FIG 2.

The viral NS1 and NS2 proteins differentially regulate viral RNA transcription and replication in the RNP reconstitution system. (A) Effects of Flag-NS1 and/or Flag-NS2 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 and 48 hpt, respectively. (B) Effects of untagged NS1 and/or NS2 on the accumulation of viral RNAs detected as described above for panel A. (C) Effects of untagged NS1 and/or NS2 on the accumulation of viral RNAs in the RNP/M 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. (D and E) Regulatory effects of Flag-NS1 and/or Flag-NS2 on protein expression levels in RNP reconstitution systems. HEK 293T cells were transfected with plasmids expressing the PB2, PB1, PA, and NP proteins of WSN virus and the pPOLI-NA/NCR-eGFP (D) or pPOLI-PB2/NCR-eGFP (E) plasmid. The protein expression plasmids expressing Flag-NS1 and/or Flag-NS2 were cotransfected. The expression levels of GFP were examined by fluorescence micrographs at 24 hpt. (F) Effects of NS1 or/and NS2 on the accumulation of viral RNAs in the RNP/NP47 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 graphs in panels A to C and F show the relative mean intensities 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).