Supplementary Figure 5: SETX deficiency inhibits viral biogenesis.

(a and b). Growth of influenza PR8 (a) and PR8ΔNS1 (b) viruses in A549 cells untreated or treated with control non-targeting siRNA (siCtrl) or SETX-specific siRNA (siSETX). Cells that were not siRNA treated (No si) were also included as controls. Viral titers were determined by plaque assays on MDCK cells (for PR8) or MDCK cells stably expressing NS1 (for PR8ΔNS1). (c) Growth of influenza PR8 virus in siSETX-, siCtrl-treated or untreated (No si) A549 cells that stably express a shRNA targeting DDX58 (RIGI). (d) Western blot showing reconstitution of SETX-deficient cells with FLAG-tagged wildtype SETX and the SETX ATPase mutant (Mut). (e) Quantification of secreted cytokine levels in SETX-deficient and control human fibroblasts 4 hours post infection with PR8ΔNS1. Note that although amounts of IP-10 levels are easily detectable via ELISA, IFNA2 levels were much lower and could result from an IFNB1-dependent induction of IFNA2. (f) Growth of influenza PR8 virus in SETX-deficient and WT human fibroblasts. Viral titers were determined by plaque assay on MDCK cells. Note that final viral titers are similar to A549 cells but the kinetics of viral growth differs between the two cell types. (g) Expression of IFNB1, IFIT1, CXCL9 and SETX in SETX-deficient and wild-type control fibroblasts 4 hours post-infection with West Nile Virus (Kunjin strain; WNV KUNV). **P < 0.005 (t-test). (h) KUNV growth in wildtype and SETX-deficient primary human fibroblast cells. Viral titers were determined by focus-forming assay on Vero cells. (i). Western blot of SETX, RIG-I, IFIT1 and Tubulin proteins isolated from human glial cells after 0, 4, 12 and 24 hours post-infection with PR8∆NS1. Data are from three independent experiments (mean and s.e.m in a-b and f; s.d in c, e, g and h). Representative western blots are shown in d and i. (j) Model of RNAPII- and activator-dependent recruitment of SETX at antiviral loci during infection. Promoter proximal termination is elicited via SETX translocation over nascent transcript as a result of sequence-specific RNA recognition (upper panel) or via helicase activity and R-loop resolution as a result of structure-specific recognition of RNA/DNA hybrids at promoters (lower panel).