PKR is required for the antiviral activity of DBR1. (A) PKR+/+ and PKR−/− BJ cells were transduced with EV or DBR1, followed by WB for indicated antibodies. GAPDH was used as a loading control. Data shown are representative of three independent experiments. (B) PKR+/+ and PKR−/− BJ cells were transduced with EV or DBR1 and infected with HSV-1 at an MOI of 0.5, followed by quantification of virus replication by RT-qPCR. Data representative of three independent experiments. Graphs depict the mean with SD and points represent biological replicates. Statistical analysis was performed with one-way ANOVA with Dunnett’s multiple comparisons test. NS, not significant, ****, P < 0.0001. (C) Similar to B, except the supernatant was collected at different time points for viral titration. Data representative of three independent experiments. Graphs depict the mean with SD, and points represent biological replicates. Statistical analysis was performed with one-way ANOVA with Dunnett’s multiple comparisons test. NS, not significant, ***, P < 0.001. (D) Similar to B, except the cells were infected with VSV at an MOI of 0.1 (n = 3 for each group). Data representative of three independent experiments. Graphs depict the mean with SD, and points represent biological replicates. Statistical analysis was performed with one-way ANOVA with Dunnett’s multiple comparisons test. ****, P < 0.0001. (E) Similar to C, except the cells were infected with VSV at an MOI of 0.1. Data are representative of three independent experiments. Graphs depict the mean with SD and points represent biological replicates. Statistical analysis was performed with one-way ANOVA with Dunnett’s multiple comparisons test. NS, not significant, ****, P < 0.0001. (F) Similar to B and D except that the cells were analyzed by WB for the indicated antibodies. Tubulin was used as a loading control. Data shown are representative of three independent experiments. (G) Similar to B except that expression of the indicated ISRs was detected by RT-qPCR. Data representative of three independent experiments. Graphs depict mean with SD, and points represent biological replicates. Statistical analysis was performed with one-way ANOVA with Dunnett’s multiple comparisons test. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (H and I) PKR+/+ and PKR−/− BJ cells were transduced with scrambled shRNA or shDBR1 and infected with VSV MOI = 0.1 (H) or HSV-1-GFP MOI = 0.5 (I), followed by quantification of virus replication by RT-qPCR. Data representative of three independent experiments. Graphs depict the mean with SD and points represent biological replicates. Statistical analysis was performed with one-way ANOVA with Dunnett’s multiple comparisons test. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001. (J) PKR+/+ and PKR−/− BJ cells were transduced with scrambled shRNA or shDBR1, and WB was then performed to detect the indicated proteins. Tubulin was used as a loading control. Data shown are representative of three independent experiments. (K and L) BJ cells transduced with the scrambled control or shDBR1 cells together with EV or PKR overexpression were infected with VSV-GFP (MOI: 0.1) (K), or HSV-1-GFP (MOI: 0.5) (L) for 24 h, followed by quantification of virus replication by RT-qPCR (n = 3 for each group). Data representative of three independent experiments. Graphs depict the mean with SD and points represent biological replicates. Statistical analysis was performed with one-way ANOVA with Dunnett’s multiple comparisons test. ***, P < 0.001; ****, P < 0.0001. (M) Similar to L, except that the cells overexpressing DBR1-copGFP and DBR1Y17H were also included, and the indicated proteins were detected by WB. Tubulin was used as a loading control. Data shown are representative of three independent experiments. Source data are available for this figure: SourceData F3.
Figure 3.

PKR is required for the antiviral activity of DBR1. (A) PKR+/+ and PKR−/− BJ cells were transduced with EV or DBR1, followed by WB for indicated antibodies. GAPDH was used as a loading control. Data shown are representative of three independent experiments. (B) PKR+/+ and PKR−/− BJ cells were transduced with EV or DBR1 and infected with HSV-1 at an MOI of 0.5, followed by quantification of virus replication by RT-qPCR. Data representative of three independent experiments. Graphs depict the mean with SD and points represent biological replicates. Statistical analysis was performed with one-way ANOVA with Dunnett’s multiple comparisons test. NS, not significant, ****, P < 0.0001. (C) Similar to B, except the supernatant was collected at different time points for viral titration. Data representative of three independent experiments. Graphs depict the mean with SD, and points represent biological replicates. Statistical analysis was performed with one-way ANOVA with Dunnett’s multiple comparisons test. NS, not significant, ***, P < 0.001. (D) Similar to B, except the cells were infected with VSV at an MOI of 0.1 (n = 3 for each group). Data representative of three independent experiments. Graphs depict the mean with SD, and points represent biological replicates. Statistical analysis was performed with one-way ANOVA with Dunnett’s multiple comparisons test. ****, P < 0.0001. (E) Similar to C, except the cells were infected with VSV at an MOI of 0.1. Data are representative of three independent experiments. Graphs depict the mean with SD and points represent biological replicates. Statistical analysis was performed with one-way ANOVA with Dunnett’s multiple comparisons test. NS, not significant, ****, P < 0.0001. (F) Similar to B and D except that the cells were analyzed by WB for the indicated antibodies. Tubulin was used as a loading control. Data shown are representative of three independent experiments. (G) Similar to B except that expression of the indicated ISRs was detected by RT-qPCR. Data representative of three independent experiments. Graphs depict mean with SD, and points represent biological replicates. Statistical analysis was performed with one-way ANOVA with Dunnett’s multiple comparisons test. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (H and I) PKR+/+ and PKR−/− BJ cells were transduced with scrambled shRNA or shDBR1 and infected with VSV MOI = 0.1 (H) or HSV-1-GFP MOI = 0.5 (I), followed by quantification of virus replication by RT-qPCR. Data representative of three independent experiments. Graphs depict the mean with SD and points represent biological replicates. Statistical analysis was performed with one-way ANOVA with Dunnett’s multiple comparisons test. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001. (J) PKR+/+ and PKR−/− BJ cells were transduced with scrambled shRNA or shDBR1, and WB was then performed to detect the indicated proteins. Tubulin was used as a loading control. Data shown are representative of three independent experiments. (K and L) BJ cells transduced with the scrambled control or shDBR1 cells together with EV or PKR overexpression were infected with VSV-GFP (MOI: 0.1) (K), or HSV-1-GFP (MOI: 0.5) (L) for 24 h, followed by quantification of virus replication by RT-qPCR (n = 3 for each group). Data representative of three independent experiments. Graphs depict the mean with SD and points represent biological replicates. Statistical analysis was performed with one-way ANOVA with Dunnett’s multiple comparisons test. ***, P < 0.001; ****, P < 0.0001. (M) Similar to L, except that the cells overexpressing DBR1-copGFP and DBR1Y17H were also included, and the indicated proteins were detected by WB. Tubulin was used as a loading control. Data shown are representative of three independent experiments. Source data are available for this figure: SourceData F3.

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