FIG 6.

Amino acid 52N in H7N9 NP contributes to resistance against human MxA. (A) MxA sensitivity of SH/1-NP(wt), SH1-NP(N52Y), and VN/04 was determined in the VN/04 polymerase reconstitution system with increasing amounts of MxA expression plasmid as described in the legend of Fig. 4D. Relative polymerase activity was calculated as the ratio of luciferase activity of wild-type MxA and MxA(T103A). The activity in the absence of MxA was set to 100%. Error bars indicate standard deviations of three independent experiments. Student's t test was performed to determine the P values. **, P < 0.01; ***, P < 0.001. Western blot analysis shows the expression levels of MxA and PB2 in the cell lysates. (B) Expression of MxA limits replication of H7N9 NP(N52Y). Control A549 cells and A549 cells constitutively expressing MxA were infected at an MOI of 2 with the indicated viruses. At 16 h p.i. cells were lysed and analyzed for viral NS1, MxA, and β-actin by Western blotting. The second panel shows a longer exposure of the anti-NS1 blot. The Western blot signals of NS1 were quantified using ImageJ program and normalized to the signals for actin in the cell lysates (graphs). (C) Viral titers in A549 cells stably expressing MxA and control cells. Cells were infected for 16 h at an MOI of 2 with indicated viruses in the absence of TPCK-trypsin. Viral titers are depicted as PFU/ml from three independent samples; error bars represent standard deviations.