Basal expression of interferon regulatory factor 1 drives intrinsic hepatocyte resistance to multiple RNA viruses

Abstract

Current models of cell-intrinsic immunity to RNA viruses centre on virus-triggered inducible antiviral responses initiated by RIG-I-like receptors or Toll-like receptors that sense pathogen-associated molecular patterns, and signal downstream through interferon regulatory factors (IRFs), transcription factors that induce synthesis of type I and type III interferons¹. RNA viruses have evolved sophisticated strategies to disrupt these signalling pathways and evade elimination by cells, attesting to their importance². Less attention has been paid to how IRFs maintain basal levels of protection against viruses. Here, we depleted antiviral factors linked to RIG-I-like receptor and Toll-like receptor signalling to map critical host pathways restricting positive-strand RNA virus replication in immortalized hepatocytes and identified an unexpected role for IRF1. We show that constitutively expressed IRF1 acts independently of mitochondrial antiviral signalling (MAVS) protein, IRF3 and signal transducer and activator of transcription 1 (STAT1)-dependent signalling to provide intrinsic antiviral protection in actinomycin D-treated cells. IRF1 localizes to the nucleus, where it maintains the basal transcription of a suite of antiviral genes that protect against multiple pathogenic RNA viruses, including hepatitis A and C viruses, dengue virus and Zika virus. Our findings reveal an unappreciated layer of hepatocyte-intrinsic immunity to these positive-strand RNA viruses and identify previously unrecognized antiviral effector genes.

Figure 1.. IRF1 restricts RNA virus infections in hepatocytes.

(a) Intracellular HAV RNA on 5 d.p.i. in PH5CH8 cells transduced with lentivirus expressing shRNAs targeting different genes. **p < 0.001 vs. control [two-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons test]. (b) Kinetics of HAV RNA replication over 5 days in PH5CH8 cells expressing IRF1 vs. IRF3 vs. RELA sgRNAs. *p<0.05, **p<0.01 vs. control (two-way ANOVA with Dunnett’s multiple comparisons test). Immunoblots of IRF1, IRF3, IRF7, and RelA in the knockout cells are shown on left. Viral titers on 5 days p.i. are shown on the right. **p<0.01 vs. control (one-way ANOVA with Dunnett’s multiple comparisons test). (c) Fecal HAV shedding on days 5 and 7 postinoculation (left, data are pooled from 2 different time points), and intrahepatic HAV RNA on days 3 and 7 (right, each symbol = one animal) in wildtype (WT) vs. Irf1^−/− C57BL/6 mice. *p<0.05 vs. WT (two-sided unpaired Mann-Whitney test). (d) HAV RNA on 5 d.p.i. in PH5CH8 cells expressing IFNAR1 or IFNLR1 sgRNAs vs. IRF1 sgRNA. Immunoblots of IFNAR1 and ISGs (MDA5 and OAS1) induced either by recombinant IFN-α (100 U ml⁻¹ for 24 h) or IFN-λ (10 ng ml⁻¹) in these knockout cells are shown on left. **p<0.01 vs. control (one-way ANOVA with Dunnett’s multiple comparisons test). (e) HAV RNA on 5 d.p.i. in PH5CH8 cells expressing STAT1 sgRNA and both STAT1 and IRF1 sgRNAs (right). **p<0.01 vs. control (one-way ANOVA with Dunnett’s multiple comparisons test). Immunoblots showing the absence of ISG expression in response to type I and type III IFNs (left). (f) HAV RNA on 5 d.p.i. in PH5CH8 cells in continued presence of JAK inhibitors, 3 μM ruxolitinib or 0.3 μM pyridone 6 (left). *p<0.05, **p<0.01 vs. control (one-way ANOVA with Dunnett’s multiple comparisons test or two-sided t-test). Knocking out IRF1 enhanced HAV replication in the presence of ruxolitinib (right). *p<0.05, **p<0.01 vs. control (two-sided unpaired t-test). (g) Effect of double-knockout of IRF1 in the absence of MAVS or IRF3 expression on HAV replication. Relative HAV RNA levels on 5 d.p.i., normalized to those without IRF1 sgRNA were set to 1 (right). Immunoblots are shown on left. **p<0.01 vs. control (two-sided unpaired t-test). (h) Immunoblots of IRF1 in control and IRF1 knockout Huh-7.5 cells (left). GLuc secreted from Huh-7.5 cells infected with JFH1-QL/GLuc virus (10³ FFU ml⁻¹) over ensuing 96 h (right). **p<0.01 vs. control (two-way ANOVA with Dunnett’s multiple comparisons test). (i) HAV RNA levels in IRF1 sgRNA-expressing vs. control Huh-7.5 cells infected at an m.o.i. of 1 over ensuing 48 h. **p < 0.01 vs. control (two-sided unpaired t-test). (j) DENV and ZIKV RNA levels in IRF1 vs. control siRNA-transfected Huh-7.5 cells infected at an m.o.i. of 1 over ensuing 48 h. **p<0.01 vs. control (two-sided unpaired t-test). Data are means ± s.d. from 3 independent experiments (a,b,d-h, j) or from 3 technical replicates representative of 2 independent experiments (c,i). The precise p-values are shown in Supplementary Table 9.

Figure 2.. IRF1 constitutively activates basal transcription of PRDIII-I- and ISRE-dependent antiviral genes.

(a) Dual luciferase reporter analysis of 4×PRDIII-I-Luc (top panels) and ISRE-Luc (bottom panels) activities in mock- (left panels) and HAV-infected (right panels) PH5CH8 cells. Promoter activities in IRF1-sgRNA (#1, #2) vs. control or IRF3 sgRNA-expressing cells differed significantly (p < 0.01, two-way ANOVA with Dunnett’s multiple comparisons test). (b) Dose-response analysis of PRDIII-I (top) and ISRE (bottom) activities in HAV and SeV-infected, wildtype PH5CH8 cells at the indicated m.o.i. *p<0.05, **p<0.01 vs. mock (one-way ANOVA with Dunnett’s multiple comparisons test). (c) Dual luciferase reporter analysis of 4×PRDIII-I-Luc (top) and ISRE-Luc (bottom) activities in mock-infected Huh-7.5 cells. Note that SeV does not activate these promoters in Huh-7.5 cells. **p<0.01 vs. control (two-way ANOVA with Dunnett’s multiple comparisons test). (d) Nuclear localization of IRF1 in two different hepatic cell lines and primary human fetal hepatocytes. Data are representative of 2 independent experiments. Scale bar, 20 μm. (e) HAV RNA at 24 h p.i. in Huh-7.5 cells expressing IRF1 sgRNA pre-treated with actinomycin D (ActD, 5 μg ml⁻¹) for 30 min before infection. **p<0.01, *p<0.05 vs. control (two-sided unpaired t-test). (f) DENV RNA at 18 h p.i. or ZIKV RNA at 24 h p.i. in IRF1-depleted Huh-7.5 cells pre-treated with actinomycin D (ActD, 5 μg ml⁻¹) for 30 min before infection. *p<0.05, **p<0.01 vs. control (two-sided unpaired t-test). Data are means ± s.d. from 3 technical replicates representative of 2 independent experiments (a-d) or from 3 independent experiments (e, f). The precise p-values are shown in Supplementary Table 9.

Figure 3.. Shared and distinct antiviral activities of IRF1 effector genes identified by high-throughput RNA sequencing.

(a) The Venn diagram showing numbers of genes with expression changes of ≥ 2-fold for each knockout. (b) List of genes reduced >2-fold in IRF1 sgRNA-expressing cells, in comparison with IRF3 sgRNA-expressing cells. Values shown are means of fold changes of genes expressed in cells transduced with 2 independent IRF1 sgRNAs (left) or an IRF3 sgRNA (right). See Supplementary Tables 5–7 for more details. (c) Validation of RNA-seq results by RT-qPCR assays of RNA extracted from noninfected vs. HAV-infected PH5CH8 cells. Scatter plots show the ratio of abundance of indicated genes between IRF1 vs. control sgRNA-expressing PH5CH8 cells in HAV-infected (y-axis) and noninfected cells (x-axis). (d) Heatmap showing the relative abundance of indicated genes in noninfected PH5CH8 cells determined by RT-qPCR. (e) Relative HAV RNA abundance on 5 d.p.i. of PH5CH8 transfected with siRNA targeting different IRF1 effector genes. **p<0.01 vs. control. (f) Independent validation of the siRNA results and the combination of 4 siRNAs. *p<0.05, **p<0.01 vs. control. (g) Relative GLuc activity on 3 d.p.i. of HCV-infected Huh-7.5 cells. *p<0.05, **p<0.01 vs. control. (h) Independent validation of the siRNA results and the combination of 3 siRNAs. *p<0.05, **p<0.01 vs. control. (i) Relative DENV RNA on 24 h p.i. of infected Huh-7.5 cells. *p<0.05 vs. control. (j) Independent validation of the siRNA results and the combination of 2 siRNAs. *p<0.05, **p<0.01 vs. control. (k) ZIKV RNA abundance on 24 h p.i. of infected Huh-7.5 cells. *p<0.05, **p<0.01 vs. control. (l) Independent validation of the siRNA results and the combination of 2 siRNAs. **p<0.01 vs. control. Data are means ± s.d. from 3 independent experiments (e-g, i-l) or from 3 technical replicates representative of 2 independent experiments (h). p-values were derived using one-way ANOVA with Dunnett’s multiple comparisons test (e, g, i-l) or two-sided unpaired t-test (f, h). The precise p-values are shown in Supplementary Table 9.

Figure 4.. IRF1-regulated RARRES3 acyl transferase restricts HAV replication by downregulating mTOR.

(a) Lentivirus transduction of catalytically-active RARRES3 restricts HAV infection in PH5CH8 cells expressing IRF1 sgRNA #2 (left panels) or Huh-7.5 cells (right panels). RARRES3, but not its catalytically-inactive RARRES3/C113S, inhibited HAV infection in both cell lines. *p<0.05, **p<0.01 vs. vector control (two-way ANOVA with Dunnett’s multiple comparisons test). (b) Huh-7.5 cells stably expressing indicated lentiviral vectors were challenged with HAV carrying NanoLuc (NLuc) with 30 μM 2’CMA (DAA) or vehicle (DMSO). NLuc activities at indicated time points post-infection are shown. **p<0.01 (two-way ANOVA with Dunnett’s multiple comparisons test). (c) Transfection of subgenomic HAV-Luc RNA or its replication-incompetent mutant (Δ3D) in Huh-7.5 cells expressing wild-type RARRES3 versus RARRES3/C113S. **p<0.01 vs. vector control (two-way ANOVA with Dunnett’s multiple comparisons test). (d) Infection of HAV/NLuc in Huh-7.5 cells expressing RARRES3 sgRNA. Immunoblots are shown on top. **p<0.01 vs. vector control (two-way ANOVA with Dunnett’s multiple comparisons test). (e) Steady-state levels of mTOR-related factors in Huh-7.5 cells stably expressing RARRES3 and RARRES3/C113S. (f) Immunoblots of P70S6K siRNA-transfected Huh-7.5/RARRES3 cells. (g) Phosphorylation of p70S6K and mTOR in Huh-7.5 cells expressing IRF1 sgRNA. (h) Impacts of mTOR inhibitors on HAV/NLuc vs. HCV/GLuc vs. DENV/NLuc replication and the cell viability. Inhibitory effects of all these inhibitors on HAV/NLuc vs. other reporter viruses differed significantly. **p<0.01 (two-way ANOVA with Dunnett’s multiple comparisons test). (i) Inhibition of transfected subgenomic HAV-Luc RNA replication in Huh-7.5 cells by the mTOR inhibitors. DAA, 30 μM 2’CMA. **p<0.01 vs. DMSO control (one-way ANOVA with Dunnett’s multiple comparisons test). Data are means ± s.d. from 3 independent experiments (b, d, i) or from 3 technical replicates representative of 2 (a, e-h) or 3 independent experiments (c). The precise p-values are shown in Supplementary Table 9.