Katalin Salánki

The necrotic pathotype of the cucumber mosaic virus (CMV) ns strain is solely determined by amino acid 461 of the 1a protein.

The unique Ns isolate of Cucumber mosaic virus (CMV) induces necrotic lesions on several Nicotiana spp. in contrast to other strains that cause systemic mosaic on these plants. By using biologically active RNA transcripts from cDNAs of Ns-CMV and a reference subgroup I strain Rs-CMV, we confined the genetic determinant solely responsible for necrosis induction to amino acid 461 of the la protein translated from genomic RNA1. An Arg to Cys change at this position (R461C) rendered Rs-CMV necrotic, whereas the reciprocal C461R mutation reverted the necrotic phenotype of Ns-CMV. Necrotic (Ns-CMV, R461C) and non-necrotic (Rs-CMV and C461R) viruses accumulated to similar levels in Nicotiana clevelandii protoplasts. Deletion of the residue at position 461 abolished replicase activity of the Ns-CMV 1a protein. The R461C mutation also was introduced into the 1a protein of Trk7-CMV, a subgroup II isolate. Symptoms induced by the Trk7/R461C mutant were identical to those caused by wild-type Trk7-CMV, even when the mutant Trk7 RNA1 was co-inoculated with RNA2 and 3 of the necrotic Ns strain.

Limited utility of blue fluorescent protein (BFP) in monitoring plant virus movement

While the green fluorescent protein (GFP) is a routinely used marker gene in higher plants, there are only a few data concerning the use of blue fluorescent protein (BFP). These proteins together are used for dual colour tagging experiments in various biological systems; however, the benefits of this technique in plant virology have not been exploited yet. In this work, our aim was to determine whether the BFP is a suitable second marker in conjunction with GFP for following the progress of virus infection. Nicotiana clevelandii, N. benthamiana and N. tabacum cv. Xanthi-nc plants were infected with potato virus X vector carrying the GFP or the Y66H type BFP gene. While GFP was brightly fluorescent in all species, the fluorescence intensity of BFP varied widely, from the bright fluorescence observed in N. clevelandii to the absence of fluorescence in N. tabacum cv. Xanthi-nc. Since at even mild acidic pH BFP rapidly fades, the more acidic cytosol of N. tabacum could be responsible for impaired in vivo fluorescence. After infiltration of the infected leaves of N. clevelandii with pH 5 phosphate buffer, the fluorescence faded thus confirming this situation.

A Cucumber Mosaic Virus Based Expression System for the Production of Porcine Circovirus Specific Vaccines

Potential porcine circovirus type 2 (PCV2) capsid protein epitopes, suitable for expression on the surface of cucumber mosaic virus (CMV) particles were determined by a thorough analysis of the predicted PCV capsid protein structure. The ab initio protein structure prediction was carried out with fold recognition and threading methods. The putative PCV epitopes were selected on the basis of PCV virion models and integrated into the plant virus coat protein, after amino acid position 131. The recombinants were tested for infectivity and stability on different Nicotiana species and stable recombinant virus particles were purified. The particles were tested for their ability to bind to PCV induced porcine antibodies and used for specific antibody induction in mice and pigs. The results showed that PCV epitopes expressed on the CMV surface were recognized by the porcine antibodies and they were also able to induce PCV specific antibody response. Challenge experiment with PCV2 carried out in immunized pigs showed partial protection against the infection. Based on these results it was concluded that specific antiviral vaccine production for the given pathogen was feasible, offering an inexpensive way for the mass production of such vaccines.

Molecular evidence for the existence of two distinct subgroups in cucumber mosaic cucumovirus.

Infectious full-length cDNA clones from the genomic RNAs of a subgroup II cucumber mosaic cucumovirus strain (Trk7) were obtained. Sequence analysis of the whole genome revealed strong homology (99%) to the genome of Q-CMV, the only subgroup II strain whose entire genomic nucleotide sequence had been available in the database, and an overall 75% homology to those of subgroup I strains. We provide sequence comparisons of different parts of 1a, 2a and 2b proteins of Cucumovirus species, and propose phylogenetic trees based on these protein sequences.

Heterologous Movement Protein Strongly Modifies the Infection Phenotype of Cucumber Mosaic Virus

ABSTRACT A hybrid virus (CMVcymMP) constructed by replacing the movement protein (MP) of cucumber mosaic cucumovirus (CMV) with that of cymbidium ringspot tombusvirus (CymRSV) was viable and could efficiently spread both cell to cell and long distance in host plants. The hybrid virus was able to move cell to cell in the absence of functional CP, whereas CP-deficient CMV was restricted to single inoculated cells. In several Chenopodium and Nicotiana species, the symptom phenotype of the hybrid virus infection was clearly determined by the foreign MP gene. In Nicotiana debneyi and Nicotiana tabacum cv. Xanthi, the hybrid virus could move systemically, contrary to CymRSV.

Complete nucleotide sequence of the RNA 3 from subgroup II of cucumber mosaic virus (CMV) strain: Trk7☆

The complete nucleotide sequence (2209 nucleotides) of the RNA 3 of the cucumber mosaic virus strain Trk7 was determined. Trk7-CMV possesses two open reading frames which encode the 3a protein (279 amino acids) and the coat protein (218 amino acids). Sequence analysis proved the earlier biological and serological classification of the RNA 3 molecule of Trk7-CMV in subgroup II. A high degree of homology was found in the strains Q and Kin of CMV, while the average similarity between the two different subgroups is much lower.

Identification a coat protein region of cucumber mosaic virus (CMV) essential for long-distance movement in cucumber

To characterise the long-distance movement determinant of cucumoviral coat proteins (CPs), five mutants were engineered into the CMV CP bearing the corresponding tomato aspermy virus (TAV) loops exposed on the surface of the virion. Both viruses can move long-distance in Nicotiana clevelandii, but only CMV can move long-distance in cucumber. Investigation of the CMV chimeras identified three amino acids of the βB-βC loop that were essential for the CMV long-distance movement in cucumber. Introducing these mutations into the TAV CP was not sufficient for long-distance movement, indicating that this is not the sole region causing long-distance movement deficiency.

Nucleotide sequence analysis of peanut stunt virus Rp strain suggests the role of homologous recombination in cucumovirus evolution

The complete nucleotide (nt) sequence of peanut stunt virus Robinia strain (PSV-Rp) was determined and compared to other PSV strains and to representatives of the genus Cucumovirus. Nt sequence comparison showed 74.1–84.6% identity with the known PSV strains. Phylogenetic analysis revealed the different origin of the two genes encoded by RNA3. While the 3a gene clustered with PSV-W, the coat protein gene clustered with PSV-Mi. Recombination breakpoint analysis revealed two recombination points on RNA3. Based on these results, the establishment of a fourth PSV subgroup is proposed. This work revealed that homologous recombination occurred during the evolution of PSV.

Characterisation of black locust isolates of Peanut stunt virus (PSV) from the Pannon ecoregion show the frequent occurrence of the fourth taxonomic PSV subgroup

Biological and molecular characterisation of ten Peanut stunt virus (PSV) isolates from Robinia pseudoacacia was carried out. The host range of these isolates was similar to that of the previously described PSV strains in most cases, but on Pisum sativum and Lens culinaris latent infection was induced. Variability in systemic symptoms was observed only on Nicotiana glutinosa. The partial RNA3 sequences were determined, including the carboxyl terminal region of the movement protein gene, the intergenic region, the entire coat protein gene and the 3′ untranslated region. Nucleotide sequence comparison of the coat protein genes showed 77.6–84.2% identity with most of the known PSV strains and 96.3–98.0% identity with PSV-Rp the typical member of subgroup IV. Phylogenetic analysis indicated the presence of the ancient homologous recombination in all of the examined black locust isolates and all the isolates were members of the fourth PSV subgroup. These results showed that the isolates of the fourth subgroup are widely distributed in black locust in this region.

Alanine Scanning of Cucumber Mosaic Virus (CMV) 2B Protein Identifies Different Positions for Cell-To-Cell Movement and Gene Silencing Suppressor Activity

The multifunctional 2b protein of CMV has a role in the long distance and local movement of the virus, in symptom formation, in evasion of defense mediated by salicylic acid as well as in suppression of RNA silencing. The role of conserved amino acid sequence domains were analyzed previously in the protein function, but comprehensive analysis of this protein was not carried out until recently. We have analyzed all over the 2b protein by alanine scanning mutagenesis changing three consecutive amino acids (aa) to alanine. We have identified eight aa triplets as key determinants of the 2b protein function in virus infection. Four of them (KKQ/22-24/AAA, QNR/31-33/AAA, RER/34-36/AAA, SPS/40-42/AAA) overlap with previously determined regions indispensable in gene silencing suppressor function. We have identified two additional triplets necessary for the suppressor function of the 2b protein (LPF/55-57/AAA, NVE/10-12/AAA), and two other positions were required for cell-to-cell movement of the virus (MEL/1-3/AAA, RHV/70-72/AAA), which are not essential for suppressor activity.