Fig 5.

ADAR1 deaminase activity of the p150 protein isoform is required for suppression of stress granule formation as well as suppression of PKR activation and IFN-β induction following measles virus infection. ADAR1^kd cells deficient in p150 and p110 were complemented by transfection with ADAR1 constructs or the vector plasmid as indicated, or ADAR1^kd and CON^kd cells were also left untransfected as controls. At 24 h after transfection, cells were either infected with WT measles virus (MV) or left uninfected (UI). Infection was with a multiplicity of infection of 1 TICD₅₀/cell for microscopy experiments or 3 TICD₅₀/cell for Western immunoblot assays, IFN-β mRNA quantitation, and yield determinations. ADAR1^kd cells complemented with ADAR1 proteins are designated p110 (WT) and p150 (WT) for wild-type protein isoforms and p150 Zα (Y177A) and p150 (cat) (H910Q, E912A) for p150 proteins with mutated domains. Vector, empty vector. (A) At 24 h after infection, cells were analyzed by immunofluorescence microscopy using G3BP1 antibody as a marker for SG formation. (B) Quantitation of SG-positive cells. Wide-field 40× images were obtained, and a minimum of 150 cells were analyzed per experiment for the presence of stress granules as shown in panel A. *, P < 0.001; **, P ≤ 3 × 10⁻⁵ by Student's t test comparing uninfected ADAR1^kd cells to infected cells. The results shown are means and standard errors from three independent experiments. (C) Western immunoblot analysis of ectopic ADAR1 expression. At 24 h postinfection, whole-cell extracts were prepared and analyzed using antibodies against ADAR1, PKR, phospho-Thr446-PKR, and α-tubulin as indicated. (D) Quantitation of PKR activation (n-fold) as measured by the level of phospho-Thr446-PKR to total PKR protein determined by Western immunoblot analysis as shown in panel C. *, P < 0.05 by Student's t test to compare the level of P-PKR in infected ADAR1^kd cells to infected cells complemented as indicated or to CON^kd cells. The results shown are means and standard errors from three independent experiments. (E) IFN-β mRNA levels. At 24 h after infection, total RNA was isolated and IFN-β transcript levels were determined by qPCR and normalized to GAPDH. Results shown are representative of 4 independent experiments.