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. 2017 Jan 10;8(1):e02150-16. doi: 10.1128/mBio.02150-16

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Copyright © 2017 Roth et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

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FIG 4 — DENV-induced host cell translation repression is independent of the PKR-eIF2α signaling pathway. (A and B) activation of PKR by DENV does not result in eIF2α phosphorylation. Cells were infected with DENV (MOI of 10) for 12, 24, 36, and 48 h (lanes 5 to 8). Naive Huh7 cells (Mock) cultured in parallel for the same time periods were used as reference (lanes 1 to 4). Shown are representative Western blot analyses (n = 4). (A) Analysis of PKR and phospho-PKR (p-PKR) abundance. Cells transfected with the synthetic dsRNA poly(I-C) (lane 9) were used as a positive control. (B) Analysis of eIF2α and phospho-eIF2α (p-eIF2α) abundance. Cells treated with arsenite (lane 9) were used as a positive control. Phosphorylation of eIF2α was analyzed by Phos-tag acrylamide gel (lower panel). (C) DENV-induced translational repression is PKR independent. Polysome profiles of Huh7 PKR ko cells (clone 2#3) left untreated (Mock) or infected with DENV (MOI of 10) were recorded at the indicated times. Shown are representative polysome profile analyses (lower panel) and mean percentages of polysomal ribosomes ± SEM (upper panel). n, number of profiles analyzed. (D) DENV-induced translational repression is eIF2α independent. Polysome profiles of Huh7 control cells (Ctrl) and Huh7 cells stably expressing GADD34 and infected with DENV (MOI of 10) were recorded 24 h p.i. Naive cells (Mock) were used as a control. Shown are representative polysome profile analyses (lower panel) and mean percentages of polysomal ribosomes ± SEM (upper panel). n, number of profiles analyzed.