FIG 1.

Mx sensitivity of PR/8 NP mutants at positions 100, 283, and 313. (A and B) Viral polymerase reconstitution assay. HEK 293T cells were transfected with expression plasmids coding for the polymerase subunits PB2, PB1, PA, and the indicated NP protein of hvPR/8, a firefly luciferase minigenome under the control of the RNA polymerase I promoter and the Mx1-encoding plasmid. In addition, a plasmid encoding Renilla luciferase under the control of the SV40 promoter was cotransfected to normalize variations in transfection efficiency. Mx1(K49A) is an inactive mutant that was used as a control to normalize the data obtained by coexpression of wild-type Mx1. Relative polymerase activity was calculated as the ratio of luciferase activity in the presence of wild-type Mx1 compared to the luciferase activity in the presence of antiviral inactive Mx1(K49A). The polymerase activity with wild-type NP was set to 100%. Bars represent mean values with standard deviations of two independent experiments performed in triplicates. Two-way analysis of variance was performed to calculate P values. ns, not significant; *, P < 0,01; ***, P < 0,001. A representative Western blot analysis shows the expression levels of wild-type Mx1 (72 kDa), NP (56 kDa), and β-actin (42 kDa) in the cell lysates. (A) Inhibition of the viral polymerase activity by Mx1. Relative luciferase activity was determined in the presence of Mx1 or Mx1(K49A). (B) Point mutations in NP influence Mx1 sensitivity of the viral polymerase. Wild-type NP or NP encoding single or combinations of mutations were used in the replicon assay. Luciferase activity obtained in the presence of wild-type Mx1 was normalized to the activity in the presence of Mx1(K49A). (C) Growth kinetics of hvPR/8 encoding either wild-type NP or NP with the indicated mutations. Calu-3 cells were infected with an MOI of 0.001 in the presence of 0.5 μg/ml trypsin and were incubated at 37°C. At the indicated time points, virus titers in the cell supernatants were determined by plaque assay. Error bars indicate the standard deviation of one experiment performed in duplicates. (D and E) Lung titers of hvPR/8 encoding wild-type NP compared to the viruses encoding the indicated NP mutants in C57BL/6 (D) or B6.A2G-Mx1 (E) mice. Mice were infected intranasally with 200 PFU for 48 h. Viral titers of the lung homogenates were determined by plaque assay. (F) MLD₅₀ values of hvPR/8 encoding wild-type NP or the indicated NP mutants determined in C57BL/6 and B6.A2G-Mx1 mice (n = 5/group). α, anti.