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A protein interaction map of Soybean mosaic virus (SMV) strain G7H was generated by the yeast twohybrid system. Clones encoding the genes P1, HC-Pro, P3, 6K1, CI, 6K2, VPg, NIa, NIb, and CP were fused downstream of the GAL4 binding domain (GAL4-BD) and of the GAL4 activation domain (GAL4-AD). The GAL4-BD and GAL4-AD fusion derivatives of each gene were co-transformed into yeast and transformants in which interaction took place were identified on selective media. Interacting fusion proteins were extracted from the yeast cells, run on SDS-PAGE gels and finally checked by Western blotting with GAL4 polyclonal antibodies. Strong interactions were detected between the pairs CP/CP, HC-Pro/HC-Pro, NIa/NIa, and CP/HC-Pro. Relatively weak but significant interaction was detected between VPg and NIa. Although not all of the protein-protein interactions previously reported in other potyviruses were detected, the interactions revealed here were, in general, similar to those reported previously.
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The 3' region of Potato virus X (PVX) has the 74 nt 3'- nontranslated region (NTR) that is conserved among all potexviruses and contains several cis-acting elements for minus-strand and plus-strand RNA accumulation. Three stem-loop structures (SL1-SL3), especially formation of SL3 and U-rich sequence of SL2, and near upstream elements in the 3' NTR were previously demonstrated as important cis-acting elements. To investigate the binding of these cis-acting elements within 3' end with host protein, we used the electrophoretic mobility shift assays (EMSA) and UV-cross linking analysis. The EMSA with cellular extracts from tobacco and RNA transcripts corresponding to the 150 nt of the 3' end of PVX RNA showed that the 3' end of PVX formed complexes with cellular proteins. The specificity of protein binding was confirmed through competition assay by using with 50-fold excess of specific and non-specific probes. We also conducted EMSA with RNAs containing various mutants on those cis-acting elements (Δ10, SL3B, SL2A and Δ21; J Mol Biol 326, 701-720) required for efficient PVX RNA accumulation. These analyses supported that these cisacting elements are required for interaction with host protein(s). UV-cross linking analysis revealed that at least three major host proteins of about 28, 32, and 42 kDa in mass bound to these cis-elements. These results indicate that cis-acting elements from 3' end which are important for minus and plus-strand RNA accumulation are also required for host protein binding.
Potato virus X (PVX) contains cis-acting elements including stem-loop 1 (SL1) RNA at the 5' region; SL1is conserved among all potexviruses. The SL1 at the positive-sense RNA, SL1(+), is required for PVX RNA replication, cell-to-cell movement, and translation. Previous research demonstrated that SL1(+) RNA also serves as the origin of assembly for encapsidation of PVX RNA. To identify the essential sequences and/or regions for capsid protein (CP) subunit recognition within SL1(+) RNA, we used electrophoretic mobility shift assays (EMSA), UV cross-linking, and yeast threehybrid analyses. The EMSA and UV cross-linking analyses with PVX CP subunits and RNA transcripts corresponding to the SL1(+) RNA showed that the SL1(+) RNA formed complexes with CP subunits. We also conducted EMSA and yeast three-hybrid analyses with RNAs containing various mutations of SL1(+)RNA elements. These analyses indicated that SL1(+)RNA is required for the interaction with PVX CP and that the RNA sequences located at the loop C and tetra loop of the SL1(+) are crucial for CP binding. These results indicate that, in addition to being important for RNA accumulation, the SL1(+) RNA from the 5' region of the PVX genome is also required for specific binding of PVX CP.
The 5′-region of Potato virus X (PVX) RNA, which contains an AC-rich, single-stranded region and stemloop structure 1 (SL1), affects RNA replication and assembly. Using Systemic Evolution of Ligands by EXponential enrichment (SELEX) and the electrophoretic mobility shift assay, we demonstrate that SL1 interacts specifically with tobacco protoplast protein extracts (S100). The 36 nucleotides that correspond to the top region of SL1, which comprises stem C, loop C, stem D, and the tetra loop (TL), were randomized and bound to the S100. Remarkably, the wild-type (wt) sequence was selected in the second round, and the number of wt sequences increased as selection proceeded. All of the selected clones from the fifth round contained the wt sequence. Secondary structure predictions (mFOLD) of the recovered sequences revealed relatively stable stem-loop structures that resembled SL1, although the nucleotide sequences therein were different. Moreover, many of the clones selected in the fourth round conserved the TL and C-C mismatch, which suggests the importance of these elements in host protein binding. The SELEX clone that closely resembled the wt SL1 structure with the TL and C-C mismatch was able to replicate and cause systemic symptoms in plants, while most of the other winners replicated poorly only on inoculated leaves. The RNA replication level on protoplasts was also similarly affected. Taken together, these results indicate that the SL1 of PVX interacts with host protein(s) that play important roles related to virus replication.
For quarantine purpose, we selected five plant RNA viruses including Cucumber vein yellowing virus (CVYV),Cucurbit yellow stunting disorder virus (CYSDV), Potato aucuba mosaic virus (PAMV), Potato yellow dwarf virus (PYDV), and Tomato chlorosis virus (ToCV), which are not reported in Korea and cause serious economic losses to the family Cucurbitaceae or Solanaceae. To detect those viruses, we employed RT-PCR technique with specific oligonucleotide primer pairs and tested their detection efficiency for each virus. To design RT-PCR primers,coat protein was used for CVYV, CYSDV, and ToCV whereas RNA polymerase and nucleocapsid regions were used for PAMV and PYDV, respectively. The development of an RT-PCR based method proved a useful tool for rapid detection and identification of quarantine virus infections.
To detect five plant viruses (Beet black scorch virus, Beet necrotic yellow vein virus, Eggplant mottled dwarf virus,Pelargonium zonate spot virus, and Rice yellow mottle virus) for quarantine purposes, we designed 15 RT-PCR primer sets. Primer design was based on the nucleotide sequence of the coat protein gene, which is highly conserved within species. All but one primer set successfully amplified the targets, and gradient PCRs indicated that the optimal temperature for the 14 useful primer sets was 51.9 oC. Some primer sets worked well regardless of annealing temperature while others required a very specific annealing temperature. A primer specificity test using plant total RNAs and cDNAs of other plant virusinfected samples demonstrated that the designed primer sets were highly specific and generated reproducible results. The newly developed RT-PCR primer sets would be useful for quarantine inspections aimed at preventing the entry of exotic plant viruses into Korea.
Fusarium graminearum virus 2 (FgV2) infects Fusarium graminearum strain 98-8-60 and has at least five segments of double-stranded RNAs (dsRNAs), denoted as dsRNA-1 to dsRNA-5. In this study, the genome of FgV2 was sequenced and its phylogenetic relationship with other mycoviruses was analyzed. The lengths of FgV2 dsRNAs 1–5 ranged from 2414 to 3580 base pairs (bp). The 5' and 3' untranslated regions (UTRs) are highly conserved, and each dsRNA segment had 78–105and 84–306 bp of 5' and 3' UTRs, respectively. Each dsRNA segment contained a single open reading frame (ORF). Computer analysis of dsRNA-1 revealed a putative open reading frame (ORF) that shows high sequence identity with an RNA-dependent RNA polymerase (RdRp) containing eight conserved motifs. dsRNAs 2−5also each contain one putative ORF coding for products of unknown function. The sequences of FgV2 dsRNA-2and dsRNA-3 have significant sequence identity with Magnaporthe oryzae chrysovirus 1 (MoCV1) dsRNA-3and -4, respectively. When compared to other dsRNA mycoviruses in a phylogenetic analysis of the putative RdRp protein, FgV2 was found to form a distinct virus clade with Aspergillus mycovirus 1816 and MoCV1 in the family Chrysoviridae.