FIG 7 .

Four arginine-to-lysine residues render the sH1N1 NP sensitive to TRIM22 restriction in a viral growth assay. (A) Recombinant viruses were generated starting from a pandemic H1N1 system. These viruses were used to infect control and human TRIM22-transduced MDCK cells at an MOI of 0.001. Substitution of pH1N1 NP with sH1N1 NP WT increased the susceptibility to TRIM22 restriction activity, whereas mutant viruses generated through the introduction of the mutation cluster composed of 4 K-to-R changes (mutant R4) into sH1N1 resulted in a gain of TRIM22 resistance. Viral titers are expressed as the means ± SD from three experiments performed in duplicate. P values were determined using two-way ANOVA (****, P < 0.0001). (B) WCEs from control and TRIM22-overexpressing MDCK cells infected with the reverse-genetics viruses were analyzed by Western blotting assay, and viral NP expression levels were evaluated. TRIM22 expression was also examined, and β-actin was used as a normalizer. These results are representative of one out of three independent experiments.