Figure 3. AtRH2 and AtRH5 bind to the RIII(−) replication enhancer element in the TBSV (−)RNA.

(A) Schematic representation of the four regions carrying cis-acting sequences in the genomic RNA and DI-72 repRNA used in the binding assay. Specific binding by the various cellular DEAD-box helicases are shown. (B) In vitro binding assay with purified AtRH2. The assay contained the ³²P-labeled DI-72 (−)repRNA (∼0.1 pmol) plus increasing amount of unlabeled competitor RNAs, each used in the same amounts, including RI(−) (3 and 6 pmol), RII(−) (2 and 4 pmol), RIII(−) (5 and 10 pmol) or RIV(−) (4 and 8 pmol). The free or AtRH2-bound ssRNA was separated on nondenaturing 5% acrylamide gels. (C) RNA gel shift analysis shows that AtRH5 binds the most efficiently to RIII(−). ³²P-labeled DI-634 (−)repRNA template (∼0.1 pmol) from FHV and unlabeled competitor RNAs (2 and 4 pmol) representing one of the four regions of TBSV DI-72 RNA from both RNA strands (see panel A) were used in the competition assay. The AtRH5 - ³²P-labeled ssRNA complex was visualized on nondenaturing 5% acrylamide gels. Each experiment was repeated at least three times. Note that we used the heterologous FHV DI-634 (−)RNA in the binding assay to allow comparison of (+) versus (−)RNA regions of TBSV RNA. The template competition assay showed efficient binding/competition by the RIII(−) and RI(+) sequences for AtRH5. (D) Comparable viral RNA binding assay reveals different binding specificity for DDX3-like AtRH20 and the DDX5-like AtRH5 DEAD-box helicases. See additional details in panel C. The template competition assay showed efficient binding/competition by the RI(−), RII(−), RIV(−) and RI(+) sequences for AtRH20. (E) Schematic representation of the long-range RNA-RNA interaction between the “base” sequence in the cPR promoter in RI(−) and the complementary “bridge” sequence in RIII(−) REN [56]. The competitor RNAs used in panel F are shown schematically. (F) The bridge sequence contributes to binding of RIII(−) to AtRH5 in vitro. Top image: RNA-binding analysis of AtRH5 – DI-72 (−)repRNA interaction after UV cross-linking. ³²P-labeled DI-72 (−)repRNA was used in the absence (lane 1) or presence of various cold competitor RNAs (lanes 2–7) as shown in panel E. Bottom image: SDS-PAGE shows the purified AtRH5 after UV cross-linking to demonstrate comparable sample loading.