Figure 2.

Dicer cleaves the EV71 5′UTR into vsRNA1. (A) vsRNA1 in SF268 cells transfected with the control plasmid (shControl) or a plasmid expressing shRNA against Dicer (shDicer) was detected using the vsRNA1 probe. The levels of Dicer and viral 3C protein in these cells were detected by western blotting. (B) In vitro EV71 5′UTR cleavage assay with recombinant Dicer. The ³²P -labelled EV71 5′UTR and 3′UTR RNA was detected after being incubated with reaction buffer only (-) or with various amounts (from 0.005 to 0.25 U) of recombinant Dicer for 1.5 h at 37°C. (C) Dicer protein contains 2 RNAse III domains (RIIIDa and RIIIDb) and a dsRBD. N-terminal FLAG-fused Dicer protein (WT) and mutated Dicer proteins with 4 point mutations (*) in the catalytic sites of RIIIDa / RIIIDb (MUT) and with a dsRBD deletion (ΔdsRBD) were used for the in vitro cleavage of the EV71 5′UTR RNA. The ³²P-labelled EV71 5′UTR RNA was detected after being incubated with reaction buffer or with these FLAG-Dicer proteins at 37°C for 1.5 h (upper right panel). The FLAG-Dicer proteins in each reaction were detected using an antibody against the FLAG peptide (lower right panel). (D) A vsRNA1 probe was used to detect vsRNA1 generated by EV71-infected cells (Cell RNA) and by synthetic EV71 5′UTR RNA after Dicer treatment (Dicer-treated). RNA isolated from mock-infected cells and from a synthetic 5′UTR without Dicer treatment (Un-treated) served as negative controls. (E) Detection of vsRNA1 in wild-type (WT) or Δ105–133 mutant EV71 replicon RNA treated with recombinant Dicer. Full-length replicon RNA and vsRNA1 were detected using a ³²P-labelled probe against nt 250–270 and nt 105–133 of the viral genome.