Figure 5.

Single amino acid mutation of the NP confers resistance to eqMx1. (A) The different AAs in the NP of H7N9_ZJ13, H3N8_JL89, and H3N8_XJ07. (B–E) HEK 293T cells were transfected with a firefly minigenome reporter, Renilla expression control, and respectively indicated point mutants of the NP from either H3N8_JL89 polymerase (B) or H7N9_ZJ13 polymerase (B) or H5N1 polymerase (C) in the presence of eqMx1 (0, 200 ng). Polymerase activity of mutants with point mutations at sites 34 and 52 of the NP from both H3N8_JL89 (D) and H7N9_ZJ13 (E) was measured against huMxA (0, 200 ng). Luciferase activity was measured at 24 hr post-transfection. The polymerase activity observed in the presence of the HA-tagged eqMx1 was normalized to an empty control vector (black bar). The resulting relative activity in the presence of either H7N9_ZJ13 (blue) or H3N8_JL89 (grey) was set to 100%. The sequence analysis of the aforementioned IAV strains is shown in C. (A–E) data are firefly Luciferase gene activity normalized to that of Renilla. (Statistical differences between cells are indicated, following a one-way ANOVA and subsequent Dunnett’s test; NS = not significant, * 0.01 ≤ p < 0.05, ** 0.01 ≤ p < 0.01, *** p < 0.001. Error bars represent the SEM of the replicates within one representative experiment). The expression levels of all NP point mutant proteins were assessed by western blotting.