Fig 9. Effects of the number of hydrogen bonds on IFN-α and β expression and viral replication.

(A) 3D structure of NS5. NS5-372 and NS5-386 are highlighted in red and green, respectively. Green broken lines represent hydrogen bonds. The hydrogen bonds formed by NS5-372 and NS5-386 with their neighboring residues are indicated by yellow arrows. (B) DEF were infected with the indicated substitution mutant viruses at 1 MOI and harvested at 24 hpi for measurement of IFN-α and β production at the mRNA level by qRT-PCR. *, p < 0.05; **, p < 0.01; ***, p < 0.001, by Student’s t-test. (C) DEF were infected with the indicated substitution mutant viruses at 0.01 MOI for analysis of replication efficiency. The supernatants were sampled at the indicated time points and titrated with TCID₅₀ assays on BHK cells. Significant difference between rGI and rGI/V372I-H386Y is labeled (***, p < 0.001; **, p < 0.01). Significant difference between rGI and rGI/V372L-H386Y is marked (###, p < 0.001; ##, p < 0.01). Significant difference between rGIII and rGIII/A372G-Y386K is indicated (&, p < 0.05). Significant difference between rGIII and rGIII/A372P-Y386R is indicated (φφ, p < 0.01; φ, p < 0.05). (D and E) Monolayers of DEF were infected with the indicated substitution viruses at 100 PFU for analysis of plaque morphology. The plaques were stained with crystal violet at 4 dpi (D) and the plaque diameters were measured and plotted €. The significant differences between groups were tested by Student’s t-test (***, p < 0.001).