Figure 4

IAV NS1 expression contributes to IAV-induced Mdm2 destabilization, and consecutively alters Mdm2/p53 interaction. (A) Stability assay in presence of NS1. A549 cells were transfected with either an empty plasmid or a plasmid expressing NS1 from A/Moscow/10/00 (H3N2), and Mdm2 stability was evaluated at 48 h post-transfection. Stability was assessed by monitoring relative protein levels (RPL) of Mdm2 during a 1 h period, after treatment with 50 µM cycloheximide (CHX). Mean values +/− standard deviation from three independent experiments are presented. ^*** for P-value < 0.001. Ku80 was used as a loading control. (B) Four different recombinant IAV were generated using reverse genetics, using the same A/PuertoRico/8/34 (H1N1) genomic background, and harboring NS segment from different IAV strains; NS from A/PuertoRico/8/34 (H1N1, PR8), swine-origin A/Lyon/0969/09 (H1N, SO), A/Moscow/10/99 (H3N2, MO), A/Finch/England/20151/94 (H5N2, EN). Human lung A549 cells were mock-infected or infected with these different IAV with an MOI of 4 and cell lysates were harvested at 8 h post-infection for western blot analysis. Mdm2 relative protein levels (Mdm2 RPL) were measured by densitometry and calculated from data from three independent experiments. An asterisk indicates a significant difference compared with the results for mock-infected cells (^**** for P-value < 0.0001). Ku80 was used as a loading control. (C) Impact of NS1 transient expression on the interaction between p53 and Mdm2. A549 cells were transfected with either an empty plasmid, or two quantities of plasmids expressing NS1 (1 and 4 µg NS1 H3N2). After 48 h post-transfection, cells lysates were analyzed using a co-immunoprecipitation assay using an anti-p53 polyclonal antibody.