aggregates on the crystalline surface were recorded. The surfaces of individual virions within crystals were visualized and seen to be quite distinctive with the hexameric and pentameric capsomers of the T = 3 capsids being clearly resolved. This, so far as we are aware, is the first direct visualization of the capsomere structure of a virus by AFM. In the course of recording the in situ development of the crystals, a profound restructuring of the surface arrangement was observed. This transformation was highly cooperative in nature, but the transitions were unambiguous and readily explicable in terms of an organized loss of classes of virus particles from specific lattice positions. In some cases areas of a single crystal surface were recorded in which were captured successive phases of the transition. We believe this provides the first visual record of a cooperative restructuring of the surface of a supramolecular crystal.

Polymorphism of turnip yellow mosaic virus empty shells and evidence for conformational changes occurring after release of the viral RNA. A differential scanning calorimetric study.

Turnip yellow mosaic virus (TYMV) is a small isometric plant virus which decapsidates by releasing its RNA through a hole in the capsid, leaving behind an empty shell [R. E. F. Matthews and J. Witz, (1985) Virology 144, 318-327]. Similar empty shells (artificial top component, ATC) can be obtained by submitting the virions to various treatments in vitro. We have used differential scanning calorimetry, analytical sedimentation, and electron microscopy to investigate the thermodenaturation of natural empty shells (NTC, natural top component) present in purified virus suspensions, and of several types of ATCs. ATCs divided in two major classes. Those obtained by alkaline titration, by the action of urea or butanol behaved as NTC: their thermograms contained only one peak corresponding to the irreversible dissociation of the shells and the denaturation of the coat protein. The temperature of this unique transition varied significantly with pH, from 71 degrees C at pH 4.5 to 84 degrees C at pH 8.5. The thermograms of ATCs obtained by freezing and thawing, or by the action of high pressure, contained two peaks: shells dissociated first into smaller protein aggregates at 57 degrees C (at pH 5.0) to 61 degrees C (at pH 8.5), which denatured at the temperature of the unique transition of NTC. Shells obtained by heating virions to 55 degrees C at pH 7.6, changed conformation after the release of the viral RNA, as upon continuous heating to 95 degrees C, their thermograms were similar to those of the shells obtained by freezing and thawing, whereas after purification they behaved like NTC. Structural implications of these observations are discussed.

Use of a mass-thickness marker to estimate systematic errors and statistical noise in the detection of phosphorus by electron spectroscopic imaging.

The element signal obtained from electron-energy-filtered micrographs depends on the systematic error in calculating the background and on the noise in the background-corrected image. Both systematic error and statistical fluctuation of the background can be assessed experimentally with a specimen that combines the element-containing feature with a mass-thickness marker. The approach is described for the mapping of phosphorus in turnip yellow mosaic viruses prepared on a supporting carbon film of variable thickness. The thickness modulations are produced by the additional deposition of heat-evaporated carbon through a second grid used as a mask. The three-window power-law method and the two-window difference method are compared. With the three-window power-law method, the mass-thickness modulations of the marker are still visible in the map, indicating a systematic error for the calculated background. In addition, the intensity profile over the area of the thick carbon film is broader than in the map corrected by the two-window method, indicating a higher level of noise. With the two-window difference method, mass-thickness contrast was practically eliminated due to an improved protocol that uses the mass-thickness marker to calculate the scaling factor: instead of scaling the grey-level of a single background feature, the pre-edge image is scaled to the contrast of the marker area in the image acquired at the element-specific energy loss.

Identification of okra mosaic virus from Indigofera spicata in Nigeria.

Okra mosaic virus (OMV, tymovirus group) was isolated from Indigofera spicata plants growing at the International Institute of Tropical Agriculture (IITA) in Ibadan, Nigeria. Its identity was established on the basis of particle morphology, analysis of viral coat protein and nucleic acid and serology. In reciprocal agar gel diffusion tests, the virus isolate from I. spicata and an OMV isolate from okra in Ibadan (OMV-Ibadan isolate) were found to be serologically identical. However, because the isolates differ in symptom induction in various host plants, the name OMV-Indigofera isolate is suggested. This is the first report on the occurence of OMV in I. spicata.

Crystal structure of turnip yellow mosaic virus.

The structure of turnip yellow mosaic virus (TYMV) has been solved to 3.2 A resolution and an R-value of 18.7%. The structure is consistent with models based on low resolution X-ray and electron microscopy studies, with pentameric and hexameric protein aggregates protruding from the surface and forming deep valleys at the quasi three-fold axes. The N-terminal 26 residues of the A-subunit are disordered, while those of the B- and C-subunits are seen to interact around the interior of the quasi six-fold cluster where they form an annulus. The three histidine residues of each protein subunit are located in the interior and accessible for interaction with the RNA genome. The appearance of the interior surface of the virus capsid, along with buried surface area calculations, suggest that a pentameric unit is lost during decapsidation.

Difference imaging reveals ordered regions of RNA in turnip yellow mosaic virus.

BACKGROUND: Turnip yellow mosaic virus (TYMV) is a small icosahedral plant virus with a capsid containing 180 subunits arranged with hexamer-pentamer clustering. Cross-linking studies have indicated extensive contacts between RNA and coat protein, suggesting that substantial parts of the RNA might be icosahedrally ordered. RESULTS: Comparison of maps computed to a Fourier cut-off of 1.5 nm from electron micrographs of ice-embedded specimens of TYMV and of empty capsids produced by freeze-thawing reveals strong inner features around the threefold axes in the virus but not in the empty capsid. Internal features of subunit packing indicate that interhexamer contacts are closer than those between pentamers and hexamers and that pentamer density in the empty capsid is reduced relative to that in the virus. CONCLUSIONS: The differences between virus and empty capsid indicate that substantial parts of the RNA are icosahedrally ordered and that the exit of RNA on freeze-thawing is accompanied by the loss of at least one pentamer unit.