RNA lariats inhibit PKR activation and weaken cellular antiviral activity. (A) HEK293T cells were transfected with 1 µg/ml RNA purified from shDBR1 HEK293T cells, and ID1 and DKK1 lariat RNA levels were determined 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 < 0.01; ***, P < 0.001. (B and C) HEK293T cells were transfected with 1 µg/ml RNA purified from HEK293T cells transduced with the scrambled shRNA, shDBR1, or DBR1 for 2 h and were then infected with VSV-GFP at a MOI of 0.01 for 24 h. Flow cytometry was then performed to analyze viral GFP levels. 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 < 0.01; ***, P < 0.001. (D and E) HEK293T cells were transfected with 1 µg/ml RNA purified from HEK293T cells transduced with scrambled shRNA or shDBR1 for 2 h and infected with HSV-1 at an MOI of 0.1 (D) or VSV at a MOI of 0.01 (E) for various time points, 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. (F) HEK293T cells were transfected with 0.8 µg/ml RnaseR-digested RNA purified from HEK293T cells transduced with scrambled control shRNA or shDBR1, and then infected with VSV at a MOI of 0.01 for 24 h, 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. (G) HEK293T PKR+/+ and PKR−/− cells were transfected with 0.8 µg/ml RnaseR-digested RNA purified from HEK293T cells transduced with scrambled control shRNA or shDBR1, and infected with HSV-1 at a MOI of 0.1 for 24 h, 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 < 0.01; ***, P < 0.001. (H) HEK293T cells were transfected with 1 µg/ml RNA purified from HEK293T cells transduced with scrambled control shRNA, shDBR1, or DBR1, and stimulated with polyI:C or infected with HSV-1 at an MOI of 0.5 or VSV at a MOI of 0.1 for 24 h. Cell lysates were detected by WB with the indicated antibodies. GAPDH was used as a loading control. Data shown are representative of three independent experiments. (I) Diagram of RNA lariat-expressing plasmid design. This graph is created in BioRender. Ru, S. (2024) https://BioRender.com/u76g969. (J) HEK293T cells were transfected with the lariat-expressing plasmids for 24 h, and lariat RNA ID1 and DKK1 levels were measured 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 < 0.001; ****, P < 0.0001. (K and L) HEK293T cells were transfected with lariat-expressing plasmids for 24 h; they were then infected with HSV-GFP (MOI: 0.5) (K) or VSV-GFP (MOI: 0.1) (L) and viral gene expression was quantified 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 < 0.001. (M) HEK293T cells were transfected with lariat ID1 or lariat DKK1 plasmids together with or without PKR overexpression. Cells were then stimulated with polyI:C and WB was then performed with the indicated antibodies. GAPDH was used as a loading control. Data shown are representative of three independent experiments. Source data are available for this figure: SourceData F4.
Figure 4.

RNA lariats inhibit PKR activation and weaken cellular antiviral activity. (A) HEK293T cells were transfected with 1 µg/ml RNA purified from shDBR1 HEK293T cells, and ID1 and DKK1 lariat RNA levels were determined 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 < 0.01; ***, P < 0.001. (B and C) HEK293T cells were transfected with 1 µg/ml RNA purified from HEK293T cells transduced with the scrambled shRNA, shDBR1, or DBR1 for 2 h and were then infected with VSV-GFP at a MOI of 0.01 for 24 h. Flow cytometry was then performed to analyze viral GFP levels. 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 < 0.01; ***, P < 0.001. (D and E) HEK293T cells were transfected with 1 µg/ml RNA purified from HEK293T cells transduced with scrambled shRNA or shDBR1 for 2 h and infected with HSV-1 at an MOI of 0.1 (D) or VSV at a MOI of 0.01 (E) for various time points, 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. (F) HEK293T cells were transfected with 0.8 µg/ml RnaseR-digested RNA purified from HEK293T cells transduced with scrambled control shRNA or shDBR1, and then infected with VSV at a MOI of 0.01 for 24 h, 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. (G) HEK293T PKR+/+ and PKR−/− cells were transfected with 0.8 µg/ml RnaseR-digested RNA purified from HEK293T cells transduced with scrambled control shRNA or shDBR1, and infected with HSV-1 at a MOI of 0.1 for 24 h, 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 < 0.01; ***, P < 0.001. (H) HEK293T cells were transfected with 1 µg/ml RNA purified from HEK293T cells transduced with scrambled control shRNA, shDBR1, or DBR1, and stimulated with polyI:C or infected with HSV-1 at an MOI of 0.5 or VSV at a MOI of 0.1 for 24 h. Cell lysates were detected by WB with the indicated antibodies. GAPDH was used as a loading control. Data shown are representative of three independent experiments. (I) Diagram of RNA lariat-expressing plasmid design. This graph is created in BioRender. Ru, S. (2024) https://BioRender.com/u76g969. (J) HEK293T cells were transfected with the lariat-expressing plasmids for 24 h, and lariat RNA ID1 and DKK1 levels were measured 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 < 0.001; ****, P < 0.0001. (K and L) HEK293T cells were transfected with lariat-expressing plasmids for 24 h; they were then infected with HSV-GFP (MOI: 0.5) (K) or VSV-GFP (MOI: 0.1) (L) and viral gene expression was quantified 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 < 0.001. (M) HEK293T cells were transfected with lariat ID1 or lariat DKK1 plasmids together with or without PKR overexpression. Cells were then stimulated with polyI:C and WB was then performed with the indicated antibodies. GAPDH was used as a loading control. Data shown are representative of three independent experiments. Source data are available for this figure: SourceData F4.

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