E3 2021: Ex-Nintendo boss doesn't think much of the digital concept

E3 2021

Due to the corona pandemic, E3 2021 will only take place in digital form. Appropriate concepts have already been worked out and are currently waiting for the green light from the major publishers and developer studios. In addition to numerous keynotes, a preview event and an award show are also planned. But when it comes to Reggie Fils-Aimé, the former head of Nintendo of America, that is not enough to actually inspire game fans.

In a video interview with Gamertag magazine, he explained that he was aware that the digital orientation was uncompromising this year. However, the previously known concept of the Entertainment Software Association (ESA) for the event does not particularly appeal to him. There must be a better way to "activate" the fans in front of the home screen and to let them "experience the content". For him, following hours of stream is not what the E3 is actually about. He elaborated on it with a purely fictional example:

"It's about playing The Last Us Part 3 together, possibly for the first time, or the next game from Breath of the Wild. Or the next great game from the new merger of Xbox studios. "

Recommended editorial content Here you will find external content from [PLATTFORM]. To protect your personal data, external integrations are only displayed if you confirm this by clicking on "Load all external content": Load all external content I consent to external content being displayed to me. This means that personal data is transmitted to third-party platforms. Read more about our privacy policy . External content More on this in our data protection declaration. It remains to be seen whether and in what form it might even be possible to involve the fans more in the action at E3 2021. In principle, however, Reggie Fils-Aimé's not a bad idea to at least rethink the previous digital concepts. What would a perfect digital edition of E3 2021 look like for you? Write your ideas in the comments!

Source: Gamertag

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E3 ligase Nedd4l promotes antiviral innate immunity by catalyzing K29-linked cysteine ubiquitination of TRAF3

Nedd4l deficiency impairs antiviral innate immunity

To investigate whether Nedd4l plays a role in antiviral innate immunity, we initially used small interfering RNA (siRNA) specific to Nedd4l to inhibit Nedd4l expression in peritoneal macrophages and found that Nedd4l knockdown significantly decreased vesicular stomatitis virus (VSV)-induced IFN-β and TNF-α production in them (Supplementary Fig. 1a, b). To confirm the role of Nedd4l in antiviral innate immunity, we used a Nedd4l-deficient mouse model with spontaneous mutation in genome to investigate the effects of Nedd4l deficiency on virus-induced type I interferon and proinflammatory cytokine production. Primary peritoneal macrophages were isolated from control wild-type mice and Nedd4l-deficient mice, and then infected with VSV. Nedd4l deficiency impaired the production of IFN-β, IL-6, and TNF-α induced by the viruses at protein level, as detected by ELISA, and also at mRNA level, as detected by real-time quantitative polymerase chain reaction (RT-qPCR) (Fig. 1a, b). Consistently, Nedd4l deficiency inhibited mRNA expression of IFN-β, IL-6, and TNF-α induced by poly(I:C) transfection (Fig. 1c), demonstrating that Nedd4l positively regulated innate immunity mediated by cytoplastic RNA sensor. Similarly, Nedd4l deficiency inhibited IFN-β, IL-6, and TNF-α mRNA expression induced by poly(dA:dT) transfection (Supplementary Fig. 1c). Nedd4l deficiency also reduced IFN-β, IL-6, and TNF-α production and mRNA expression in macrophages stimulated with LPS and poly(I:C) (Supplementary Fig. 1d,e,f). However, Nedd4l deficiency did not affect IL-6 and TNF-α mRNA expression in macrophages stimulated with LTA (Supplementary Fig. 1g), suggesting Nedd4l selectively regulated innate immunity triggered by TLR family members.

Fig. 1: Nedd4l deficiency inhibited antiviral innate immunity in macrophages.

a ELISA assay of IFN-β, IL-6, and TNF-α production in wild-type (Nedd4l+/+) and Nedd4l-deficient homozygous (Nedd4l−/−) peritoneal macrophages infected with VSV (MOI = 10) for 12 and 24 h. b RT-qPCR analysis of IFN-β, IL-6, and TNF-α mRNA expression in peritoneal macrophages in (a) infected with VSV (MOI = 10) for 6 and 12 h. c RT-qPCR analysis of IFN-β, IL-6, and TNF-α mRNA expression in wild-type (Nedd4l+/+) and Nedd4l-/- peritoneal macrophages transfected with poly(I:C) (10 μg/ml) for 6 and 9 h. Data in (a–c) are presented as mean ± SD (n = 3 per group) and p-values by two-tailed unpaired Student’s t-test are indicated. Results in (a–c) are representative of three independent experiments.

The spontaneous Nedd4l-deficient mice display reduced body weights compared to wild-type control mice. To investigate the role of Nedd4l in antiviral innate immunity in vivo, we generated Nedd4lfp/fp mice (Supplementary Fig. 2a), crossed the mice with Lyz2-Cre transgene mice to delete Nedd4l in macrophages. Expression of Nedd4l in peritoneal macrophages and bone-marrow-derived macrophages was efficiently deleted (Supplementary Fig. 2b), while expression of Nedd4l in B cells and T cells from spleen and bone marrow were similar between Nedd4lfp/fpLyz2-Cre+/+ mice and wild-type mice (Supplementary Fig. 2c). We assessed the frequency of immune cell populations in spleen, bone marrow, and blood of the mice, and found that the abundance of macrophages, granulocytes, B cells, and T cells was similar between Nedd4lfp/fpLyz2-Cre+/+ and wild-type control Nedd4lfp/fpLyz2-Cre−/−mice (Supplementary Fig. 2d). These results suggest that Nedd4l myelogenous deficiency did not affect the development of major immune cell populations. The Nedd4lfp/fpLyz2-Cre+/+ and wild-type control Nedd4lfp/fpLyz2-Cre−/− mice were infected with VSV by intraperitoneal injection. IFN-β, IL-6, and TNF-α concentrations in serum of the mice were then detected. As shown in Fig. 2a, conditional Nedd4l deficiency inhibited VSV-induced IFN-β and IL-6 production in vivo. Meanwhile, VSV replication and TCID50 increased in spleen and liver in Nedd4lfp/fpLyz2-Cre+/+ mice than in control Nedd4lfp/fpLyz2-Cre−/− mice (Fig. 2b, c). Consistently, the survival of the Nedd4lfp/fpLyz2-Cre+/+ mice was much lower than the survival of control Nedd4lfp/fpLyz2-Cre−/− mice (Fig. 2d). These results demonstrate that Nedd4l positively regulates type I interferon and proinflammatory cytokine production in macrophages and is critical for antiviral innate immunity in vivo.

Fig. 2: Conditional Nedd4l deficiency inhibited antiviral innate immunity in vivo.

a ELISA assay of IFN-β, IL-6, and TNF-α in serum of control Nedd4lfp/fpLyz2-Cre−/− and Nedd4lfp/fpLyz2-Cre+/+ mice intraperitoneally infected with VSV (4 × 105 PFU/g) for 6 and 12 h (n = 8 per group). b, c RT-qPCR analysis of VSV mRNA and TCID50 in spleen (b) and liver (c) in control Nedd4lfp/fpLyz2-Cre−/− and Nedd4lfp/fpLyz2-Cre+/+ mice intraperitoneally infected with VSV (4 × 105 PFU/g) for 24 h (RT-qPCR analysis n = 8 and TCID50 n = 6). d The survival rates of control Nedd4lfp/fpLyz2-Cre−/− and Nedd4lfp/fpLyz2-Cre+/+ mice were monitored for 3 days after intraperitoneally infected with VSV (8 × 105 PFU/g) (Nedd4lfp/fpLyz2-Cre−/− n = 7 and Nedd4lfp/fpLyz2-Cre+/+ n = 8, and p-values by two-tailed unpaired Student’s t-test are indicated). Data are presented as mean ± SD and p-values by two-tailed unpaired Student’s t-test are indicated in (a–c) and p-value by Gehan-Breslow-Wilcoxon test are indicated in (d). Results in (a–d) are representative of three independent experiments.

Nedd4l promotes TRAF3-dependent signaling

Upon VSV infection, RLRs activate IRF3, NF-κB, and MAPKs to induce type I interferon and proinflammatory cytokine production. We detected VSV-induced phosphorylation of IRF3, NF-κB, and MAPKs in Nedd4l-deficient and wild-type control macrophages. As shown in Fig. 3a, Nedd4l deficiency inhibited VSV-induced IRF3 phosphorylation without affecting the phosphorylation of ERK1/2, p38, JNK1/2, IκBα, and NF-κB p65. Nedd4l deficiency inhibited phosphorylation of TBK1, the upstream molecule that activates IRF3, suggesting that Nedd4l positively regulated RLR signaling by functioning upstream TBK1. In RLR signaling, TBK1 is activated downstream of TRAF3, thus we observed the effect of Nedd4l expression on TRAF3/TBK1 complex formation, which is required for TBK1 activation. In macrophages, Nedd4l deficiency decreased VSV infection-induced TRAF3/TBK1 complex formation (Fig. 3b). In 293T cells, overexpression of Nedd4l increased the association between TRAF3 and TBK1 (Fig. 3c). In contrast, it did not affect the association between TRAF3 and MAVS (Fig. 3d), indicating that Nedd4l regulates VSV-induced interferon production by targeting TRAF3.

Fig. 3: Nedd4l promotes TRAF3-dependent signaling.

a Peritoneal macrophages from wild-type (Nedd4l+/+) and Nedd4l-deficient (Nedd4l−/−) mice were infected with VSV (MOI = 10) for indicated time. Phosphorylated and total TBK1, IRF3, p38, JNK, ERK1/2, NF-kB p65, and IκBα were detected by western blot. Intensities of p-TBK1, TBK1, p-IRF3, and IRF3 signals in the three independent experiments were quantified by ImageJ and averages of the signals were shown in graphs. b Wild-type (Nedd4l+/+) and Nedd4l-deficient (Nedd4l−/−) peritoneal macrophages were infected with VSV (MOI = 10) for indicated time, followed by immunoprecipitation (IP) with anti-TRAF3 and immunoblot (IB) analysis with antibodies specific for TBK1, cIAP1/2, HECTD3, TRAF3, Nedd4l, and GAPDH (WCL: whole-cell lysates). c IB of Myc-TBK1, Flag-TRAF3, and Myc-Nedd4l co-immunoprecipitated with anti-Flag from lysates of 293T cells transfected with indicated plasmids. d IB of HA-MAVS, Flag-TRAF3, and Myc-Nedd4l co-immunoprecipitated with anti-Flag from lysates of 293T cells transfected with indicated plasmids. Results in (a–d) are representative of three independent experiments.

TRAF3 not only functions in RLR-mediated type I interferon production, but also functions in TLR-mediated type I interferon production in macrophages. Similar as in RLR signaling, Nedd4l deficiency inhibited LPS-induced phosphorylation of TBK1 and IRF3 without visibly affecting LPS-induced MAPK and NF-κB phosphorylation (Supplementary Fig. 3a). TRAF3 regulates c-Rel expression in LPS-stimulated macrophages21. Nedd4l deficiency decreased c-Rel expression in LPS-stimulated macrophages (Supplementary Fig. 3a), whereas Nedd4l deficiency did not obviously affect c-Rel expression in VSV-infected macrophages (Supplementary Fig. 3b).

Nedd4l directly interacts with TRAF3

To illuminate the mechanism by which Nedd4l promotes the interaction between TRAF3 and TBK1, we investigated whether Nedd4l interacts with MAVS, TRAF3, or TBK1. Nedd4l-expressing plasmid was transfected into 293T cells together with plasmids expressing MAVS, TRAF3, or TBK1. Among the three molecules, only TRAF3 was co-immunoprecipitated with Nedd4l (Fig. 4a). Interaction between endogenous TRAF3 and Nedd4l was also detectable in macrophages following virus infection (Fig. 4b). Furthermore, recombinant Nedd4l bound to glutathione S-transferase (GST)-fused TRAF3 in pull-down experiment (Fig. 4c), demonstrating direct interaction between Nedd4l and TRAF3. Mapping the domain that mediated the binding of Nedd4l to TRAF3, a series of truncated Nedd4l mutants were constructed (Supplementary Fig. 4). Deletion of the C2 or HECT domain in Nedd4l did not affect the interaction between Nedd4l and TRAF3, however, deletion of both C2 and WW domain in Nedd4l disrupted the association between Nedd4l and TRAF3, suggesting that Nedd4l bound TRAF3 through its WW domain (Fig. 4d).

Fig. 4: Nedd4l interacts with TRAF3.

a IB of HA-MAVS, HA-TBK1, HA-TRAF3, and Myc-Nedd4l co-immunoprecipitated with anti-Myc from lysates of 293T cells transfected with indicated plasmids with HA-tag, Myc-tag, or GAPDH antibodies. b IB of TRAF3 and Nedd4l co-immunoprecipitated with anti-TRAF3 from lysates of C57BL/6 mice peritoneal macrophages infected with VSV (MOI = 10) for indicated time. c In vitro GST-pull-down assay of Nedd4l with GST or GST-fused TRAF3. d IB of TRAF3 and Nedd4l co-immunoprecipitated with Myc-tag-specific antibody from lysates of 293T cells co-transfected with plasmids expressing Flag-TRAF3 and Myc-tagged wild-type Nedd4l or mutant Nedd4l with CW (∆CW),C2 (∆C2), or HECT (∆HECT) domain deleted. Results in (a–d) are representative of three independent experiments.

Nedd4l mediates K29-linked ubiquitination of TRAF3

Ubiquitination of TRAF3 is required for virus-induced TRAF3/TBK1 complex formation. Since Nedd4l interacts with TRAF3, we supposed that Nedd4l increases TRAF3/TBK1 complex formation by promoting TRAF3 ubiquitination. As shown in Fig. 5a, Nedd4l deficiency decreased VSV infection-induced TRAF3 ubiquitination in macrophages. By using antibodies specific to K48- and K63-linked ubiquitin chains, we found that Nedd4l deficiency decreased both K48- and K63-linked ubiquitination of TRAF3. Nedd4l deficiency also reduced LPS-induced TRAF3 ubiquitination, including K48- and K63-linked ubiquitination in macrophages (Supplementary Fig. 5a). Consistently, Nedd4l overexpression increased TRAF3 ubiquitination, including K48- and K63-linked ubiquitination in VSV-infected 293T cells (Fig. 5b). However, Nedd4l deficiency did not reduce LPS-induced TRAF2 ubiquitination (Supplementary Fig. 5b). In vitro ubiquitin ligase activity assay confirmed the ability of Nedd4l to enhance TRAF3 ubiquitination (Fig. 5c). The ability of Nedd4l to increase ubiquitination of TRAF3 was dependent on the ligase activity of Nedd4l, since mutation of C942A or deletion of HECT domain of Nedd4l, both of which disrupted ligase activity of Nedd4l, eliminated Nedd4l-induced increase of TRAF3 ubiquitination (Fig. 5d). On the other hand, deletion of C2 domain increased Nedd4l-mediated ubiquitination, consistently with previous report that C2 domain inhibited Nedd4l ligase activity22. A series of HA-tagged Ub mutants (K11R, K29R, K33R, K48R, K63R), in which one of the seven lysine residues was mutated, were used to identify the lysine residue in ubiquitin that was required for Nedd4l-catalyzed polymeric Ub chain formation. As shown in Fig. 5e, only the mutation of K29 disrupted Nedd4l-mediated TRAF3 ubiquitination. Another series of Ub mutants, in which only one of the seven lysine residues was reserved and the other lysine residues were mutated, were also used to characterize the type of Nedd4l-catalyzed poly-ubiquitin chain. Consistent with the above observation, there was only one mutant, Ub(K29 only), which could be linked to TRAF3 as efficiently as wild-type ubiquitin (Fig. 5f), demonstrating that Nedd4l directly catalyzed K29-linked TRAF3 ubiquitination rather than directly catalyzing K48- or K63-linked TRAF3 ubiquitination. We hypothesize that Nedd4l-catalyzed K29-linked ubiquitination might facilitate K48- and K63-linked self-ubiquitination of TRAF3 or ubiquitination of TRAF3 mediated by other E3 ligases.

Fig. 5: Nedd4l-catalyzed K29-linked ubiquitination of TRAF3.

a IB of total ubiquitination, K48- and K63-linked ubiquitination of TRAF3 in VSV-infected wild-type (Nedd4l+/+) and Nedd4l-deficient (Nedd4l−/−) peritoneal macrophages. b Plasmids expressing Flag-TRAF3, HA-Ub, and Myc-Nedd4l were co-transfected in 293T cells. After 24 h, cells were infected with VSV (MOI = 10) for 12 h. IB analysis of total ubiquitination, K48- and K63-linked ubiquitination of Flag-tagged TRAF3 with indicated antibodies. c IB analysis of TRAF3 incubated with Nedd4l recombinant proteins and ubiquitination reaction components in vitro. d IB analysis of total ubiquitination of Flag-tagged TRAF3 in 293T cells co-transfected with plasmids expressing Flag-TRAF3, HA-Ub, and Myc-tagged wild-type Nedd4l or mutant Nedd4l with indicated antibodies. e IB analysis of ubiquitination of Flag-TRAF3 in 293T cells co-transfected with plasmids expressing Flag-TRAF3, Myc-Nedd4l, and HA-tagged wild-type Ub or HA-tagged mutant Ub (K11R, K29R, K33R, K48R, and K63R) with indicated antibodies. f IB analysis of ubiquitination of Flag-TRAF3 in 293T cells co-transfected with plasmids expressing Flag-TRAF3, Myc-Nedd4l, and HA-tagged wild-type Ub or HA-tagged mutant Ub (K6 only, K11 only, K27 only, K29 only, K33 only, K48 only, K63 only, Ub-K null) with indicated antibodies. Results in (a–f) are representative of three independent experiments.

Nedd4l ubiquitinates cysteine residues in TRAF3

We tried to identify Nedd4l-catalyzed ubiquitination sites in TRAF3. TRAF3 plasmid was transfected into 293T cells with or without Nedd4l plasmid, and then TRAF3 was immunoprecipitated for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The LC-MS/MS data suggested that Nedd4l overexpression increased ubiquitination at K273 and K315 lysine residues (Supplementary Fig. 6a, b). We mutated these candidate ubiquitination sites, transfected the mutants into 293T cells together with Nedd4l and mutant HA-tagged Ub (K29 only). K50 residue was also mutated as a negative control. As shown in Fig. 6a, mutation of K50, K273, and K315 did not affect K29-linked ubiquitination of TRAF3. Interestingly, LC-MS/MS analysis showed that ubiquitination at two cysteine residues, C56 and C124, also increased in Nedd4l overexpressing cells (Supplementary Fig. 6c, d). TRAF3 contains six zinc fingers, two of them (RZ1 and RZ2) are located in the RING (really interesting gene) domain, the other four (Z1, Z2, Z3, and Z4) are located between the RING domain and C-terminal TRAF domain (Supplementary Fig. 6e). C56, together with C53, C73, and C76, forms a zinc finger in the N-terminal RING domain (RZ1). Meanwhile, C124, together with C117, H136, and C141, forms a zinc finger (Z1) between the RING domain and C-terminal TRAF domain (Supplementary Fig. 6e). We constructed mutant TRAF3(C56R) and TRAF3(C124R) plasmids. Surprisingly, mutation of C56 and C124 dramatically decreased Nedd4l-catalyzed K29-linked ubiquitination of TRAF3 (Fig. 6a), suggesting that the C56 and C124 residues were the ubiquitination sites to which Nedd4l-catalyzed k29-linked ubiquitin chains were attached. However, when the TRAF3 mutants were transfected with Nedd4l and wild-type HA-tagged ubiquitin (HA-Ub), mutation of K273 and K315 slightly decreased TRAF3 ubiquitination, but mutation of C56 and C124 remarkably increased TRAF3 ubiquitination, including K48- and K63-linked ubiquitination (Fig. 6b). To illuminate the mechanism underlying the phenomenon, we observed the effects of C56 and C124 mutation on TRAF3 ubiquitination without Nedd4l overexpression. Interestingly, mutation of C56 or C124 increased K48- and K63-linked ubiquitination of TRAF3 even in the absence of Nedd4l overexpression (Fig. 6c). Since C56 and C124 are residues that constitute RZ1 and Z1 zinc fingers, we suppose that C56 and C124 mutation might increase K48- and K63-linked ubiquitination of TRAF3 via affecting the formation of the zinc fingers. To further investigate the role of RZ1 and Z1 zinc fingers in the regulation of TRAF3 ubiquitination, we mutated the other residues (C53R, C73R, C76R, C117R, H136R, and C141R) which formed RZ1 and Z1 zinc fingers together with C56 and C124. Mutating each of the residues significantly increased TRAF3 ubiquitination (Fig. 6d), suggesting that modulation or disruption of RZ1 and Z1 zinc fingers increases TRAF3 ubiquitination.

Fig. 6: Nedd4l ubiquitinates cysteine residues in TRAF3.

a IB analysis of ubiquitination of Flag-tagged TRAF3 in 293T cells co-transfected with plasmids expressing Myc-Nedd4l, HA-tagged mutant Ub (K29 only), and Flag-tagged wild-type TRAF3 or mutant TRAF3 (K50R, K273R, K315R, C56R, or C124R). b IB analysis of total ubiquitination, K48- and K63-linked ubiquitination of Flag-tagged TRAF3 in 293T cells co-transfected with plasmids expressing Myc-Nedd4l, HA-tagged wild-type Ub, and Flag-tagged wild-type TRAF3 or mutant TRAF3 (K50R, K273R, K315R, C56R, or C124R). c IB analysis of total ubiquitination, K48- and K63-linked ubiquitination of Flag-tagged TRAF3 in 293T cells co-transfected with plasmids expressing HA-tagged wild-type Ub and Flag-tagged TRAF3, TRAF3 (C56R), TRAF3 (C124R), TRAF3(C56R/C68A/H70A), or TRAF3(C124R/C68A/H70A). d IB analysis of total ubiquitination, K48- and K63-linked ubiquitination of Flag-tagged TRAF3 in 293T cells co-transfected with plasmids expressing HA-tagged wild-type Ub and Flag-tagged wild-type TRAF3 or mutant TRAF3 (C56R, C124R, C53R, C73R, C76R, C117R, H136R, C141R). Results in (a–d) are representative of three independent experiments.

TRAF3 is an E3 ligase and proposed to be capable of self-ubiquitination. Mutation of C68A/H70A inactivates the ligase activity of TRAF312. To examine whether self-ubiquitination was responsible for the increased ubiquitination in TRAF3(C56R) and TRAF3(C124R), we introduced C68A/H70A mutation to construct ligase activity dead TRAF3(C56R) and TRAF3(C124R). As shown in Fig. 6c, catalytic dead mutant TRAF3(C56R/C68A/H70A) and TRAF3(C124R/C68A/H70A) were similarly ubiquitinated as TRAF3(C56R) and TRAF3(C124R), suggesting that self-ubiquitination was not the major cause for the increased TRAF3(C56R) and TRAF3(C124R) ubiquitination.

Nedd4l promotes TRAF3 to interact with cIAP1/2 and HECTD3

TRAF3 is ubiquitinated by other E3 ligases such as cIAP1/2 and HECTD312,18. We examined whether Nedd4l affected the interaction between TRAF3 and these E3 ligases. As shown in Fig. 3b, Nedd4l deficiency decreased VSV infection-induced TRAF3-cIAP1/2 and TRAF3-HECTD3 complex formation in macrophages. Consistently, in 293T cells, overexpression of Nedd4l promoted TRAF3 to associate with cIAP1/2 and HECTD3 (Fig. 7a, b). Similar to the results in ubiquitination assay, mutation of C56 also increased the interaction of TRAF3 with cIAP1/2 and HECTD3 (Fig. 7c, d). The effects of C56 and C124 mutation on TRAF3-mediated signaling were examined. When overexpressed in HEK293T cells, mutant TRAF3(C56R) and TRAF3(C124R) more efficiently recruited TBK1 to form TRAF3/TBK1 complex compared with wild-type TRAF3 (Supplementary Fig. 7a). We co-transfected wild-type TRAF3, mutant TRAF3(C56R), or TRAF3(C124R) together with NF-κB or IRF3 luciferase reporter gene into HEK293 cells. Compared with wild-type TRAF3, mutant TRAF3(C56R) and TRAF3(C124R) increased NF-κB and IRF3 luciferase reporter gene expression in a dose-dependent manner (Supplementary Fig. 7b, c). Consistently, mutation of C56 and C124 increased VSV-induced IFN-β, IL-6, and TNF-α mRNA expression (Fig. 7e). C61 is near to C56. However, mutation of C61 did not affect VSV-induced IFN-β, IL-6, and TNF-α mRNA expression (Fig. 7e). TRAF3, TRAF3(C56R), and TRAF3(C124R) plasmids were also transfected into TRAF3-deficient HEK293T cells. The doses of the transfected plasmids were optimized so that expression level of TRAF3, TRAF3(C56R), and TRAF3(C124R) in the cells was comparable with that of endogenous TRAF3 in wild-type parent HEK293T cells (Supplementary Fig. 7d). In these cells, mutation of C56 and C124 also increased VSV-induced IFN-β mRNA expression (Supplementary Fig. 7e), providing further evidence that C56 and C124 residues are important in regulating virus-induced innate immunity.

Fig. 7: Nedd4l promotes TRAF3 to associate with cIAP1/2 and HECTD3.

a IB of Flag-cIAP1, Flag-cIAP2, Myc-Nedd4l, and HA-TRAF3 co-immunoprecipitated with anti-HA from lysates of 293T cells transfected with indicated plasmids. b IB of Flag-HECTD3, Myc-Nedd4l and HA-TRAF3 co-immunoprecipitated with anti-HA from lysates of 293T cells transfected with indicated plasmids. c IB of Flag-cIAP1, Flag-TRAF3, and Flag-TRAF3(C56R) co-immunoprecipitated with anti-TRAF3 from lysates of 293T cells transfected with indicated plasmids. d IB of Flag-HECTD3, Flag-TRAF3 and Flag-TRAF3(C56R) co-immunoprecipitated with anti-TRAF3 from lysates of 293T cells transfected with indicated plasmids. e RT-qPCR analysis of IFN-β, IL-6, and TNF-α mRNA expression in 293 cells transfected with plasmids expressing TRAF3 or mutant TRAF3 and then infected with VSV (MOI = 10) for 12 h. Data are presented as mean ± SD (n = 3 per group) and p-values by two-tailed unpaired Student’s t-test are indicated in e. Results in (a–e) are representative of three independent experiments.