(A) Alignment of human and mouse REUL amino acid sequences. The RING, PRY and SPRY domains are indicated by green, blue and red lines individually. (B) Expression of REUL in mammalian cells. Lysates of the indicated cells were analyzed by Western blot with a mouse polyclonal anti-REUL antibody. (C) REUL localization in HEK293 and Hela cells. Upper panels: HEK293 cells transfected with an expression plasmid for HA-tagged REUL, immunofluorescent staining was performed with anti-HA (αHA) or mouse IgG; untransfected HEK293 cells stained with preimmune serum (preserum) or anti-REUL serum (αREUL); lower panels: transfected or untransfected Hela cells stained as in the upper panels. The experiments were repeated twice, and similar results were obtained.

(A) REUL interacts with RIG-I but not MDA5 and VISA. HEK293 cells (1×10⁶) were transfected with HA-REUL alone or together with Flag-RIG-I, Flag-MDA5 and Flag-VISA plasmids (5 µg each). Cell lysates were immunoprecipitated (IP) with anti-Flag (αF), anti-HA (αHA) or mouse IgG antibody (IgG). The immunoprecipitates were analyzed by Western blot (IB) with anti-HA or anti-Flag antibody. Whole-cell lysates (WCL) were analyzed by Western blots with anti-HA and anti-Flag to determine the expression of REUL, RIG-I, MDA5 and VISA. (B) REUL is colocalized with RIG-I but not VISA in HEK293T and Hela cells. Upper panels: HEK293T cells were transfected with HA-REUL, Flag-RIG-I or Flag-VISA. Immunofluorescent staining was performed with anti-HA (red) and anti-Flag (green); lower panels: Hela cells were transfected and stained as in the upper panels. The experiments were repeated twice, and similar results were obtained. (C) Endogenous interaction of REUL and RIG-I. HEK293T (3×10⁶) cells were treated with SV, transfected with polyI∶C (100 µg) or left untreated for 4 h before lysate. Cell lysates were immunoprecipitated (IP) and Western blot (IB) by indicated antibodies. (D) Identification of domains of REUL mediating the interaction with RIG-I. HEK293T cells (1×10⁶) were transfected with expression plasmids for HA-RIG-I alone or together with Flag-REUL or its mutants (5 µg each). Cell lysates were immunoprecipitated with indicated antibodies. The immunoprecipitates were analyzed by Western blots with anti-HA or anti-Flag antibody. Whole-cell lysates were analyzed by Western blots with anti-Flag and anti-HA to determine the expression levels of transfected plasmids. Upper panel: schematic representation of REUL. (E) Identification of domains of RIG-I mediating the interaction with REUL. HEK293T cells (1×10⁶) were transfected with F-REUL (left panel), F-TRIM25 (middle panel), F-TRIM5-α (right panel) alone or together with HA-RIG-I or its mutants. Immunoprecipitation and Western blot analysis were performed with indicated antibodies.

(A), (B), (C) REUL potentiates RIG-I-, but not MDA5-mediated activation of ISRE (A), NF-κB (B) and IFN-β (C) promoter in a dose-dependent manner. HEK293T cells (1×10⁵) were transfected with the indicated reporter plasmid (50 ng), pRL-TK Renilla luciferase plasmid (50 ng), expression plasmid for RIG-I or MDA5 (200 ng) and the indicated amounts of an expression plasmid for REUL or empty plasmids. Luciferase assays were performed 24 h after transfection. (D) REUL potentiates RIG-I-, but not MDA5-mediated IRF3 dimerization. HEK293 cells (2×10⁵) were transfected with indicated plasmids. 24 h after transfection, cell lysates were separated by native PAGE and analyzed with IRF3 antibody. The experiments were repeated three times with similar results. (E) REUL potentiates RIG-I-, but not MDA5-mediated IRF3 translocation. HEK293-RIG-I stable cell line and HEK293-MDA5 stable cell line (2×10⁵ cells) were transfected with indicated plasmids. 24 h after transfection, immunofluorescent staining was performed with anti-HA antibody (red) and anti-IRF3 antibody (green). The cells expressed REUL or VISA was counted, and the percentage of cells with nuclear IRF3 was calculated. The experiments were repeated twice, and similar results were obtained. (F) REUL potentiates RIG-I-, but not MDA5-mediated gene expression. HEK293T cells (2×10⁵) were transfected with indicated plasmids. 24 h after transfection, total RNA was isolated and RT-PCR was performed using indicated primers. (G), (H), (I) RING domain deletion mutant REULΔRING suppresses RIG-I-, but not MDA5-mediated activation of ISRE (H), NF-κB (I) and IFN-β (G) promoter in a dose-dependent manner. Transfection and luciferase assay were performed as in (A), (B) and (C).

(A), (B), (C) RING domain deletion mutant REULΔRING suppresses SV-induced activation of ISRE (A), NF-κB (B) and IFN-β (C) promoter in a dose-dependent manner. HEK293T cells (1×10⁵) were transfected with the indicated reporter plasmid (50 ng), pRL-TK Renilla luciferase plasmid (50 ng) and the indicated amounts of an expression plasmid for REULΔRING or empty plasmids. 24 h after transfection, cells were left uninfected or infected with SV for 8 h before luciferase assays were performed. (D) REULΔRING has no effect on polyI:C-induced IFN-β promoter. The transfections were done as in (A), (B) and (C). 16 h after transfection, cells were further transfected with polyI∶C (4 µg) or untransfected for 12 h before luciferase assays were performed. (E) Effects of REUL RNAi plasmids on the expression of transfected REUL. HEK293T cells (2×10⁵) were transfected with expression plasmids for HA-REUL and HA-WDR34 as control (0.5 µg each), and the indicated RNAi plasmids (1 µg). At 48 h after transfection, cell lysates were analyzed by Western blot with anti-HA and anti-GAPDH antibody. (F), (G), (H) REUL RNAi plasmids suppress SV-induced activation of ISRE (F), NF-κB (G) and IFN-β (H) promoter. HEK293T cells (1×10⁵) were transfected with the indicated reporter plasmid (50 ng), pRL-TK Renilla luciferase plasmid (50 ng), and the indicated REUL RNAi (1 µg). At 40 h after transfection, cells were left uninfected or infected with SV for 8 h before luciferase assays were performed. (I) REUL RNAi suppresses RIG-I-, but not MDA5-mediated gene expression. HEK293T cells (2×10⁵) were transfected with indicated plasmids. At 40 h after transfection, cells were left uninfected or infected with SV for 8 h before total RNA was isolated and RT-PCR was performed using indicated primers. (J) REUL suppresses NDV-eGFP replication. HEK293T cells (1×10⁵) were transfected with the indicated plasmids. At 20 h after transfection, cells were infected with NDV-eGFP at MOI 0.001. At 40 h after infection, virus titer and replication were determined by plaque assay or GFP expression visualized by fluorescence microscopy. Pfu, plaque-forming unit. (K) REUL accelerates RIG-I-mediated anti-VSV response. HEK293T cells (2×10⁵) were transfected with indicated plasmids (0.5 µg each). At 30 h after transfection, cells were infected with VSV (MOI = 0.001) and supernatants were harvested at 12, 18, 24 and 30 h post infection. Supernatants were analyzed for VSV production using standard plaque assays. Plaques were counted and titers calculated as plaque-forming units (pfu/ml). (L) Knockdown of REUL inhibits RIG-I-mediated anti-VSV response. The experiments were carried out as in (K).

(A) REUL increases the ubiquitination levels of RIG-I in overexpression system. HEK293T cells were transfected with indicated REUL, REULΔRING, RIG-I or ubiquitin plasmids. Cell lysates were immunoprecipitated (IP) with anti-HA (αHA). The immunoprecipitates were analyzed by Western blot (IB) with anti-Flag antibody (αF). Whole-cell lysates were analyzed by Western blots with anti-HA or anti-Flag to determine the expression of REUL, REULΔRING and RIG-I. (B) REUL increases the ubiquitination levels of RIG-I in TRIM25 knock down cells. HEK293T cells were transfected with TRIM25 RNAi and indicated RIG-I, REUL and REULΔRING plasmids. 48 h after transfection, cells were lysated. Immunoprecipitation and western blot were performed as in (A). (C) REUL delivered ubiquitin to RIG-I in vitro. In vitro expressed REUL and RIG-I were used to ubiquitination assay, Biotinylated ubiquitin was detected by HRP-streptavidin. (D) REUL effects on ubiquitination of endogenous RIG-I. HEK293T cells transfected with indicated REUL, REULΔRING, and ubiquitin plasmids. 24 h after transfection, cells were left uninfected or infected with SV for 24 h before lysate. Immunoprecipitation and Western blot were performed as in (A) except that anti-RIG-I antibody (αRIG-I) were used to detect endogenous RIG-I. (E) REUL does not increase the ubiquitination levels of RIG-I ΔCARD. HEK293T cells transfected with indicated REUL, RIG-I, RIG-I ΔCARD and ubiquitin plasmids. Cell lysates were immunoprecipitated with anti-HA (αHA). The immunoprecipitates were analyzed by Western blot with anti-Flag antibody (αF). Whole-cell lysates were analyzed by Western blot with anti-HA or anti-Flag to determine the expression of REUL, RIG-I and RIG-IΔCARD.

(A) K154R, K164R and K172R are the essential sites for RIG-I induced activation of IFN-β promoter. HEK293T cells (1×10⁵) were transfected with IFN-β promoter reporter plasmid (50 ng), pRL-TK Renilla luciferase plasmid (50 ng), and the indicated expression plasmids of RIG-I wild type (WT) and mutations. Reporter assays were performed 24 h after transfection. Lower panel: expression of RIG-I wild type (WT) and mutations. (B) K154R, K164R and K172R are the essential sites for REUL effect. Transfection and luciferase assay were performed as in (A), except cotransfection with REUL. (C) K154R, K164R and K172R are the essential sites for REUL-mediated RIG-I ubiquitination. HEK293T cells were transfected with indicated expression plasmids of RIG-I wild type, mutations and ubiquitin, together with or without REUL expression plasmid. Cell lysates were immunoprecipitated with anti-HA (αHA). The immunoprecipitates (IP) were analyzed by Western blot (IB) with anti-Flag antibody (αF). Whole-cell lysates (WCL) were analyzed by Western blots with anti-HA or anti-Flag to determine the expression of REUL, RIG-I wild type and mutations.