Fig 2: DHX16 interacts with the E3-Ub ligase TRIM6(A) Interaction between DHX16 and TRIM6 following co-expression in HEK293Ts (coIP).(B) Diagram of TRIM6 deletion mutants.(C) Interaction between DHX16 and the SPRY domain of TRIM6 following co-expression in HEK293Ts (coIP).(D) Co-localization between DHX16 and TRIM6 following co-expression in A549s. Scale bar, 20 µm (confocal microscopy).(E) Nuclear/cytoplasmic distribution of DHX16 following TRIM6 co-expression in A549s (n = 17).(F) Interaction between DHX16 and K48-poly-Ub following co-expression with either WT or C15A TRIM6 in HEK293Ts (coIP).(G) Interaction between endogenous DHX16 and K48-poly-Ub in WT or TRIM6 KO A549s (coIP).(H) IFN-ß, IFITM1, and IFI27 expression from WT or TRIM6 KO A549s transfected with DHX16 for 24 h (qRT-PCR).Data are expressed as means (n = 3) ± SD. ***p < 0.001; ****p < 0.0001 (one-way or two-way ANOVA with Tukey's multiple comparisons). Data are representative of 2–3 independent experiments.See also Figures S4 and S5.

Fig 3: DHX16 is required for efficient induction of ISGs(A–C) A549s treated with siNTC or siDHX8/15/16 followed by IAV infection for 24 h (PR8 MOI = 0.1). (A) Silencing efficiency of siRNAs and (B) IFN-ß expression (qRT-PCR). (C) Silencing efficiency of DHX16 protein (immunoblot).(D–F) Next-generation sequencing (NGS) analysis of A549s treated with siRNA and followed by IAV infection as described in (A). (D) Volcano plot of ISGs significantly downregulated in siDHX16 versus siNTC mock samples. (E) Enriched GO terms and pathways (Enrichr) of downregulated transcripts. (F) Heatmap of transcriptomic changes.(G) Validation of selected ISGs identified in NGS (qRT-PCR).Data are expressed as means (n = 3) ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 (Student's t test or one-way ANOVA with Dunnett's or Sidak's multiple comparisons). Data are representative of 2–3 independent experiments.See also Figure S1.

Fig 4: DHX16 interacts with unanchored Ub and RNA to promote IFN-I production(A) The IsoT de-ubiquitination assay.(B) De-coupling of unanchored K48-poly-Ub from DHX16 following co-expression of IsoT in HEK293Ts (coIP).(C) IFN-ß expression following co-expression of DHX16 and IsoT in HEK293Ts infected with IAV for 24 h (PR8 MOI = 0.1) (qRT-PCR).(D) In vitro interactions between DHX16 and RIG-I ± recombinant poly-Ub chains (coIP).(E) Effects of recombinant poly-Ub (1 µg) and purified IAV vRNA (100 ng) on DHX16 ATPase activity in an in vitro ATPase assay.(F) Interaction between DHX16 and unanchored K48-poly-Ub following co-expression in HEK293Ts in the presence of RNase A (100 µg/mL) (coIP).(G) Immunoblot quantification of K48-Ub in (F) (densitometry).(H) Interactions between DHX16 and RIG-I following co-expression in HEK293Ts in the presence of RNase A (0–100 µg/mL) (coIP).(I) DHX16-RIG-I complex formation mediated through vRNA and unanchored poly-Ub.Data are expressed as means (n = 3) ± SD. *p < 0.05; **p < 0.01; ***p < 0.001 (one-way ANOVA with Tukey's multiple comparisons). Data are representative of 2–3 independent experiments.

Fig 5: DHX16 enhances activation of the IFN-I production pathway(A–D) A549s treated with siNTC or siDHX16 followed by IAV infection for 24 h (PR8 MOI = 0.1). (A) Phosphorylation of TBK1 and IRF3 (immunoblot). (B) IFN-ß and IFIT2 expression (qRT-PCR). (C) IFN-ß protein in supernatants (ELISA). (D) IAV viral titers (plaque assay).(E–G) A549s transfected with empty vector or DHX16 followed by IAV infection for 24 h (PR8 MOI = 0.1). (E) Phosphorylation of TBK1 and IRF3 (immunoblot). (F) IFN-ß and IFITM1 expression (qRT-PCR). (G) IAV viral titers (plaque assay).(H–M) PPMO-mediated in vivo silencing of DHX16 in BALB/c mice followed by IAV infection (PR8, 1000 PFU) (n = 3–4/group). (H) Schematics of experiment. (I) Validation of PPMO-mediated silencing in murine lung tissue (immunoblot). (J) IAV viral titers (plaque assay). (K) mIFN-ß and mIFITM1 expression (qRT-PCR). (L) IFN-ß protein in lung tissue (ELISA) (n = 5–8/group). (M) Weight loss from NTC or DHX16 PPMO-treated mice infected with IAV (PR8, 100 PFU) (n = 5/group).(N–Q) IFN-ß expression from siNTC or siDHX16 treated A549s infected with IAV (PR8 WT MOI = 0.1) (N) or ZIKV (FSS MOI = 0.1) (O), Calu-3s infected with SARS-CoV-2 (icSARS-CoV-2-mNG MOI = 1) (P) or A549s transfected with 3p-hpRNA (100 ng/mL) (Q) (qRT-PCR).Data are expressed as means (n = 3) ± SD or ± SEM as in (M). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 (Student's t test or one-way ANOVA with Tukey's multiple comparisons). Data are representative of 2–3 independent experiments.See also Figures S2 and S3.