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. 2020 Nov 9;94(23):e01229-20. doi: 10.1128/JVI.01229-20

FIG 4.

FIG 4

The kinase and RNase activities of IRE1α are required for ZIKV replication. (A) IRE1α phosphorylation levels upon ZIKV infection in A549 cells. A549 cells were infected with ZIKV at an MOI of 3. The cells were collected at the indicated time points for Western blotting using antibodies against IRE1α, p-IRE1α, ZIKV E, or GAPDH. (B) XBP1 splicing levels upon ZIKV infection in IRE1αKo and IRE1αRes cells. Control cells, IRE1αKo cells, and IRE1αRes cells were infected with ZIKV at an MOI of 3 and harvested at 24 h p.i. for qRT-PCR to measure XBP1 splicing levels. (C) IRE1α mutant restoration levels. Control cells, IRE1αKo cells, IRE1αRes cells, IRE1α-K599A cells, and IRE1α-K907A cells were harvested for Western blotting. Primary antibodies against IRE1α or GAPDH were used. (D) Phosphorylation levels of IRE1α in IRE1α-K599A cells. Control cells, IRE1αKo cells, IRE1αRes cells, and IRE1α-K599A cells were infected with ZIKV at an MOI of of 3 and collected at 24 h p.i. for Western blotting to examine the phosphorylation levels of IRE1α. (E) XBP1 splicing levels in IRE1α-K907A cells. Control cells, IRE1αKo cells, IRE1αRes cells, and IRE1α-K907A cells were infected with ZIKV at an MOI of 3 and harvested for qRT-PCR to measure the splicing of XBP1. (F to H) Viral replication levels in IRE1α-K599A and IRE1α-K907A cells. Control cells, IRE1αKo cells, IRE1αRes cells, IRE1α-K599A cells, and IRE1α-K907A cells were infected with ZIKV at an MOI of 3. The cells and supernatants were collected at 24 h p.i. for the measurement of viral RNA levels (F), viral E protein levels (G), and viral yields (H). (I) Impact of GSK163 on phosphorylation levels of IRE1α. A549 cells were treated with 30 μM GSK163 and infected with ZIKV at an MOI of 3 at 1 h posttreatment. The cells were then collected at 24 h p.i. for Western blotting to detect the phosphorylation levels of IRE1α. (J) Effect of GSK163 or 4μ8c on XBP1 splicing levels. A549 cells were treated with 30 μM GSK163 or 4μ8c and infected with ZIKV at an MOI of 3 at 1 h posttreatment. The cells were harvested at 24 h p.i. for qRT-PCR to measure XBP1 splicing levels. (K to M) Impact of IRE1α inhibitors on ZIKV replication levels. A549 cells were treated with 30 μM GSK163 or 4μ8c and infected with ZIKV (MOI, 3) at 1 h posttreatment. The cell samples were collected at 24 h p.i. for the examination of viral RNA levels (K), viral E protein levels (L), and viral titers (M). Human β-ACTIN mRNA level was measured as an internal control for qRT-PCR. Data are representative of those from three independent experiments. NS, no statistical significance. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (two-tailed Student t test).