U5 snRNP Core Proteins Are Key Components of the Defense Response against Viral Infection through Their Roles in Programmed Cell Death and Interferon Induction

Figures

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  Figure 1. EFTUD2 and SNRNP200 KD protect cells from death after MRV infection. (A) RTCA of MRV-infected L929 cells (MOI of 3) transfected with a control siRNA or EFTUD2, PRPF8, or SNRNP200 specific siRNA. Measurements were made every 10 min. n = 3, biological replicates. (B) RTCA of mock-infected L929 cells transfected with a control siRNA or EFTUD2, PRPF8, or SNRNP200 specific siRNA. Measurements were made every 10 min. n = 3, biological replicates. (C) Cell viability in mock- or MRV-infected L929 cells. Cells were treated with the siRNA for 55 h before being mock-infected or infected at a MOI of 3 for 65 h. Cell viability was measured using FACS by staining with Hoechst and propidium iodide; live cells were selected as Hoechst positive and propidium iodide negative. n = 3, biological replicates, two-way ANOVA with Dunnett’s multiple comparisons test against the siCTRL treated cells for each condition (mock in black, infected in red; ns, p > 0.05; ****, p ≤ 0.0001). (D) Representative images of methylene blue staining of control or EFTUD2, PRPF8, or SNRNP200-depleted L929 cells infected with 1:1 dilutions of MRV at 48 h or 96 h post-infection. (E) Quantification of cell-bound methylene…

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  Figure 2. EFTUD2 and SNRNP200 depletion hamper apoptosis. (A) Representative set of images for the high-throughput multiplex microscopy-based apoptosis assay. On the left, Annexin-V signal is shown in red, representative of apoptotic cells. On the right, propidium iodide signal is shown in red, representative of dead cells. On both sides, the nuclei are stained with Hoechst and shown in blue. (B) Percent of mock- or MRV-infected L929 cells staining positive for Annexin-V upon depletion of EFTUD2, PRPF8, or SNRNP200. Cells were infected (MOI of 3) or mock-infected, and the percent of Annexin-V positive cells was calculated 40 h later. n = 6, biological replicates, one-way ANOVA with Dunnett’s multiple comparisons test against the siCTRL treated cells (ns, p > 0.05; ****, p ≤ 0.0001). (C) Percent of dead cells upon depletion of EFTUD2, PRPF8, or SNRNP200. Cells were infected (MOI of 3) or mock-infected, and the percent of propidium iodide positive cells was calculated at 40 h post infection. n = 6, biological replicates, one-way ANOVA with Dunnett’s multiple comparisons test against the siCTRL treated cells (ns, p > 0.05; *, p ≤ 0.05; ***, p ≤ 0.001; ****, p ≤ 0.0001). (D) Relative Cas3/7…

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  Figure 3. Depletion of EFTUD2 and SNRNP200 impacts necroptosis. (A) Western blot of phosphorylated MLKL (pMLKL) and total MLKL in mock- or MRV-infected L929 cells treated with control or EFTUD2, PRPF8, or SNRNP200 specific siRNA. Cells were infected at an MOI of 3 and incubated for 40 h before proteins were harvested. Actin was used as a loading control and the MRV σ3 viral protein was used to validate the infection. Relative quantitation is shown for pMLKL/MLKL and MLKL/Actin. (B) Quantification and statistical analysis of three independent Western blots. For pMLKL/MLKL, the infected cells were compared to their respective non-infected siRNA condition using a two-way ANOVA with Šídák’s multiple comparisons test; for MLKL/Actin, all conditions were compared to the siCTRL mock condition using a one-way ANOVA with Dunnett’s multiple comparisons test. n = 3, biological replicates (ns, p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01). (C) RTCA of zVAD-fmk/TNFα treated L929 cells transfected with a control siRNA or EFTUD2, PRPF8, or SNRNP200 specific siRNA. A siCTRL condition was treated with only zVAD-fmk to confirm no cell death was induced in the absence of TNFα. Measurements were made every 15 min for…

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  Figure 4. Depletion of U5 snRNP core components impacts the mRNA level, protein levels, and alternative splicing of necroptotic regulators. (A) Relative mRNA levels for RIPK1, RIPK3, and MLKL in control or EFTUD2, PRPF8, or SNRNP200-depleted L929 cells as determined by qPCR. L929 cells were transfected with the appropriate siRNA for 72 h before RNA was harvested, reverse transcribed, and subjected to qPCR. PSMC4, PUM1, and TXNL4B were used as housekeeping genes for normalization. n = 3, biological replicates, one-way ANOVA with Dunnett’s multiple comparisons test against the control siRNA condition (ns, p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001). (B) Western blot of the main necroptotic regulators (RIPK1, RIPK3, MLKL) in L929 cells treated with control or EFTUD2, PRPF8, or SNRNP200 specific siRNA. Cells were incubated for 72 h after transfection before proteins were harvested for Western blot. Actin was used as a loading control. Relative quantitation is shown compared to the siCTRL condition. (C) Quantification and statistical analysis of three independent Western blot for RIPK1, RIPK3 and MLKL. n = 3, biological replicates, one-way ANOVA with Dunnett’s multiple comparisons test…

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  Figure 5. EFTUD2 and PRPF8 control the interferon response during MRV infection. (A) Relative mRNA levels for DDX60, IFNB1, MX1, and MX2 in control or EFTUD2, PRPF8, or SNRNP200-depleted L929 cells. L929 cells were transfected with the appropriate siRNA for 72 h before RNA was harvested, reverse transcribed, and subjected to qPCR. PSMC4, PUM1, and TXNL4B were used as housekeeping genes for normalization. n = 3, biological replicates, one-way ANOVA with Dunnett’s multiple comparisons test against the control siRNA condition (ns, p > 0.05; *, p ≤ 0.05; ****, p ≤ 0.0001). (B) Relative fold induction upon MRV infection for DDX60, IFNB1, MX1, and MX2 in control or EFTUD2, PRPF8, or SNRNP200-depleted L929 cells. L929 cells were transfected with the appropriate siRNA for 55 h before being either mock-infected or infected with MRV at a MOI of 3 for 24 h. Each replicate was arbitrarily attributed an uninfected control sample to calculate a fold-change upon infection. n = 3, biological replicates, Brown-Forsythe and Welch one-way ANOVA with Dunnett’s T3 multiple comparisons test against the control siRNA condition (ns, p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001). (C) Relative fold induction…

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  Figure 6. EFTUD2 restricts MRV’s replication in both single cycle and multiple cycles of replication. (A) MRV viral titers from siCTRL, siEFTUD2, siPRPF8, and siSNRNP200-treated L929 cells infected cells at 24 h, 48 h, and 72 h. Cells were infected at a MOI of 3, and viral titers were determined by TCID50 on L929 cells. n = 3, biological replicates, two-way ANOVA with Dunnett’s multiple comparisons test against the siCTRL matched timepoint (ns, p > 0.05; ****, p ≤ 0.0001). (B) MRV viral titers from siCTRL, siEFTUD2, siPRPF8 and siSNRNP200-treated L929 cells infected cells at 96 h. Cells were infected at a MOI of 0.1, and viral titers were determined by TCID50 on L929 cells. n = 3, biological replicates, one-way ANOVA with Dunnett’s multiple comparisons test against the siCTRL condition (ns, p > 0.05; **, p ≤ 0.01). (C) MRV viral titers in L929 cells untreated, transfected with a siRNA against EFTUD2 or RIG-I, or treated with GSK872 (3 μM) or zVAD-fmk (50 μM) to block necroptosis and apoptosis, respectively. L929 cells were infected at a MOI of 3, and viral titers were determined by TCID50 on L929 cells at 72 h post-infection. n = 3, biological replicates, one-way ANOVA with Dunnett’s…

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  Figure 7. Summary of the involvement of U5 snRNP core proteins in virus–host interactions. (A) Table depicting the respective roles of EFTUD2, PRPF8, and SNRNP200 in the interferon response pathway, apoptosis, and necroptosis. (B) Model depicting the roles of EFTUD2 during MRV infection. EFTUD2 (either cytoplasmic or nuclear; and either in the U5 snRNP or by itself when located in the nucleus) exerts an antiviral role on MRV replication (red arrow). MRV infection elicits both apoptosis and necroptosis (black arrow), which require the presence of EFTUD2 to normally proceed (dotted arrow). EFTUD2 is also required for the basal expression of ISG (thin dotted arrow), but its principal impact on the IFN response pathway during infection lies somewhere between the sensing of viral RNA and the production of IFN (dotted arrow). ISG expression exerts an antiviral effect on MRV (red arrow), and might explains, at least in part, the antiviral effect of EFTUD2, since MRV actively trigger this pathway through the sensing of its viral RNA (black arrow).