FIG 6.

Amino acids R₃₈ and K₄₁ of NS1 are the key functional sites mediating transcription promotion. (A) Schematic representation of NS1 C-terminal deletion mutants and NS1 point mutants. (B) Effects of NS1 mutants on the accumulation of viral RNAs in the RNP/NA reconstitution system in HEK 293T cells. The steady-state levels of RNAs and proteins were determined by primer extension analysis (top) and Western blotting (bottom) at 24 hpt. The graph shows the relative mean intensity of viral RNA normalized to 5S rRNA. The data represent the means ± SD from three biological replicates. Asterisks represent a significant difference from the control group (by one-way ANOVA with Dunnett’s post hoc test) (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). (C) Dose-dependent effects of NS1_WT (left) and NS1_R38A/K41A (right) on the accumulation of viral RNAs in the RNP/NA reconstitution system in HEK 293T cells. The steady-state levels of RNAs and proteins were determined by primer extension analysis (top) and Western blotting (bottom) at 24 hpt. The graph shows the relative intensity of viral RNA normalized to 5S rRNA. (D) Effects of NS1 on the accumulation of viral RNAs in the RNP/NA reconstitution system in IFN-deficient systems. (Left) HEK 293T cells were pretreated with S-ruxolitinib (4 μM) or dimethyl sulfoxide (DMSO) (as a control) for 1 h and then transfected with the plasmids of the RNP/NA reconstitution system of WSN virus, together with a plasmid expressing either the NS1_WT or NS1_R38A/K41A protein. (Right) HEK 293T cells (293T WT) and HEK 293T RIG-I knockout (293T RIG-I KO) cells were transfected with the same plasmids as the ones described above. The steady-state levels of RNAs and proteins were determined by primer extension analysis (top) and Western blotting (bottom) at 24 hpt. The graph shows the relative intensity of viral RNA normalized to 5S rRNA.