SNRNP200 spliceosome protein is required for virus-induced IFNB1 production to control viral replication.

<p>(A) HEK 293T cells stably expressing an IFNB1promoter-driven luciferase gene (HEK 293T pIFNB1-Luc) are transduced with different lentiviral-expressing shRNA targeting SNRNP200, SFRS1, SNRNP35, SF3A1, PHF5A and NHP2L1 genes. Left panel—Heat map (log2 scale) indicating the modulation of IFNB1 promoter activity following silencing of spliceosome genes and infection with SeV or transfection of poly I:C, MAVS or IRF3(5D) expression plasmids for 16 hours. Right panel—qRT-PCR validation data of the endogenous IFNB1 mRNA levels and target gene KD efficiency of cells transduced with shRNA. (B) HEK 293T are transduced with lentiviral-expressing shRNA control (shNT) or targeting SNRNP200 (shSNRNP200) or DDX58 (shDDX58) for three days and infected cells with SeV for 8, 24 or 48 hours. Supernatants are harvested and IFN-β secretion levels are measured by ELISA. (C) Immunoblot analysis of HEK 293T cells infected with SeV for 8, 24 or 48 hours following treatment with shNT or shSNRNP200 for three days. SeV, IFIT1 and actin proteins are resolved by immunobloting at the indicated time. (D) Infectivity titers of SeV particles produced as indicated in (C) are determined by harvesting supernatants at the indicated time and infecting VERO cells in virus plaque assays. (E) HEK 293T pIFNB1-Luc cells are transduced with four different shSNRNP200 at a multiplicity of infection (MOI) of 5 and 20 for three days. Relative IFNB1 promoter activity are reported as percentage of the control shRNA NT after infection with SeV or transfection of poly I:C, MAVS or IRF3(5D) expression plasmids for 16 hours (left). Simplified schematic of RLR signaling pathway leading to IFN-β promoter induction (right). Deduced points of action of SNRNP200 are marked with asterisks (blue and green for MOI = 5 and 20, respectively). Knockdown efficiencies at the various MOI are determined by immunobloting analysis of SNRNP200 protein levels. (F) Immunoblot analysis of HEK 293T cells transduced with shNT or shSNRNP200 for three days and subjected to SeV infection for 16 hours. Plasmids encoding eYFP, IRF3 and IRF3(5D) are transfected for 48 hours. Following cell harvesting, IRF3 and IFIT1 protein levels are resolved by immunobloting analysis of cell lysates.