A local strain of Paprika mild mottle virus breaks L3 resistance in peppers and is accelerated in Tomato brown rugose fruit virus-infected Tm-22-resistant tomatoes.

During October 2014, unfamiliar mild mosaic and mottling symptoms were identified on leaves of pepper (Capsicum chinense cv. Habanero) seedlings grown in the Arava valley in Israel 2-3 weeks post planting. Symptomatic plants were tested positive by ELISA using laboratory-produced antisera for tobamovirus species. Typical tobamovirus rod-shaped morphology was observed by transmission electron microscopy (TEM) analysis of purified virion preparation that was used for mechanical inoculation of laboratory test plants for the completion of Koch's postulates. The complete viral genome was sequenced from small interfering RNA purified from symptomatic pepper leaves and fruits by next-generation sequencing (NGS) using Illumina MiSeq platform. The contigs generated by the assembly covered 80% of the viral genome. RT-PCR amplification and Sanger sequencing were employed in order to validate the sequence generated by NGS technology. The nucleotide sequence of the complete viral genome was 99% identical to the complete genome of Paprika mild mottle virus isolate from Japan (PaMMV-J), and the deduced amino acid sequence was 99% identical to PaMMV-J protein. Amplicons from seed RNA showed 100% identity to the viral isolate from the collected symptomatic pepper plants. Partial host range analysis revealed a slow development of systemic infection in inoculated tomato plants (Lycopersicon esculentum). Interestingly, double inoculation of susceptible wild-type tomato plants and Tm-22-resistant tomato plants with the PaMMV-IL and Tomato brown rugose fruit virus (ToBRFV) resulted in accelerated viral expression in the plants.

The length of an internal poly(A) tract of hibiscus latent Singapore virus is crucial for its replication.

Hibiscus latent Singapore virus (HLSV) mutants were constructed to study roles of its internal poly(A) tract (IPAT) in viral replication and coat protein (CP) expression. Shortening of the IPAT resulted in reduced HLSV RNA accumulation and its minimal length required for HLSV CP expression in plants was 24 nt. Disruption of a putative long range RNA-RNA interacting structure between 5' and 3' untranslated regions of HLSV-22A and -24A resulted in reduced viral RNA and undetectable CP accumulation in inoculated leaves. Replacement of the IPAT in HLSV with an upstream pseudoknot domain (UPD) of Tobacco mosaic virus (TMV) or insertion of the UPD to the immediate downstream of a 24 nt IPAT in HLSV resulted in drastically reduced viral RNA replication. Plants infected with a TMV mutant by replacement of the UPD with 43 nt IPAT exhibited milder mosaic symptoms without necrosis. We have proposed a model for HLSV replication.

Tracking viral movement in plants by means of chlorophyll fluorescence imaging.

Many techniques have been applied to understand viral cell-to-cell movement in host plants, but little progress has been made in understanding viral vascular transport mechanisms. We propose the use of chlorophyll fluorescence imaging techniques, not only to diagnose the viral infection, but also to follow the movement of the virus through the vascular system and its subsequent spread into the leaves. In Nicotiana benthamiana plants, imaging of chlorophyll fluorescence parameters such as Ф(PSII) and NPQ proved useful to follow infections with Pepper mild mottle virus. The results demonstrate a correlation between changes in the chlorophyll fluorescence parameters and the viral distribution analyzed by tissue printing.

Immunoabsorbent nanoparticles based on a tobamovirus displaying protein A.

Earlier attempts to express peptides longer than 20 aa on the surface of tobamoviruses such as tobacco mosaic virus have failed. Surprisingly, we found that a functional fragment of protein A (133 aa) can be displayed on the surface of a tobamovirus as a C-terminal fusion to the coat protein via a 15-aa linker. The macromolecular nature of these nanoparticles allowed the design of a simple protocol for purification of mAbs with a recovery yield of 50% and > 90% product purity. The extremely dense packing of protein A on the nanoparticles (> 2,100 copies per viral particle) results in an immunoadsorbent with a binding capacity of 2 g mAb per g. This characteristic, combined with the high level of expression of the nanoparticles (> 3 g adsorbent per kg of leaf biomass), provides a very inexpensive self-assembling matrix that could meet the criteria for a single-use industrial immunoadsorbent for antibody purification.

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.

Construction of an infectious cDNA clone of Ribgrass mosaic virus Shanghai isolate and its modification to express an epitope of Mycobacterium tuberculosis.

Infectious cDNA clones of the Shanghai isolate of Ribgrass mosaic virus (RMV) were produced by joining four overlapping cDNA fragments and also in a single step by long template PCR. After inoculation of Nicotiana glutinosa with either RNA transcripts or the cDNA under the control of the CaMV 35S promoter, plants developed typical symptoms, and viral coat protein could be detected in them by Western blot analysis. However, compared to plants inoculated with purified viral RNA, lesions were fewer and appeared more slowly. An epitope of the Mycobacterium tuberculosis 31-kDa protein was inserted at the C-terminus of the viral coat protein by PCR using two overlapping fragments. The modified clone was also infectious and the foreign epitope could be detected serologically in the electron microscope and by Western blot analysis. The results demonstrate the potential of RMV as a viral gene vector.

Imaging viral infection: studies on Nicotiana benthamiana plants infected with the pepper mild mottle tobamovirus.

We have studied by kinetic Chl-fluorescence imaging (Chl-FI) Nicotiana benthamiana plants infected with the Italian strain of the pepper mild mottle tobamovirus (PMMoV-I). We have mapped leaf photosynthesis at different points of the fluorescence induction curve as well as at different post-infection times. Images of different fluorescence parameters were obtained to investigate which one could discriminate control from infected leaves in the absence of symptoms. The non-photochemical quenching (NPQ) of excess energy in photosystem II (PSII) seems to be the most adequate chlorophyll fluorescence parameter to assess the effect of tobamoviral infection on the chloroplast. Non-symptomatic mature leaves from inoculated plants displayed a very characteristic time-varying NPQ pattern. In addition, a correlation between NPQ amplification and virus localization by tissue-print was found, suggesting that an increase in the local NPQ values is associated with the areas invaded by the pathogen. Changes in chloroplast ultrastructure in non-symptomatic leaf areas showing different NPQ levels were also investigated. A gradient of ultrastructural modifications was observed among the different areas.

Cactus mild mottle virus is a new cactus-infecting tobamovirus.

A new cactus-infecting tobamovirus, Cactus mild mottle virus (CMMoV), was isolated from diseased grafted cactus, Gymnocalycium mihanovichii and its molecular properties were characterized. CMMoV is distantly related to known species of the genus Tobamovirus on the basis of serological and sequence analyses. Western blot analysis showed that CMMoV is serologically unrelated to Sammon's Opuntia virus, which is the only known species of the genus Tobamovirus found in cactus plants. The 3'-terminal 2,910 nucleotides of CMMoV have been sequenced. The coat protein (CP) and movement protein (MP) genes encode 161 and 306 amino acids residues, respectively, and the 3' untranslated region (UTR) consists of 229 nucleotides long. The nucleotide and amino acid sequences of the CP of CMMoV were 39.6% to 49.2% and 25.8% to 40.3% identical to other seventeen tobamoviruses, respectively. The MP shared 34.9% to 40.6% and 16.3% to 27.0% and 44.6% to 63.4% identities, respectively, at the amino acid and nucleotide levels with other members of the genus. Percentage identities of nucleotides of the 3' UTR ranged from 42.5% to 63.4%. Phylogenetic tree analyses of the CP and MP suggest the existence of the fifth cactus-infecting subgroup in the genus Tobamovirus. Sequence analyses of these two viral proteins revealed that the highest amino acid sequence identity between the virus and seventeen other tobamoviruses was 40.6%, supporting the view that CMMoV is a new definite species of the genus Tobamovirus.

Expression and purification of a neuropeptide nocistatin using two related plant viral vectors.

Both odontoglossum ringspot virus (ORSV) and tobacco mosaic virus (TMV) were investigated as expression viral vectors for the expression of a neuropeptide nocistatin. Chimeras of ORSV and TMV were constructed by fusion of 17 amino acids of mouse nocistatin (mNST) to the C-terminal of the coat protein (CP) gene via a Factor Xa cleavage linker to yield ORSV-mNST and TMV-mNST. Expression of the mNST peptide was demonstrated by immuno-transmission electron microscopy, western blot, mass spectrometry and radioimmunoassay. Serial passaging of the chimeric viruses revealed loss of mNST from TMV-mNST by the fifth passage. The mNST was maintained in ORSV-mNST throughout six passages. The mNST peptide could be effectively cleaved and purified from chimeric ORSV CP. To our knowledge, this is the first successful attempt in obtaining a complete peptide with no additional amino acid sequence after expression and purification through the use of either ORSV or TMV as vectors.

Rapid, high-level expression of glycosylated rice alpha-amylase in transfected plants by an RNA viral vector.

Tobamoviral vectors have been developed for the heterologous expression of glycoproteins in plants. The rice alpha-amylase gene (OS103) was placed under the transcriptional control of a tobamovirus subgenomic promoter in a RNA viral vector. One to two weeks after inoculation, transfected Nicotiana benthamiana plants accumulated glycosylated alpha-amylase to levels of at least 5% total soluble protein. The 46kDa recombinant enzyme was purified, and its structural and biological properties were analyzed. Post-translational modifications of the secreted protein were compared to rice alpha-amylase expressed in amylolytic strains of Pichia pastoris and Saccharomyces cerevisiae. Endo-H analysis revealed that the alpha-amylase was moderately glycosylated in transfected plants and hyperglycosylated in yeast.

Rapid simultaneous detection of two orchid viruses using LC- and/or MALDI-mass spectrometry.

Liquid chromatography/mass spectrometry (LC/MS) and matrix-assisted laser desorption-ionization (MALDI) mass spectrometry are capable of providing molecular mass information on biological samples with high speed, accuracy and sensitivity. With mass spectrometry, identifying a virus based on the molecular weight of its coat protein is relatively simple and accurate. The technique can be applied to all viruses with known coat protein molecular weights. Using the LC/MS and/or MALDI, this paper describes rapid simultaneous detection of the two most prevalent orchid viruses, namely cymbidium mosaic potexvirus (CymMV) and odontoglossum ringspot tobamovirus (ORSV). The coat protein molecular weights of CymMV and ORSV were detected accurately using an extract from 1 g of virus-infected Oncidium orchid flower. Because LC/MS and MALDI allow automated analyses of multiple samples with simple preparation steps, both techniques are ideal for rapid identification of viruses from a large number of samples. This is the first report on the application of LC/MS and/or MALDI for simultaneous detection of two plant viruses from an infected plant extract.

Zucchini green mottle mosaic virus is a new tobamovirus; comparison of its coat protein gene with that of kyuri green mottle mosaic virus.

A novel virus we call zucchini green mottle mosaic virus (ZGMMV) was isolated from zucchini squash and its properties were determined. The size and shape of its virions, and other properties suggest that the virus is a tobamovirus. The coat protein (CP) genes of ZGMMV and kyuri green mottle mosaic virus (KGMMV), which also infects zucchini squash plants, were cloned and their nucleotides sequences were determined. The CP genes of ZGMMV and KGMMV are composed of 161 amino acid residues, and they share 77.6% amino acid identity. Western blot analysis showed that the two viruses are serologically related but not identical. Comparison of the sequences with those of sixteen other tobamoviruses revealed that the two viruses had much higher identity to cucumber green mottle mosaic virus (CGMMV), another tobamovirus infectious to cucurbit plants, than other tobamoviruses. The nucleotide and amino acid sequences of ZGMMV were from 29.5 to 78.4% and from 29.3 to 77.6% identical, respectively, to those of other tobamoviruses. The predicted virion assembly origins of the two tobamoviruses were located in the CP region of the genomic RNAs, and the predicted secondary structures were more similar to that of CGMMV than those of other tobamoviruses. The seventeen tobamo-viruses could be classified into three main subgroups based on the phylogenetic tree analysis on the CP gene, and ZGMMV and KGMMV formed a third subgroup together with CGMMV and sunn-hemp mosaic virus (SHMV). These results show that ZGMMV is a previously unknown member of the Tobamovirus genus.

Preservation of plant cell ultrastructure during immunolocalization of virus particles.

Most immunoelectron microscopy techniques used for ultrastructural analyses of virus-infected plant tissues significantly compromise cellular membranous structures as well as overall contrast and resolution of the image. Here, we describe a protocol which avoids these flaws but retains full antigenicity of the sample. A direct comparison of the conventional and the improved electron immunostaining procedures is presented using tobacco and Arabidopsis thaliana plants infected with turnip vein clearing virus.

Caspar carboxylates: the structural basis of tobamovirus disassembly.

Carboxylate groups have been known for many years to drive the disassembly of simple viruses, including tobacco mosaic virus (TMV). The identities of the carboxylate groups involved and the mechanism by which they initiate disassembly have not, however, been clear. Structures have been determined at resolutions between 2.9 and 3.5 A for five tobamoviruses by fiber diffraction methods. Site-directed mutagenesis has also been used to change numerous carboxylate side chains in TMV to the corresponding amides. Comparison of the stabilities of the various mutant viruses shows that disassembly is driven by a much more complex set of carboxylate interactions than had previously been postulated. Despite the importance of the carboxylate interactions, they are not conserved during viral evolution. Instead, it appears that during evolution, patches of electrostatic interaction drift across viral subunit interfaces. The flexibility of these interactions confers a considerable advantage on the virus, enabling it to change its surface structure rapidly and thus evade host defenses.

Structure of ribgrass mosaic virus at 2.9 A resolution: evolution and taxonomy of tobamoviruses.

Ribgrass mosaic virus (RMV) is a member of the tobamovirus group of plant viruses. The structure has been determined at 2.9 A resolution by fiber diffraction methods, and refined by molecular dynamics methods to an R-factor of 0.095. The carboxyl-carboxylate interactions that drive disassembly in tobamoviruses are present in RMV, but are very different from those in other tobamoviruses. RMV has some of the structural features of a subgroup I tobamovirus, a smaller number from subgroup II, and a number that appear to be unique to the RMV cluster of viruses. The structural studies confirm the evolutionary and taxonomic separation of the RMV cluster from both subgroup I and subgroup II tobamoviruses.

A conserved, precise RNA encapsidation pattern in Tobamovirus particles.

The bidirectional RNA encapsidation pathway in nine sequenced Type 1 Tobamovirus genomes will result in RNA-coat protein assembly, up to and including the first transcribed G, adjacent to the 5'-cap structure (m7 Gppp). This precision is highly conserved, despite wide interstrain variations in the absolute position of the phase-determining core of the origin-of-assembly sequence (Gxx)n and in overall genome length (6311-6507 nts). A Type 2 Tobamovirus genome did not comply with this pattern. All genomes had a statistically significant bias for G at every third (or 3n) position, resulting in a preponderance of GNN codons and hence a high Val, Ala, Gly, Asp, Glu content, at least in the large (126/183 kDa) and amino-coterminal replicase protein genes. Contrary to predictions from the X-ray fibre diffraction structure of tobacco mosaic virus (TMV, U1 strain), only one (pepper mild mottle virus) of the nine Type 1 Tobamoviruses positioned the preferred G-repeat in the most favourable (5') position of the trinucleotide binding site on each coat protein (CP) subunit. In all but one of the eight remaining Type 1 Tobamovirus genomes, G would predominate in the CP 3'-site. The significance of these observations for TMV particle assembly, disassembly and host cell interactions are discussed.

Structure determination of cucumber green mottle mosaic virus by X-ray fiber diffraction. Significance for the evolution of tobamoviruses.

Cucumber green mottle mosaic virus (CGMMV) is a rod-shaped virus of the tobacco mosaic virus (TMV) group. The structure of cucumber green mottle mosaic virus has been determined by fiber diffraction methods at 3.4 A resolution, and refined by molecular dynamics methods to an R factor of 0.093. Disassembly of TMV is driven by the mutual repulsion of intersubunit carboxyl-carboxylate pairs, but one of these pairs is not conserved in CGMMV. An alternative pair, located about 5 A from the site of the TMV pair, has been found in CGMMV. Comparison of the two structures suggests that the carboxylate groups are free to migrate in the subunit interfaces during evolution.

Immunosorbent electron microscopic studies on a tobamovirus causing brinjal necrotic mosaic.

Serological relationships of brinjal necrotic mosaic virus (BNMV), a strain of tobacco mosaic virus (TMV) causing necrotic mosaic disease of brinjal in India to other TMV strains was investigated by immunosorbent electron microscopy (ISEM). The intensity of trapping and decoration revealed a close relationship of BNMV to TMV-D, TMV-U1 and TMV-WU1 strains, and a distant relationship to TMV-A1 and TMV-P11 strains. There was a negligible relationship to TMV-P14, tomato mosaic virus (ToMV) and cucumber green mottle mosaic virus (CGMMV). Therefore, BNMV is proposed to be distinct from the previously reported TMV-A1 strain of brinjal.

Electron microscopic and molecular characterization of turnip vein-clearing virus.

We recently isolated turnip vein-clearing virus (TVCV), a tobamovirus which causes vein clearing in Brassica rapa (turnip) and a mosaic in Nicotiana tabacum (tobacco). We present an electron microscopic and molecular characterization of TVCV. Viral particles from lower epidermis peel contained rod-shaped viral particles, typical of tobamoviruses. Viral RNA extracted from infected turnip leaves was used as template for cDNA synthesis prior to cloning in a plasmid vector. Inserts of selected cDNA clones were sequenced to obtain the nucleotide sequence of the 126 K replicase component. The nucleotide and predicted amino acid sequences were 56 to 59% identical to those of most other sequenced tobamoviruses. The least related sequence, that of cucumber green mottle mosaic virus, was more related to the TVCV lineage than it was to those of the other sequenced tobamoviruses. UV spectroscopy suggested a tryptophan content characteristic of the ribgrass mosaic virus (RMV) group. Fragmentation of the TVCV coat protein by cyanogen bromide treatment produced a profile of fragments indistinguishable from those generated from the coat protein of RMV. Thus, while symptoms of TVCV infection on Nicotiana tabacum cv. Samsun and Nicotiana clevelandii differ from those reported for RMV, TVCV appears to be closely related to RMV.

Developments in the understanding of the particle structure of tobraviruses.

Particles of tobraviruses resemble those of tobacco mosaic tobramovirus (TMV) in having helical symmetry and in being rod-shaped. However, isolated tobravirus coat protein and TMV coat protein respond to changes in the ionic strength and pH of the solute in contrasting ways. The types of aggregate formed in solutions of coat protein also differ which may be related to differences in the apparent mechanism of reconstitution of virus particles from isolated protein and RNA. The amino acid sequences of tobravirus and tobramovirus coat proteins have been shown to be similar in some regions known to be important for the structure of TMV particles. These alignments also show that tobravirus proteins are larger than tobramoviral proteins in part because of extra residues at the C-terminus. Tobravirus particles give a signal in proton NMR spectroscopy but TMV particles do not. The signal is caused by segmental mobility of the C-terminal peptide. This difference between TMV and tobraviruses may be related to a property not shared by tobraviruses and TMV and it is therefore speculated that the mobile C-terminal peptide of tobravirus coat proteins may be important in the transmission of tobravirus particles by nematode vectors.