FIG 9.
DHX9 expression regulates nonstructural protein translation. (A) Mock-transfected HEK293T cells (WT) and cells transfected with the indicated shRNAs were analyzed for DHX9 and GAPDH expression. The cells were infected with CHIKV-nsP1W258A-NanoLuc (depicted in the upper panel). (B) After 4 h at a nonpermissive temperature (37°C), the nanoluciferase activity was determined in cell lysates and normalized to the protein concentration in the samples. ****, P < 0.0001; **, P < 0.01. (C) HEK293T cells transfected to overexpress HA-DHX9 were infected CHIKV-nsP1W258A-NanoLuc and maintained at 37°C for 4 h. NsP translation was monitored by quantification of the nanoluciferase activity in the cell lysate, and values were normalized according to the protein content in the sample. The expression of the HA-DHX9 transgene was controlled by immunoblotting the total cell extracts with the indicated antibodies. (D) Parental (WT) or DHX9 CRISPR/Cas9-edited HEK293T cells were transfected with the ΔnsP4 CHIKV replicon (depicted in the upper panel). At 8 h after transfection, cell lysates were analyzed for DHX9, GAPDH, and nsP expression. The band intensity was determined, and the nsP2/GAPDH ratios are indicated.