Comparative Study of Non-Enveloped Icosahedral Viruses Size.

Now, as before, transmission electron microscopy (TEM) is a widely used technique for the determination of virions size. In some studies, dynamic light scattering (DLS) has also been applied for this purpose. Data obtained by different authors and using different methods could vary significantly. The process of TEM sample preparation involves drying on the substrate, which can cause virions to undergo morphology changes. Therefore, other techniques should be used for measurements of virions size in liquid, (i.e. under conditions closer to native). DLS and nanoparticle tracking analysis (NTA) provide supplementary data about the virions hydrodynamic diameter and aggregation state in liquid. In contrast to DLS, NTA data have a higher resolution and also are less sensitive to minor admixtures. In the present work, the size of non-enveloped icosahedral viruses of different nature was analyzed by TEM, DLS and NTA: the viruses used were the encephalomyocarditis virus (animal virus), and cauliflower mosaic virus, brome mosaic virus and bean mild mosaic virus (plant viruses). The same, freshly purified, samples of each virus were used for analysis using the different techniques. The results were compared with earlier published data and description databases. DLS data about the hydrodynamic diameter of bean mild mosaic virus, and NTA data for all examined viruses, were obtained for the first time. For all virus samples, the values of size obtained by TEM were less than virions sizes determined by DLS and NTA. The contribution of the electrical double layer (EDL) in virions hydrodynamic diameter was evaluated. DLS and NTA data adjusted for EDL thickness were in better agreement with TEM results.

Ultrastructural insights into tomato infections caused by three different pathotypes of Pepino mosaic virus and immunolocalization of viral coat proteins.

This paper presents studies on an ultrastructural analysis of plant tissue infected with different pathotypes of Pepino mosaic virus (PepMV) and the immunolocalization of viral coat proteins. Because the PepMV virus replicates with a high mutation rate and exhibits significant genetic diversity, therefore, isolates of PepMV display a wide range of symptoms on infected plants. In this work, tomato plants of the Beta Lux cultivar were inoculated mechanically with three pathotypes representing the Chilean 2 (CH2) genotype: mild (PepMV-P22), necrotic (PepMV-P19) and yellowing (PepMV-P5-IY). The presence of viral particles in all infected plants in the different compartments of various cell types (i.e. spongy and palisade mesophyll, sieve elements and xylem vessels) was revealed via ultrastructural analyses. For the first time, it was possible to demonstrate the presence of crystalline inclusions, composed of virus-like particles. In the later stage of PepMV infection (14 dpi) various pathotype-dependent changes in the structure of the individual organelles (i.e. mitochondria, chloroplasts) were found. The strongest immunogold labeling of the viral coat proteins was also observed in plants infected by necrotic isolates. A large number of viral coat proteins were marked in the plant conductive elements, both xylem and phloem.

The molecular basis for flexibility in the flexible filamentous plant viruses.

Flexible filamentous plant viruses cause more than half the viral crop damage in the world but are also potentially useful for biotechnology. Structural studies began more than 75 years ago but have failed, owing to the virion's extreme flexibility. We have used cryo-EM to generate an atomic model for bamboo mosaic virus, which reveals flexible N- and C-terminal extensions that allow deformation while still maintaining structural integrity.

[The characterization of bean yellow mosaic virus isolated from soybean].

The comparative characteristics of biological, physico-chemical and antigenic properties of bean yellow mosaic virus isolated from soybeans are shown. The obtained results indicate the existence of individual properties of the isolate which help to distinguish it from the typical strains and isolates studied previously in Ukraine by other authors.

The backbone model of the Arabis mosaic virus reveals new insights into functional domains of Nepovirus capsid.

Arabis mosaic virus (ArMV) and Grapevine fanleaf virus (GFLV) are two picorna-like viruses from the genus Nepovirus, consisting in a bipartite RNA genome encapsidated into a 30 nm icosahedral viral particle formed by 60 copies of a single capsid protein (CP). They are responsible for a severe degeneration of grapevines that occurs in most vineyards worldwide. Although sharing a high level of sequence identity between their CP, ArMV is transmitted exclusively by the ectoparasitic nematode Xiphinema diversicaudatum whereas GFLV is specifically transmitted by the nematode X. index. The structural determinants involved in the transmission specificity of both viruses map solely to their respective CP. Recently, reverse genetic and crystallographic studies on GFLV revealed that a positively charged pocket in the CP B domain located at the virus surface may be responsible for vector specificity. To go further into delineating the coat protein determinants involved in transmission specificity, we determined the 6.5 Å resolution cryo-electron microscopy structure of ArMV and used homology modeling and flexible fitting approaches to build its pseudo-atomic structure. This study allowed us to resolve ArMV CP architecture and delineate connections between ArMV capsid shell and its RNA. Comparison of ArMV and GFLV CPs reveals structural differences in the B domain pocket, thus strengthening the hypothesis of a key role of this region in the viral transmission specificity and identifies new potential functional domains of Nepovirus capsid.

The crystallographic structure of Panicum Mosaic Virus (PMV).

The structure of Panicum Mosaic Virus (PMV) was determined by X-ray diffraction analysis to 2.9Å resolution. The crystals were of pseudo symmetry F23; the true crystallographic unit cell was of space group P2(1) with a=411.7Å, b=403.9Å and c=412.5Å, with ß=89.7°. The asymmetric unit was two entire T=3 virus particles, or 360 protein subunits. The structure was solved by conventional molecular replacement from two distant homologues, Cocksfoot Mottle Virus (CfMV) and Tobacco Necrosis Virus (TNV), of ∼20% sequence identity followed by phase extension. The model was initially refined with exact icosahedral constraints and then with icosahedral restraints. The virus has Ca(++) ions octahedrally coordinated by six aspartic acid residues on quasi threefold axes, which is completely different than for either CfMV or TNV. Amino terminal residues 1-53, 1-49 and 1-21 of the A, B and C subunits, respectively, and the four C-terminal residues (239-242) are not visible in electron density maps. The additional ordered residues of the C chain form a prominent "arm" that intertwines with symmetry equivalent "arms" at icosahedral threefold axes, as was seen in both CfMV and TNV. A 17 nucleotide hairpin segment of genomic RNA is icosahedrally ordered and bound at 60 equivalent sites at quasi twofold A-B subunit interfaces at the interior surface of the capsid. This segment of RNA may serve as a conformational switch for coat protein subunits, as has been proposed for similar RNA segments in other viruses.

A new isolate of Iris severe mosaic virus causing yellow mosaic in Iris ensata Thunb.

Severe mosaic disease was observed on Iris ensata Thunb. in Spring 2008, in Hangzhou, China and it was found to be widely distributed in that region. Detection of viruses by electron microscopy resulted in the occurrence of a potyvirus in most symptomatic seedlings. Sequencing 1745 nucleotides of the 3'-terminal region of the genome of the typical viral isolate revealed that it was a new isolate of Iris severe mosaic potyvirus (ISMV), tentatively named ISMV-PHz .This strain shared high nucleotide identity with ISMV. Phylogenetic analysis also showed that this isolate clustered with iris severe mosaic potyvirus (ISMV) and onion yellow dwarf virus (OYDV) into a monophyletic group, and was closest in similarity to ISMV. The divergence of potyvirus-infecting iris species had a higher degree of relevance with natural host but not with the region from which it was isolated. This is the first report of ISMV isolated from I. ensata in China.

Primary changes of the mechanical properties of Southern Bean Mosaic Virus upon calcium removal.

The mechanical properties of viral shells are crucial determinates for the pathway and mechanism by which the genetic material leaves the capsid during infection and have therefore been studied by atomic force microscopy as well as by atomistic simulations. The mechanical response to forces from inside the capsid are found to be relevant, especially after ion removal from the shell structure, which is generally assumed to be essential during viral infection; however, atomic force microscopy measurements are restricted to probing the capsids from outside, and the primary effect of ion removal is still inaccessible. To bridge this gap, we performed atomistic force-probe molecular dynamics simulations of the complete solvated icosahedral shell of Southern Bean Mosaic Virus and compared the distribution of elastic constants and yielding forces on the icosahedral shell for probing from inside with the distribution of outside mechanical properties obtained previously. Further, the primary effect of calcium removal on the mechanical properties on both sides, as well as on their spatial distribution, is quantified. Marked differences are seen particularly at the pentamer centers, although only small structural changes occur on the short timescales of the simulation. This unexpected primary effect, hence, precedes subsequent effects due to capsid swelling. In particular, assuming that genome release is preceded by an opening of capsomers instead of a complete capsid bursting, our observed weakening along the fivefold symmetry axes let us suggest pentamers as possible exit ports for RNA release.

Diagnosis of virus infection in orchid plants with high-resolution optical coherence tomography.

This work investigates the use of optical coherence tomography (OCT) to identify virus infection in orchid plants. Besides revealing the cross-sectional structure of orchid leaves, highly scattering upper leaf epidermides are detected with OCT for virus-infected plants. This distinct feature is not observable under histological examination of the leaf samples. Furthermore, the leaf epidermides of stressed but healthy plants, which exhibit similar visual symptoms as virus-infected plants, are not highly scattering and are similar to those of healthy plants. The results suggest that virus-infected orchid plants can be accurately identified by imaging the epidermal layers of their leaves with OCT. The OCT modality is suitable for fast, nondestructive diagnosis of orchid virus infection, which may potentially lead to significant cost savings and better control of the spread of viruses in the orchid industry.

Mechanical properties of the icosahedral shell of southern bean mosaic virus: a molecular dynamics study.

The mechanical properties of viral shells are crucial for viral assembly and infection. To study their distribution and heterogeneity on the viral surface, we performed atomistic force-probe molecular dynamics simulations of the complete shell of southern bean mosaic virus, a prototypical T = 3 virus, in explicit solvent. The simulation system comprised more than 4,500,000 atoms. To facilitate direct comparison with atomic-force microscopy (AFM) measurements, a Lennard-Jones sphere was used as a model of the AFM tip, and was pushed with different velocities toward the capsid protein at 19 different positions on the viral surface. A detailed picture of the spatial distribution of elastic constants and yielding forces was obtained that can explain corresponding heterogeneities observed in previous AFM experiments. Our simulations reveal three different deformation regimes: a prelinear regime of outer surface atom rearrangements, a linear regime of elastic capsid deformation, and a rearrangement regime that describes irreversible structural changes and the transition from elastic to plastic deformation. For both yielding forces and elastic constants, a logarithmic velocity dependency is evident over nearly two decades, the explanation for which requires including nonequilibrium effects within the established theory of enforced barrier crossing.

Rubus chlorotic mottle virus, a new sobemovirus infecting raspberry and bramble.

The complete nucleotide sequence of a new member of the unassigned genus Sobemovirus, isolated from raspberry and bramble plants in north east Scotland and given the name Rubus chlorotic mottle virus (RuCMV), was obtained. The virus has a single, positive-strand RNA genome of 3,983 nucleotides and, in common with other sobemoviruses, contains four open reading frames (ORFs) encoding, from 5' to 3', the P1 protein that is likely to be a suppressor of RNA silencing, ORF2a that has homology to serine-proteases, ORF2b that is the probable RNA dependent RNA polymerase, and ORF3 that is the coat protein. ORF2b protein is potentially expressed as a fusion with ORF2a protein by a -1 frameshift at the heptanucleotide sequence UUUAAAC. Phylogenetic analyses showed that RuCMV is a distinct virus not closely related to any of the other sequenced sobemoviruses. Based on the obtained sequence a full-length cDNA copy of RuCMV was cloned and in vitro transcripts derived from this clone were shown to be fully infectious.

Rapid and efficient purification of Cowpea chlorotic mottle virus by sucrose cushion ultracentrifugation.

A simple, economical and efficient method for isolation of Cowpea chlorotic mottle virus (CCMV) particles was developed. Abundant viral particles could be obtained with three steps of 10 min duration each of conventional differential centrifugations and one sucrose cushion ultracentrifugation step of 2 h. Characterization of purified virus assessed by transmission electron microscopy, polyacrylamide gel electrophoresis (PAGE) and Western blot revealed the typical isometric particles of CCMV without any visible contamination or degradation. RNA extracted from purified CCMV particles showed all four RNA components by agarose gel electrophoresis. Both purified virus particles and RNA were biologically active and initiated successful infection upon inoculation to cowpea.

Aspects of adaptive answering formation in virus-host plant pathosystem for different wheat cultivars under simulating microgravity condition.

Investigations of prolonged clinorotation effect on some morphological and physiological parameters under Wheat streak mosaic virus WSMW-infection of Apogee and Lada wheat cultivars were carried out. Experiments were held on universal clinostat CYCLE-2. Clinorotation caused changing of WSMV virions shape and reducing of the virus reproduction. Apogee wheat plants grown under two stress factors (infection and clinorotation) produced more kernels than stationary (motionless) plants, but the average weight of kernel was lower. Under clinorotation changes in host plant-virus system take place and adaptive reactions for simulated microgravity conditions form. These lead to reduction of potyvirus replication.

Molecular evidence supporting the confirmation of maracuja mosaic virus as a species of the genus Tobamovirus and production of an infectious cDNA transcript.

The complete genome sequence of maracuja mosaic virus (MarMV) was determined and analyzed. The full MarMV genome consisted of 6794 nucleotides, and this is the largest genome size among known tobamoviruses. The MarMV genome RNA contained four open reading frames (ORFs) coding for proteins of M(r) 126, 181, 34 and 18 kDa from the 5' to 3' end, respectively. The lengths of the 5' nontranslated region (NTR) and the 3' NTR were 54 and 177 nucleotides, respectively. Phylogenetic tree analysis revealed that these MarMV-encoded proteins are related to members of the Malvaceae- and Cucurbitaceae-infecting tobamoviruses. MarMV is different from other tobamoviruses and forms a new Passifloraceae-infecting subgroup. Western blot analysis showed that MarMV cross-reacted strongly with antibodies against Kyuri green mottle mosaic virus and Hibiscus latent Singapore virus. Synthesized capped transcripts from full-length cDNA of MarMV were infectious. These data clearly indicate that MarMV belongs to a separate species of the genus Tobamovirus.

Occurrence of viruses infecting pea in Iran.

A survey was conducted to determine the incidence of Alfalfa mosaic virus (AMV), Bean yellow mosaic virus (BYMV), Broad bean wilt virus-1 (BBWV), Pea leafroll virus (PLRV), Pea enation mosaic virus (PEMV), Pea seed borne mosaic virus (PSbMV), Potato virus x(PVX), Tomato mosaic virus (ToMV), Tomato spotted wilt virus (TSWV) on pea (Pisum sativum) in Iran. A Total of 1276 random and 684 symptomatic pea samples were collected during the spring and summer of 2002-2004 in Tehran province of Iran, where pea is grown, and tested by enzyme-linked immunosorbent assay (ELISA) using specific polyclonal antibodies. Serological diagnoses were confirmed by electron microscopy and host range studies. Incidence of viruses in decreasing order was PVX (69%), ToMV (59%), PSbMV (36.6%), BBWV-1 (26.1%), BYMV (20.3%), AMV (17.77%), TSWV (12.6%), PEMV (10.9%), PLRV (6.78%). In this survey, natural occurrence of AMV, BBWV-1, PSbMV, TSWV, PVX and ToMV was reported for the first time on the pea in Iran.

Role of metal ion-mediated interactions in the assembly and stability of Sesbania mosaic virus T=3 and T=1 capsids.

Sesbania mosaic virus (SeMV) capsids are stabilized by RNA-protein, protein-protein and calcium-mediated protein-protein interactions. The removal of calcium has been proposed to be a prerequisite for the disassembly of the virus. The crystal structure of native T=3 SeMV capsid revealed that residues D146 and D149 from one subunit and Y205, N267 and N268 of the neighboring subunit form the calcium-binding site (CBS). The CBS environment is found to be identical even in the recombinant CP-NDelta65 T=1 capsids. Here, we have addressed the role of calcium and the residues involved in calcium co-ordination in the assembly and stability of T=3 and T=1 capsids by mutational analysis. Deletion of N267 and N268 did not affect T=3 or T=1 assembly, although the capsids were devoid of calcium, suggesting that assembly does not require calcium ions. However, the stability of the capsids was reduced drastically. Site-directed mutagenesis revealed that either a single mutation (D149N) or a double mutation (D146N-D149N) of SeMV coat protein affected drastically both the assembly and stability of T=3 capsids. On the other hand, the D146N-D149N mutation in CP-NDelta65 did not affect the assembly of T=1 capsid, although their stability was reduced considerably. Since the major difference between the T=3 and T=1 capsids is the absence of the N-terminal arginine-rich motif (N-ARM) and the beta-annulus from the subunits forming the T=1 capsids, it is possible that D149 initiates the N-ARM-RNA interactions that lead to the formation of the beta-annulus, which is essential for T=3 capsid assembly.

Evidence that soilborne wheat mosaic virus moves long distance through the xylem in wheat.

Soilborne wheat mosaic virus (SBWMV) is a member of the genus Furovirus of plant viruses. SBWMV is transmitted to wheat roots by the plasmodiophorid vector Polymyxa graminis. Experiments were conducted to determine the path for SBWMV transport from roots to leaves. The results of immunogold labeling suggest that SBWMV enters and moves long distance through the xylem. SBWMV may enter primary xylem elements before cell death occurs and then move upward in the plant after the xylem has matured into hollow vessels. There is also evidence for lateral movement between adjacent xylem vessels.

Leginon: an automated system for acquisition of images from vitreous ice specimens.

We have developed a system to automatically acquire cryo-electron micrographs. The system is designed to emulate all of the decisions and actions of a highly trained microscopist in collecting data from a vitreous ice specimen. These include identifying suitable areas of vitreous ice at low magnification, determining the presence and location of specimen on the grid, automatically adjusting imaging parameters (focus, astigmatism) under low-dose conditions, and acquiring images at high magnification to either film or a digital camera. This system is responsible for every aspect of image acquisition and can run unattended, other than requiring periodic refilling of the cryogens, for over 24 h. The system has been tested out on a variety of specimens that represent typical challenges in the field of cryo-electron microscopy. The results show that the overall performance of the system is equivalent to that of an experienced microscopist.

Molecular characterization and interviral relationships of a flexuous filamentous virus causing mosaic disease of sugarcane (Saccharum officinarum L.) in India.

A virus isolate causing mosaic disease of commercial sugarcane was purified to homogeneity. Electron microscopy revealed flexuous filamentous virus particles of ca 890 x 15 nm. The virus isolate reacted positively with heterologous antiserum to narcissus latent virus form UK, but failed to react with potyvirus group specific antiserum. N-terminal sequencing of the intact coat protein (CP) and the tryptic peptides indicated that the virus was probably a potyvirus but distinct from several reported potyviruses. Comparison of the 3'-terminal 1084 nucleotide sequence of the RNA genome of this virus revealed 93.6% sequence identity in the coat protein coding region with the recently described sugarcane streak mosaic virus (Pakistani isolate). The molecular weight of the coat protein (40 kDa) was higher than that deduced from the amino acid sequence (34 kDa). The apparent increase in size was shown to be due to glycosylation of the coat protein which has not been reported thus far in the family, Potyviridae. This is the first report on the molecular characterization of a virus causing mosaic disease of sugarcane in India and the results demonstrate that the virus is a strain of sugarcane streak mosaic virus, a member of the Tritimovirus genus of the Potyviridae. We have named it sugarcane streak mosaic virus--Andhra Pradesh isolate (SCSMV-AP).

Mutations in viral movement protein alter systemic infection and identify an intercellular barrier to entry into the phloem long-distance transport system.

Viral systemic infection of a plant host involves two processes, cell-to-cell movement and long-distance transport. Molecular determinants associated with these two processes were probed by investigating the effects that alanine scanning mutations in the movement protein (MP) of red clover necrotic mosaic virus (RCNMV) had on viral infection in the plant hosts Nicotiana edwardsonii, Vigna unguiculata (cowpea), and the experimental plant Nicotiana benthamiana. Plants were inoculated with RCNMV expressing wild-type and mutant forms of the MP. Immunocytochemical studies at the light and electron microscope levels were performed on these plants, using a polyclonal antibody raised against the RCNMV capsid protein to identify the cells/tissues that RCNMV could infect. These experiments demonstrated that one cellular boundary at which the RCNMV MP functions to facilitate entry into the phloem long-distance transport system is located at the interfaces between the bundle sheath and phloem parenchyma cells and the companion cell-sieve element complex. Interestingly, in Nicotiana tabacum, a host that only allows a local infection, RCNMV cell-to-cell movement was found to be blocked at this same intercellular boundary. Four mutants that were able to systemically infect N. benthamiana were partially or completely defective for systemic infection of N. edwardsonii and cowpea, which indicated that these MP mutants exhibited host-specific defects. Thus, the roles of the RCNMV MP in cell-to-cell movement and in long-distance transport appear to be genetically distinct. These results are discussed in terms of the mechanism by which RCN MV enters the phloem to establish a systemic infection.