Identification of residues in the NP of the pandemic 1918 influenza A virus responsible for resistance to murine Mx1.

<p>(<b>A</b>) Amino acid differences between NP of H5N1, 1918 and pH1N1. Deviant amino acids of pH1N1 or 1918 NP are highlighted in red and blue, respectively. The 1918/H5N1 chimera (1918*-NP) comprises the N-terminal 365 amino acids of 1918 NP and the C-terminal 133 amino acids of the H5N1 NP and thus lacks 4 1918 NP-specific amino acids. (<b>B</b>) Viral polymerase activity in the presence of increasing concentrations of Mx1. HEK293T cells were transfected with expression plasmids coding for the PB2, PB1 and PA of H5N1, the indicated NP proteins, the firefly luciferase encoding minigenome, increasing amounts of Mx1-coding plasmid and a Renilla-expressing plasmid to normalize variation in transfection efficiency. Polymerase activity (relative activity) in the presence of antivirally inactive Mx1-K49A was used to normalize the data obtained with Mx1. Error bars indicate the standard error of the mean of three independent experiments. Western blot analysis was performed to determine the expression levels of Mx1 and H5N1 NP. (<b>C–E</b>) H5N1 polymerase activity was determined as in (B) after co-transfection of the expression plasmids coding for Mx1 (200 ng) and the indicated NP mutants (100 ng). The polymerase activity (relative activity) observed in the presence of Mx1 was normalized to Mx1-K49A. The resulting relative activity in the presence of either 1918*NP (C–D) or 1918 NP (E) was set to 100%. Western blot analysis shown in panel (E) was performed to determine the expression levels of NP. Error bars indicate the standard error of the mean of three independent experiments. Student's <i>t</i>-test was performed to determine the <i>P</i> value. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001; NS, not significant.