Fig. 5.

Translation is enhanced in virally infected and poly(I:C)-transfected cells lacking STING. (A) UV absorbance spectra of polysome sedimentation from shCTRL and shSTING cells infected at MOI 2, 12 h postinfection. (B) Overlay of VSV-infected shCTRL and shSTING profiles from A. (C) qRT-PCR analysis of VSV N mRNA from each fraction. Inset is virus titer calculated by pfu assays of supernatant virus at time of RNA isolation. Data are represented as mean ± SEM. (D) The MEFs indicated were infected or mock-infected with VSV-GFP for 10 h at MOI 5. Cells were depleted of methionine and labeled with AHA. Cells were fixed and a fluorophore was attached to AHA. Infected and mock-infected cells were analyzed by flow cytometry. VSV-infected cells were gated by GFP signal. (E) AHA MFI of all populations was determined from the data in D. Data are shown as percent MFI of mock-infected cells. Data are represented as mean ± SEM. (F) UV absorbance spectra of polysome sedimentation from shCTRL and shSTING MEFs 6 h after transfection with 2 μg/mL of poly(I:C) or mock-transfected. (G) Overlay of poly(I:C)-transfected shCTRL and shSTING profiles from F. (H) The MEFs indicated were transfected with 2 μg/mL of poly(I:C) and then labeled with AHA. Six hours after transfection, cells were fixed and AHA incorporation was measured with flow cytometry. AHA MFI of all populations was determined. Data are shown as percent MFI of mock-transfected cells. Data are represented as mean ± SEM. (I) MEFs were transfected with 2 μg/mL of poly(I:C) or mock-transfected. Lysates were separated by SDS/PAGE, and endogenous phospho-eIF2α and total eIF2α were detected by western analysis. (J) The MEFs indicated were incubated with 1 μM ISRIB for 10 min before infection with VSV-LUC at the indicated MOI, in the presence of ISRIB. Viral gene expression was determined by luciferase activity assay 12 h postinfection. *P < 0.05; **P < 0.01; ***P < 0.0001 (Student’s t test).