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

The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/442996 doi: bioRxiv preprint Figure S4 (related to Figure 5 ). IFN-λ2 mRNA is expressed and exported at similar levels from both a genomic and a cDNA reporter constructs. (A) The attomoles of IFN-λ2 mRNA in cells infected with wt IAV or IAV PA(ΔX) are plotted. Each point represents a biological replicate. The IFN-λ2 mRNA was undetectable in mock-infected cells (n.d. = not detected). N ≥ 2 (B) HEK293T cells were transfected with reporters expressing IFN-λ2 mRNA from cDNA or the genomic locus. RNA samples were collected 24 h after transfection and analyzed by RT-qPCR. The levels of IFN-λ 2 in the absence of PA-X are plotted relative to 18S cellular rRNA. All values represent mean ± standard deviation. N = 4. (C) HEK293T cells were transfected with reporters expressing a luciferase mRNA, a GFP mRNA ending in a hammerhead ribozyme (GFP-HR) and IFN-λ2 mRNAs from cDNA or the genomic locus. Cells were fractionated into nuclear and cytoplasmic fraction, and the ratio between cytoplasmic and nuclear RNA levels was calculated after normalization to 18S rRNA. The cytoplasmic/nuclear ratio for the well-exported endogenous GAPDH mRNA and transfected luciferase mRNA, the nuclear-retained endogenous MALAT1 non-coding RNA, the poorly exported transfected GFP-HR mRNA and the test IFN-λ2 mRNAs are plotted. All values represent mean ± standard deviation. N = 4. (D) The cDNAs from vector-transfected cells from Figure 5C were PCR amplified across the different introns to confirm splicing. 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 serves as a control to check that both products could be simultaneously amplified. Figure S5 (related to Figure 6 ). BirA*-X-ORF mediates biotinylation of cellular proteins. (A) Immunofluorescent staining of biotinylated proteins in HEK293A cells transfected with BirA*-myc (-myc), BirA*-X61-myc (-X61-myc), BirA*-X61(4A)-myc (-X61(4A)-myc), or BirA*-X41(Ca7) expression constructs. The localization of myc-tagged fusion proteins was visualized with anti-myc antibody (red), biotinylated proteins were stained with Streptavidin-Alexa-Fluor-488 conjugate (Biotin, green), and nuclei were stained with Hoechst dye (blue). Open arrows indicate strong nucleolar biotin staining in BirA*-X61-myc expressing cells. (B) Flow through (FT), wash 1 (W1), wash 4 (W4), and elution fractions of neutravidin agarose pulldown of proteins biotinylated using BirA*-X61-myc (X61), BirA*-X61(4A)-myc (4A), or BirA*-myc (BA) were analyzed by western blot using Streptavidin-HRP conjugate (top panel). Total protein in the same fractions was detected using Bio-Rad Stain-free chemiluminescent reagent (bottom panel). Arrow indicates position of a major band corresponding to the size of nucleolin (apparent molecular weight ~110 kDa) that is enriched in BirA*-X61-myc biotinylated protein mix compared to proteins biotinylated by the other two baits. (C) Immunofluorescent staining of biotinylated proteins in cells transfected with BirA*-myc or the BirA* fused to full-length catalytically inactive PA-X mutant, BirA*-PA-X(108A)-myc. Biotinylated proteins were stained with Streptavidin-Alexa-Fluor-488 conjugate (Biotin, green), and nucleoli were stained with anti-nucleolin antibody (NCL, red). Filled arrows indicate strong biotinylation of nucleolar proteins, o