Figure 4.

RNA-binding activity of DHX30N is necessary and sufficient for binding the NS1 protein and inhibiting virus replication. (A) Model of the DHX30N RNA-binding domain bound to dsRNA based on DHX9 RNA-binding domain 2 bound to dsRNA (3VYX). The structure of the complex shown corresponds to the single representative conformer of the top-ranked modeling cluster bound to dsRNA. The numbering of amino-acid residues starts with the Met-Ala-Ala-Ser-Arg N-terminal sequence of DHX30 isoform 3 (44). (B) Decreasing concentrations (36, 12, 4, 1.3 μM) of wt or the indicated mutant DHX30N protein were incubated with a 140-bp dsRNA for 1 h at 0°C, and the mixture was subjected to electrophoresis on a 1% agarose gel at 4°C. The gel was stained with SYBR-gold to identify RNA. (C) Bacteria-expressed GST or GST-tagged NS1-RBD was bound to glutathione magnetic beads, which were then incubated for 4 h with 293T cell extracts containing plasmid-expressed Flag-DHX30wt or Flag-DHX30 containing the indicated mutations in the N-terminal domain. These extracts had been treated with RNase A. Proteins eluted from the beads were analyzed by immunoblots probed with Flag Ab. (D) HeLa cells were transfected for 24 h with an empty (E) pcDNA3 plasmid, or a pcDNA3 plasmid expressing the indicated wt or mutant DHX30N protein. Cells were then infected for 6 h with 2 pfu/cell of Ud virus. Cell extracts were RNase A treated and then immunoblotted with the indicated Abs.