FIG 6.

Disruption of the PKR-eIF2α pathway impairs DENV4 and ZIKV replication. (A) Viral titration from A549 WT or PKR^−/− cells infected with DENV4 (MOI 2) or ZIKV (MOI 3) and harvested at 24 h.p.i. for analysis (three independent experiments). Plaque size comparison of virus in A549 WT or PKR^−/− cells infected with DENV4 and incubated for 8 days or infected with ZIKV and incubated for 5 days: measurements of (B) plaque areas and (C) number of cells per plaque from immunofluorescence. (D) Quantification by RT-qPCR of DENV4 or ZIKV viral RNA from A549 WT or PKR^−/− cells infected with DENV4 (MOI 2) or ZIKV (MOI 3) and harvested at 24 h.p.i. for analysis (three independent experiments). Relative expression calculated by 2^−ΔΔCt method using mock cells as a reference for cell genes and WT cells as reference for viral genome. Statistical analysis was performed by paired t test comparing the two cell lineages under the same conditions. A549 cells infected with ZIKV, treated with the indicated concentration of integrated stress response inhibitor (ISRIB) ordimethyl sulfoxide (DMSO), and harvested at 24 h.p.i. (two independent experiments). (E) Quantification by flow cytometry of infected population labeled with anti-E protein. (F) Viral titration from experiment supernatant in VERO cells. Statistical analysis was performed by one-way analysis of variance with Dunnett’s multiple comparisons test. In all charts, bars represent the means ± standard error of the mean. Statistical analysis was performed by paired t test comparing the two cell lineages under the same conditions. *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001. FFU, focus forming unit; ns/no markup, no statistical difference.