FIG 4.

Sensitivity of A/SH/01/13 to murine and human Mx. (A) Amino acid differences in NP of A/BM/1/18 (BM/18), A/PR/8/34 (PR/8), A/WSN/33, A/AH/1/13 (AH/1), A/SH/01/13 (SH/1), and A/Vietnam/1203/04 (VN/04). Blue boxes highlight positions increasing Mx resistance; red boxes indicate differences between AH/1 and SH/1 relative to VN/04. (B) Structural models of NP with the positions critical for Mx resistance highlighted in blue for BM/18 and PR/8 and the positions of SH/1 that differ from VN/04 highlighted in red. (C and D) Polymerase reconstitution assay with the polymerase subunits of VN/04 combined with expression plasmids encoding NP (100 ng) of SH/1, VN/04, or WSN/33. The assays were performed as described in the legend of Fig. 1B with increasing amounts of cotransfected expression plasmids for mouse Mx1 (C) or human MxA (D). Relative polymerase activity was calculated as the ratio of wild-type Mx1 or MxA to the inactive Mx1(K49A) or MxA(T103A), respectively. The activity with wild-type NP in the absence of Mx 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 < 0.05; **, P < 0.01; ***, P < 0.001. Western blot analysis shows the expression levels of Mx proteins, viral PB2 and beta-actin in the cell lysates.