FIG 1.

Influenza viruses and the viral NS1 protein alone modulate TP53-i9 minigene alternative splicing. (A) Schematic of the TP53-i9 minigene. Intron 9 of p53 was inserted into pCDNA3 plasmids with part of exon 9 and 10 on both sides, and transcription of mRNA corresponding to each spliced variant was under the control of the CMV promoter (47). (B) Levels of mRNA corresponding to p53 spliced variants α, β, and γ were measured in H1299 cells (p53 null) in which the TP53-i9 minigene plasmid was transfected 36 h before mock infection or infections by H1N1 or H3N2 influenza viruses. Cells lysates were harvested at 8 hpi for infection at an MOI of 4 or at 24 hpi for infection at an MOI of 0.1, and relative levels of mRNA corresponding to α, β, and γ p53 spliced variants were measured by specific RT-qPCR. The mRNA level of each variant was normalized against the neomycin resistance gene, which is also present in the TP53-i9 minigene. Viral protein expression (NP and NS1) was monitored by Western blotting. (C) The relative spliced mRNA level of p53α, p53β, and p53γ isoforms was measured in H1299 cells 48 h after cotransfection of the TP53-i9 minigene and 1 μg of an empty pCI plasmid or a plasmid containing wt NS1 (pCI NS1 wt, from influenza strain A/Moscow/10/99 [H3N2]) and was used to calculate the proportion of β+γ isoforms out of the total α, β, and γ variant p53 mRNA expression. Mean values ± standard deviations of results for at least three independent experiments are shown, and statistical tests compared each condition with its control condition using two-way analysis of variance (ANOVA) (**, P < 0.01; ***, P < 0.001). (D and E) In H1299 cells, the TP53-i9 minigene was cotransfected with 1 μg of empty pCI, pCI NS1wt (results extracted from Fig. 1C), or pCI NS1-Y89F, pCI NS1-R38A/K41A, and pCI NS1-CPSF4b mutants (D) or with increasing amounts (0.1, 0.5, or 1 μg) of pCI NS1 wt or pCI NS1-CPSF4b plasmid (E). Forty-eight hours after cotransfection, levels of α, β, and γ variant mRNA were measured (Fig. S1) and used to estimate the β+γ proportion. The efficacy of NS1 transient expression was validated by Western blotting. Mean values ± standard deviations of results from more than three independent experiments are shown, and statistical tests compared each condition with its control empty condition or NS1 wt condition using Student's t test (*, P < 0,05; **, P < 0.01; ***, P < 0.001).