The influenza A virus endoribonuclease PA-X usurps host mRNA processing machinery to limit host gene expression

Lea Gaucherand; Brittany K. Porter; Summer K. Schmaling; Christopher Harley Rycroft; Yuzo Kevorkian; Craig McCormick; Denys A. Khaperskyy; Marta Maria Gaglia

(A) Diagram of IFN-λ2 constructs. Int = intron. (B-E) HEK293T cells were transfected for 24 hrs with reporters expressing a luciferase control mRNA and IFN-λ2 mRNA from cDNA, the genomic locus or cDNA with one of the five IFN-λ2 introns added back (D only). Cells were also transfected with PA-X (PR8 variant in B and D, PR8, CA/7 and Udorn variants in C) or vector. The levels of luciferase and IFN-λ2 mRNAs were measured by RT-qPCR and plotted as relative levels in PA-X expressing cells vs. vector-transfected cells, after normalization to 18S rRNA. In E, the cDNA from control cells was PCR amplified across intron 4 to test splicing of intron 4. The amplified PCR products are shown (gel image is representative of four experiments). A 1:1 mix of the constructs for IFN-λ2 cDNA and genomic served as a control, to check that both products could be simultaneously amplified. (F) Cells were transfected with an intronless ("(-)") or an intron-containing ("+ intron") luciferase reporter and PR8 PA-X. Luciferase RNA levels were measured by RT-qPCR, normalized by 18S rRNA and are plotted relative to vector transfected cells. Values in this figure represent mean ± standard deviation. *,**: p < 0.05, 0.01, B,C: ANOVA followed by Tukey's pairwise test; D: ANOVA followed by Dunnett's test, p values relative to cDNA construct-transfected cells; F: Student's t test. N ≥ 4, all panels. See also Figure S4 .