DBR1 influences PKR antiviral function by modulating the level of RNA lariats. (A and B) HEK293T cells with or without PKR overexpression were infected with HSV-1-GFP at an MOI of 0.5 (A), or VSV at an MOI of 0.1 (B) for 24 h. RT-qPCR was performed to confirm viral infection. Data representative of three independent experiments. Graphs depict mean with SD, and points represent biological replicates. Statistical analysis was performed with t tests. **, P < 0.01; ****, P < 0.0001. (C) PKR+/+ and PKR−/− BJ cells were infected with VSV-GFP at an MOI of 0.1, or HSV-1-GFP at an MOI of 0.5 for 24 h, and fluorescence imaging was then performed. Scale bars, 100 µm. Data shown are representative of three independent experiments. (D and E) HEK293T cells transduced with EV or PKR were infected with VSV at an MOI of 0.1 (D), or HSV-1 at an MOI of 0.5 (E); WB was then performed with the indicated antibodies, followed by the p-PKR/PKR WB grayscale analysis of three independent experiments. Tubulin was used as a loading control. Data shown are representative of three independent experiments. Graphs depict the mean with SD and points represent biological replicates. (F) PKR+/+ and PKR−/− BJ cells were transduced with EV or DBR1 and infected with VSV-GFP (MOI: 0.1), or HSV-1-GFP (MOI: 0.5) for 24 h, and fluorescence imaging was then performed for viral GFP. Scale bars, 100 µm. Data shown are representative of three independent experiments. (G) shDBR1 BJ cells transduced with EV or PKR were infected with HSV-1-GFP at an MOI of 0.5, or VSV at an MOI of 0.1 for 24 h. Scale bars, 100 µm. Data shown are representative of three independent experiments. (H) shDBR1 HEK293T cells transduced with EV or PKR were infected with HSV-1-GFP at an MOI of 0.1 for 24 h or VSV at an MOI of 0.1, RT-qPCR was then performed to detect the expression of viral genes. Data representative of three independent experiments. Graphs depict mean with SD and points represent biological replicates. Statistical analysis was performed with unpaired t tests. **, P < 0.01; ***, P < 0.001. (I and J) RNA was extracted from HEK293T cells transduced with scrambled shRNA, shDBR1, or DBR1. The purified RNA was then used to transfect HEK293T cells for 4 h. Cells were infected with VSV at an MOI of 0.01 for 24 h. Viral titers were assessed by fluorescence imaging (I). Scale bars, 100 µm. Data shown are representative of three independent experiments. Viral titers were assessed RT-qPCR (J). 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. (K) HEK293T cells were transfected with 1 µg/ml RNA purified from HEK293T cells transduced with the scrambled control shRNA, or shDBR1 for different time periods, and were then infected with VSV-GFP at a MOI of 0.01 for 24 h. And the viral gene expression was quantified by RT-qPCR. Data representative of three independent experiments. Graphs depict mean with SD, and points represent biological replicates. Statistical analysis was performed with unpaired t tests one-way ANOVA with Dunnett’s multiple comparisons test. NS, not significant, ***, P < 0.001; ****, P < 0.0001. (L) Diagram of RNA lariat-expressing plasmid design and fluorescence imaging. Scale bars, 20 µm. Data shown are representative of three independent experiments. (M and N) HEK293T cells expressing ID1/DKK1-Lariats or ID1/DKK1-Lariats/DBR1 were infected with HSV-1 at an MOI of 0.05 (M) or VSV at an MOI of 0.01 (N), and viral replication was then determined by RT-qPCR. Data representative of three independent experiments. Graphs depict mean with SD, and points represent biological replicates. Statistical analysis was performed with t tests. **, P < 0.01. (O and P) HeLa cells transduced with the scrambled control, shDBR1, or ID1/DKK1-Lariats were infected with VSV at an MOI of 0.1 for 24 h, and the viral GFP was then detected by FACS. 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 < 0.0001. (Q and R) RT-qPCR assays for circRNA quantification in HEK293T cells transduced with shDBR1 (Q) or DBR1 (R) cells relative to control. Statistical analysis was performed with unpaired t tests. NS, not significant. Data representative of three independent experiments. Graphs depict mean with SD, and points represent biological replicates. (S) RT-qPCR assays for circRNA in cells from patients relative to controls. Statistical analysis was performed with unpaired t tests. NS, not significant. Data representative of three independent experiments. Graphs depict mean with SD, and points represent biological replicates. Source data are available for this figure: SourceData FS2.
Figure S2.

DBR1 influences PKR antiviral function by modulating the level of RNA lariats. (A and B) HEK293T cells with or without PKR overexpression were infected with HSV-1-GFP at an MOI of 0.5 (A), or VSV at an MOI of 0.1 (B) for 24 h. RT-qPCR was performed to confirm viral infection. Data representative of three independent experiments. Graphs depict mean with SD, and points represent biological replicates. Statistical analysis was performed with t tests. **, P < 0.01; ****, P < 0.0001. (C) PKR+/+ and PKR−/− BJ cells were infected with VSV-GFP at an MOI of 0.1, or HSV-1-GFP at an MOI of 0.5 for 24 h, and fluorescence imaging was then performed. Scale bars, 100 µm. Data shown are representative of three independent experiments. (D and E) HEK293T cells transduced with EV or PKR were infected with VSV at an MOI of 0.1 (D), or HSV-1 at an MOI of 0.5 (E); WB was then performed with the indicated antibodies, followed by the p-PKR/PKR WB grayscale analysis of three independent experiments. Tubulin was used as a loading control. Data shown are representative of three independent experiments. Graphs depict the mean with SD and points represent biological replicates. (F) PKR+/+ and PKR−/− BJ cells were transduced with EV or DBR1 and infected with VSV-GFP (MOI: 0.1), or HSV-1-GFP (MOI: 0.5) for 24 h, and fluorescence imaging was then performed for viral GFP. Scale bars, 100 µm. Data shown are representative of three independent experiments. (G) shDBR1 BJ cells transduced with EV or PKR were infected with HSV-1-GFP at an MOI of 0.5, or VSV at an MOI of 0.1 for 24 h. Scale bars, 100 µm. Data shown are representative of three independent experiments. (H) shDBR1 HEK293T cells transduced with EV or PKR were infected with HSV-1-GFP at an MOI of 0.1 for 24 h or VSV at an MOI of 0.1, RT-qPCR was then performed to detect the expression of viral genes. Data representative of three independent experiments. Graphs depict mean with SD and points represent biological replicates. Statistical analysis was performed with unpaired t tests. **, P < 0.01; ***, P < 0.001. (I and J) RNA was extracted from HEK293T cells transduced with scrambled shRNA, shDBR1, or DBR1. The purified RNA was then used to transfect HEK293T cells for 4 h. Cells were infected with VSV at an MOI of 0.01 for 24 h. Viral titers were assessed by fluorescence imaging (I). Scale bars, 100 µm. Data shown are representative of three independent experiments. Viral titers were assessed RT-qPCR (J). 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. (K) HEK293T cells were transfected with 1 µg/ml RNA purified from HEK293T cells transduced with the scrambled control shRNA, or shDBR1 for different time periods, and were then infected with VSV-GFP at a MOI of 0.01 for 24 h. And the viral gene expression was quantified by RT-qPCR. Data representative of three independent experiments. Graphs depict mean with SD, and points represent biological replicates. Statistical analysis was performed with unpaired t tests one-way ANOVA with Dunnett’s multiple comparisons test. NS, not significant, ***, P < 0.001; ****, P < 0.0001. (L) Diagram of RNA lariat-expressing plasmid design and fluorescence imaging. Scale bars, 20 µm. Data shown are representative of three independent experiments. (M and N) HEK293T cells expressing ID1/DKK1-Lariats or ID1/DKK1-Lariats/DBR1 were infected with HSV-1 at an MOI of 0.05 (M) or VSV at an MOI of 0.01 (N), and viral replication was then determined by RT-qPCR. Data representative of three independent experiments. Graphs depict mean with SD, and points represent biological replicates. Statistical analysis was performed with t tests. **, P < 0.01. (O and P) HeLa cells transduced with the scrambled control, shDBR1, or ID1/DKK1-Lariats were infected with VSV at an MOI of 0.1 for 24 h, and the viral GFP was then detected by FACS. 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 < 0.0001. (Q and R) RT-qPCR assays for circRNA quantification in HEK293T cells transduced with shDBR1 (Q) or DBR1 (R) cells relative to control. Statistical analysis was performed with unpaired t tests. NS, not significant. Data representative of three independent experiments. Graphs depict mean with SD, and points represent biological replicates. (S) RT-qPCR assays for circRNA in cells from patients relative to controls. Statistical analysis was performed with unpaired t tests. NS, not significant. Data representative of three independent experiments. Graphs depict mean with SD, and points represent biological replicates. Source data are available for this figure: SourceData FS2.

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