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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 6 — Activation of the p38-Mnk1 signaling pathway is required for virus production. (A) eIF4E phosphorylation levels increase in DENV-infected cells. Shown is representative Western blot analysis (n = 3) of phospho-eIF4E (p-eIF4E) abundance in naive and DENV-infected Huh7 cells at 12, 24, 36, and 48 h p.i. Phosphorylation of eIF4E was analyzed by Phos-tag acrylamide gel (lower panel). (B) Analysis of MAPK phosphorylation levels in Huh7 cells inoculated with UV-inactivated DENV (Ctrl) or infected with DENV for 24 and 43 h by using the Proteome Profiler human phospho-MAPK array. Shown in the lower panel are mean relative pixel densities normalized to the control of two independent experiments with two measurements each. (Upper panel) Representative Western blot analysis (n = 2) of Huh7 cell extracts used for analysis of MAPK phosphorylations. Naive Huh7 cells, Huh7 cells infected with DENV for 24 and 43 h, and Huh7 cells inoculated with UV-inactivated DENV for 43 h were analyzed. (C) Representative Western blot analysis of phospho-p38α (p-p38α) abundance in naive and DENV-infected Huh7 cells (n = 3). (D) Representative Western blot analysis of phospho-Mnk1 (p-Mnk1) abundance in naive and DENV-infected Huh7 cells (n = 2). Lysates of naive and murine norovirus (MNV)-infected mouse leukemic monocytes/macrophages (RAW264.7) served as a positive control (72). (E, F, and G) Phosphorylation of eIF4E is dispensable for DENV-induced repression of translation. (E) Ectopic expression of HA-tagged eIF4E in Huh7 cells. Shown is representative Western blot (WB) analysis (n = 3) of endogenous eIF4E and HA-eIF4E abundance in Huh7 HA-eIF4E, Huh7 HA-eIF4E(S209A), and Huh7 HA-eIF4E(S209D). Huh7 control (Ctrl) cells served as a control. (Upper panel) Immunoblotting using an anti-eIF4E antibody. (Lower panel) Immunoblotting using an anti-HA antibody. (F and G) DENV-induced translational repression is phospho-eIF4E independent. (F) Polysome profiles of Huh7 cells stably expressing HA-eIF4E (WT) and the mutant HA-eIF4E(S209A) 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. (G) Naive Huh7 cells (Mock) and cells infected with DENV (MOI of 10) for 8 h (DENV) were treated with CGP57380, an inhibitor of Mnk1 phosphorylation, for 16 h. Treatment with DMSO was 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. (H) Inhibition of p38α reduces DENV virus production. Huh7 cells were infected with DENV (MOI of 0.1) and cotreated with DMSO or 50 µM SB203580, an inhibitor of p38α activity. Virus titers were determined by limiting dilution assay. Shown is the mean ± SD (n = 3). *, P < 0.05. (I) Inhibition of Mnk1 phosphorylation severely diminishes DENV virus production. Huh7 cells were infected with DENV (MOI of 0.1) for 8 h and subsequently treated with DMSO or 50 µM CGP57380 for 16 h. Virus titers were determined by limiting dilution assay (TCID₅₀ per milliliter). Shown is the mean ± SD (n = 4). *, P < 0.05.