Figure 4.

RISC-mediated RNA silencing targets viral (+)RNA. (A) Schematic representations of the DI RNA variants DI-GFP1 and DI-GFP2 that contained the target sequence of the ‘gf698’ siRNA at different positions. The target sequence, which is indicated by a black line, corresponds to a short sequence of GFP mRNA and is complementary to the ‘gf698’ siRNA guide strand. The dashed line in DI-GFP2 represents a sequence that is complementary to the target sequence. (B) With both constructs shown in (A), the ‘gf698’ target sequence was introduced in either sense (s) or antisense (as) orientation. Accordingly, as it is shown in the schematic representation of DI RNA replication, RISC programmed with the ‘gf698’ siRNA was supposed to target either (+) or (−)RNA molecules. (C) ‘RISC formation/cleavage assay’ with DI-GFP RNAs. The assay was performed as described in Figure 1B using ‘gf698’ siRNA and ³²P-labeled RNA transcripts of the respective (+) and (−)DI-GFP RNAs. The target RNA (indicated) and the cleavage products (asterisks) were analyzed by denaturing PAGE and autoradiography. Lanes 1, 3, 5 and 7; assays performed in the absence of siRNA (negative controls). Lanes 2, 4, 6 and 8; assays performed in the presence of ‘gf698’ siRNA. (D) ‘Replication inhibition assay’ with DI-GFP RNAs. The assays were performed as described in Figure 3B (variant 1), i.e. RISC programmed with ‘gf698’ siRNA was added to a translation/replication reaction performed with the (+)RNA of the different DI-GFP variants. RP and cleavage products (asterisks) are indicated. Lanes 1, 4, 7 and 10; assays in the absence of p92 (no replication). Lanes 2, 5, 8 and 11; assays in the absence of siRNA (negative controls). Lanes 3, 6, 9 and 12; assays in the presence of ‘gf698’ siRNA. (E) SiRNA-programmed RISC also interferes with ongoing viral replication. ‘Replication inhibition assays’ were performed with (+)DI-GFP1(s) and ‘gf698’ siRNA as depicted in Figure 3B following experimental variants 1 or 2. That is, the RISC- and replicase-forming reactions were combined either before the initiation of RNA replication (0 h) or 1 h (1 h) after starting the replication reaction. The RP and cleavage products (asterisks) are indicated. Lane 1; assay performed in the absence of p92 (no replication). Lanes 2 and 3; assays where the reaction mixtures were combined before the initiation of replication in the absence (lane 2) and presence (lane 3) of ‘gf698’ siRNA. Lanes 4 and 5; assays where the reaction mixtures were combined after 1 h of viral replication in the absence (lane 4) and presence (lane 5) of ‘gf698’ siRNA.