Fig 2: Knockdown of IFIT genes partially reverses attenuation of dNSP16.(a) Replication of WT (black) and dNSP16 (blue) in the context of siRNA treatment. 1.25 × 105 Vero E6 cells/well were reverse transfected with 2 pmol total of the indicated siRNA construct(s) 2 days prior to infection and also pre-treated with 100 U IFN-I a day prior to infection, multiplicity of infection (MOI) = 0.01. Data shown at 48 hours post-infection (HPI). Statistical comparisons on graph are with respect to siRNA control treatment (“siNC”). (b) Baseline IFIT1 protein expression in Calu-3 2B4 and Vero E6 cells, or Vero E6 cells 1 day post-stimulation with IFN-I. (c) Viral replication kinetics for WT (black) or dNSP16 (blue) following treatment with anti-IFIT1 (dashed) or control siRNA (solid). 1.25 × 105 Vero E6 cells were reverse transfected with 1 pmol of the indicated siRNA construct 2 days prior to infection and also pre-treated with 100 U IFN-I a day prior to infection, MOI = 0.01. (d) Comparison of the viral titers at 48 HPI from panel (c), black = WT, blue = dNSP16. (e) Viral replication kinetics for WT (black) or dNSP16 (blue) following treatment with anti-IFIT3 (dashed) or control siRNA (solid). 1.25 × 105 Vero E6 cells/well were transfected with 1 pmol of the indicated siRNA construct 2 days prior to infection and also pre-treated with 100 U IFN-I a day prior to infection, MOI = 0.01. (f) Comparison of the viral titers at 48 HPI from panel (e), black = WT, blue = dNSP16. For panels (a), (d) and (f), ****p<0.001: results of two-way ANOVA with Tukey’s multiple comparison test (a = 0.05). Means are plotted with error bars denoting standard deviation. For all panels, n = 3 biological replicates for all data points. Dotted lines represent limits of detection. PFU = plaque-forming units.

Fig 3: Linear ubiquitination sustains STAT1 signaling homeostasis.a Western blot analysis of endogenous STAT1 in HEK293T cells transfected with Flag-LUBAC. b Western blot analysis of endogenous STAT1 in HEK293T cells transfected with control shRNAs (–) or shHOIP (#1, #2). c Western blot analysis of pY701-STAT1 (pY-STAT1) levels in HEK293T cells transfected with increasing amounts of Flag-LUBAC. d Western blot analysis of pY-STAT1 in HOIP-WT or HOIP-KO HEK293T cells treated with IFNα (1000 IU/ml). e Western blot analysis of pY-STAT1 in HEK293T cells cotransfected with Flag-Sharpin and Flag-HOIL-1L, together with Flag-HOIP wild-type or its catalytically inactivated mutants (C885A and C916A), and then treated with IFNα (1000 IU/ml) for 30 min. f Dual-luciferase reporter assay of the ISRE activity in HEK293T cells cotransfected with control shRNAs (shCON) or shHOIP (#1, #2), together with ISRE luciferase and Renilla, and then stimulated with IFNα (1000 IU/ml) for 20 h. g RT-qPCR analysis of the representative ISG (Ifit1) mRNA in HEK293T cells transfected with either shCON or shHOIP (#1, #2), and then stimulated with IFNα or IFNβ (1000 IU/ml) for 4 h. h RT-qPCR analysis of Ifit1 mRNA in HOIP-WT or HOIP-KO HEK293T cells treated with IFNβ (1000 IU/ml) for 4 h. i RT-qPCR analysis of VSV viral RNA in 2fTGH cells transfected with shHOIP and then stimulated with IFNα (60 IU/ml) for 20 h, followed by infection with VSV (MOI = 0.1) for 24 h. j Western blot analysis of VSV-encoded proteins VSV-G in HeLa cells transfected with Flag-LUBAC and then treated as i. k RT-qPCR analysis of IFN (Ifnɑ, Ifnγ, and Ifnλ) mRNA in HEK293T cells transfected with LUBAC. l Immunoprecipitation analysis of pY-STAT1 in HEK293T cells transfected with shCON (–) or shHOIP (#1, #2). m RT-qPCR analysis of a representative ISG (Isg54) mRNA in HEK293T cells transfected with shHOIP (#1, #2). N.S., not significant (p > 0.05) and *p < 0.05, **p < 0.01, ***p < 0.001 (two-tailed unpaired Student’s t test). Data are shown as mean and s.d. of three biological replicates (f–i, k, m), or are representative of three independent experiments (a–e, j, l).

Fig 4: IFIT1 mediated PD-L1 stabilization via regulating PD-L1 ubiquitination. A. Co-immunoprecipitation (Co-IP) assay detecting IFIT1 in RKO cell lysates immunoprecipitated with anti-PD-L1. B. RKO cells were overexpressed with FLAG_IFIT1 and HA_PD-L1. Co-IP assay detecting exogenous IFIT1 (FLAG) in RKO cell lysates immunoprecipitated with anti-HA. C-D. RKO cells transfected with control siRNAs and IFIT1 siRNAs were treated with cycloheximide (CHX) (50 µg/mL) at the indicated time points. Immunoblot of PD-L1 at indicated time points. E. Quantification of relative remaining PD-L1 in C-D. F. Immunoblot of PD-L1 in RKO cells transfected with IFIT1 siRNAs and treated with MG132 (10uM, 6 hours). G. Immunoblot of PD-L1 in RKO cells transfected with IFIT1 siRNAs and treated with CQ (25uM, 6 hours). H. Immunoblot of PD-L1 in RKO cells transfected with IFIT1 siRNAs and treated with 3-MA (6 hours). I-J. Immunoblot of cell lysates immunoprecipitated with anti-PD-L1 in RKO cells transfected with the indicated constructs.

Fig 5: IFN Resistance of C9-Deleted VACV in IFIT KO Cells(A) Reduction of IFIT expression in IFIT KO cells. Lysates of A549 or A549 IFIT KO cells untreated (−) or treated (+) with IFN-β were analyzed by WB.(B) Effects of IFIT KO on IFN resistance. A549 and KO cells were untreated (−) or pretreated (+) with IFN-β and infected with 3 PFUs per cell of VACV WR or vΔC9. After 8 h, lysates were analyzed by WB.(C) Effect of IFIT KO on RNA levels. A549 and KO cells were infected in triplicate as in (B) for 6 h. RNA was extracted, reverse transcribed, and quantified by ddPCR. Error bars: SEMs.(D) Antiviral activity of IFIT1 is reduced by the mutation of the RNA-binding motif. Untreated (−) or IFN-β-pretreated (+) A549, IFIT1 KO cells, and IFIT1 KO cells trans-complemented with vector, FLAG-IFIT1, or FLAG-IFIT1R187H by retrovirus transduction were mock infected or infected with 3 PFUs per cell of VACV WR or vΔC9. After 8 h, cell lysates were analyzed by WB.(E) Antiviral activity of IFIT3 is reduced by mutation of IFIT1-association motif. Untreated (−) or IFN-β-pretreated (+) A549 IFIT3 KO or A549 IFIT3 KO cells transcomplemented with vector, FLAG-IFIT3, or FLAG-IFIT3ΔCTD by retrovirus transduction were mock infected or infected with 3 PFUs per cell of VACV WR or vΔC9. After 8 h, lysates were analyzed by WB.Representative blots from three biological repeats in (A) and (B) and two in (D) and (E).