The 2b protein of Cucumber mosaic virus has a role in nearly all steps of the viral cycle including cell-to-cell movement, symptom induction and suppression of antiviral RNA silencing. Previous studies demonstrated the presence of 2b protein in the nucleus and in cytoplasm as well. Phosphorylation site of 2b protein is conserved in all CMV isolates, including proposed constitute motifs for casein kinase II and cyclin-dependent kinase 2. To discern the impact of 2b protein phosphorylation, we created eight different mutants to mimic the non-phosporylated (serine to alanine) as well as the phosphorylated state (serine to aspartic acid) of the protein. We compared these mutants to the wild-type (Rs-CMV) virus in terms of symptom induction, gene silencing suppressor activity as well as in cellular localization. Here, in this study we confirmed the phosphorylation of 2b protein in vivo, both in infected N. benthamiana and in infiltrated patches. Mutants containing aspartic acid in the phosphorylation site accumulated only in the cytoplasm indicating that phosphorylated 2b protein could not enter the nucleus. We identified a conserved dual phosphorylation switch in CMV 2b protein, which equilibrates the shuttling of the 2b protein between the nucleus and the cytoplasm, and regulates the suppressor activity of the 2b protein.

The nonstructural protein (NSs) of Tomato spotted wilt virus (TSWV) was previously identified as an avirulence determinant for Tsw-based resistance on pepper. The NSs of wild-type (WT) and resistance-breaking (RB) TSWV strains isolated in Hungary had only two amino acid substitutions (104, 461). We have analysed the ability of the NSs and their point mutant variants to trigger Tsw-mediated hypersensitive responses and RNA silencing suppressor (RSS) activity in patch assays. We identified a single amino acid change at position 104 (T-A) that was responsible for the necrosis induction or loss, while a significant difference was not detected in the RSS activity of the two parental strains. We have successfully complemented the infection of the WT strain on resistant pepper cultivar with the infectious S RNA transcript of the RB strain and the WT-T104A point mutant. Our work provides direct evidence that a single amino acid change can induce an RB phenotype.

The pharmacokinetic properties of deramciclane fumarate (EGIS-3886), a new potential anxiolitic agent, and its N-desmethyl metabolite have been investigated in Wistar rats after 10 mgkg(-1) deramciclane fumarate was administered orally, intraperitoneally or intravenously. A highly sensitive, validated and optimized gas chromatographic method with nitrogen selective detection (GC-NPD) using a solid-phase extraction technique was used to determine plasma levels of the parent compound and its N-desmethyl metabolite. After oral administration the absorption of the parent compound was very fast (t(max) 0.5h). The maximum plasma concentration (C(max)) was detected at 44.9, > or =177.8 and > or =2643.0 ngmL(-1) after oral, intraperitoneal and intravenous administration of deramciclane, respectively. For the metabolite the respective Cmax values were 32.0, > or =25.4 and 51.0 ngmL(-1). The pharmacokinetic curves of both the parent compound and its metabolite showed enterohepatic recirculation for all administration routes. The biological half-life (tbeta 1/2) for deramciclane ranged from 3.42 to 5.44 h and for the N-desmethyl metabolite the range was 2.90-5.44 h, after administration of the drug by the three different routes. After intravenous administration AUC0-infinity, of deramciclane was 29.2- and 5.4-times higher than that observed after oral and intraperitoneal treatment, respectively. These AUC0-infinity ratios were only 2.1- and 1.5-times higher for the metabolite. The absolute bioavailability of deramciclane in rats was 3.42% after oral and 18.49% after intraperitoneal administration. The comparative pharmacokinetic study of deramciclane in rat after the different administration routes showed fast absorption. Furthermore, plasma levels were found to be administration route-dependent, low bioavailability of the parent compound indicated an extremely fast and strong first-pass metabolism. The apparent volume of distribution suggested strong tissue binding after administration of the drug by any of the three routes studied.

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.

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Resurgence of Tomato spotted wilt virus (TSWV) worldwide as well as in Hungary causing heavy economic losses directed the attention to the factors contributing to the outbreak of this serious epidemics. The introgression of Tsw resistance gene into various pepper cultivars seemed to solve TSWV control, but widely used resistant pepper cultivars bearing the same, unique resistance locus evoked the rapid emergence of resistancebreaking (RB) TSWV strains. In Hungary, the sporadic appearance of RB strains in pepper-producing region was first observed in 2010-2011, but in 2012 it was detected frequently. Previously, the non-structural protein (NSs) encoded by small RNA (S RNA) of TSWV was verified as the avirulence factor for Tsw resistance, therefore we analyzed the S RNA of the Hungarian RB and wild type (WT) isolates and compared to previously analyzed TSWV strains with RB properties from different geographical origins. Phylogenetic analysis demonstrated that the different RB strains had the closest relationship with the local WT isolates and there is no conserved mutation present in all the NSs genes of RB isolates from different geographical origins. According to these results, we concluded that the RB isolates evolved separately in geographic point of view, and also according to the RB mechanism.

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