Fig 2: TRIM25 blocks TRAF6-mediated ubiquitination of EZH2.A Endogenous interaction between TRIM25 and EZH2 was determined using co-immunoprecipitation with anti-TRIM25 or anti-EZH2 antibodies in SW480 cells. B Exogenous interaction between TRIM25 and EZH2 was determined using co-immunoprecipitation with anti-Flag or anti-HA antibodies in HEK293T cells co-transfected with Flag-EZH2 and HA-TRIM25. C Immunofluorescence images showing colocalization of endogenous EZH2 and TRIM25 in SW480 cells. D Flag-EZH2, siTRIM25, His-tagged ubiquitin wild-type (WT) or mutation plasmids (K48 or K63 mutants) were transfected into HEK293T cells as the indicated combinations. Immunoprecipitation with anti-Flag antibodies was performed to detect the ubiquitination level of EZH2. E Immunoprecipitation assay using IgG and anti-EZH2 antibodies in SW480 cells transfected with siNC or siTRIM25, followed by western blotting analysis with the indicated antibodies. WCE: Whole-cell extracts. F Flag-EZH2, siTRIM25, siTRAF6, His-tagged ubiquitin wild-type (WT) were transfected into HEK293T cells as the indicated combinations. Immunoprecipitation with anti-Flag antibodies and western blotting with anti-His antibodies were performed to detect the ubiquitination level of EZH2 under different conditions. G Western blotting analysis of EZH2 in SW480 cells transfected with siTRIM25 and/or siTRAF6.

Fig 3: TRAF6 KD reduces NF-κB and p38 activation in SAMHD1 KO cells. (A) Stable THP-1 control or SAMHD1 KO cells lines with either control or TRAF6 KD were cultured in standard media and mock treated or stimulated with IL-1β. Cells were harvested 10 min poststimulation and lysed for immunoblotting. Relative TRAF6 protein levels were calculated by densitometry analysis. The TRAF6 signal was normalized to GAPDH. Untreated control cells were set as 1. (B) Samples for RT-qPCR and mRNA analysis were collected 2 h poststimulation. Data represent triplicate samples, and error bars depict SEM. Statistical analysis was performed by two-way ANOVA. (C) THP-1 control or SAMHD1 KO cells lines with either control or TRAF6 KD were cultured in standard media and mock treated or stimulated with LPS. After 6 h, cells were harvested and lysed for immunoblotting. Relative levels of phosphorylated p38 (p-p38) were calculated by densitometry analysis. The p-p38 signal was normalized to total p38 protein and GAPDH. Mock-treated, unstimulated control cells were set as 1. (D) TNF-α mRNA levels were quantified by RT-qPCR and normalized to spliced GAPDH. The graph depicts data derived from triplicate samples, and error bars represent SEM. Statistical analysis was performed by two-way ANOVA with Tukey’s multiple-comparison posttest. ****, P ≤ 0.0001.

Fig 4: NEMO and TAK1 were necessary for NSP6- and ORF7a-mediated NF-κB activation. (A) Scheme of canonical NF-κB pathway. (B to D) HEK293T-TAK1-KO (B), HEK293T-NEMO-KO (C), and HEK293T-TRAF2-KO, HEK293T-TRAF6-KO, and HEK293T-RIP2-KO (D) cells were transfected with pcDNA3.1-HA, pcDNA3.1-HA-NSP6, or pcDNA3.1-HA-ORF7a, along with pGL4-NF-κB-LucP2 and pNull-RLuc. The level of luciferase activity was determined at 24 h posttransfection. The firefly luciferase activity was normalized to Renilla luciferase activity. An empty plasmid was used as a control and set to 1. Results are shown as the mean ± SD of three independent experiments. *, P < 0.05; **, P < 0.01 (Student’s t test). (E) HEK293T cells were transfected with pcDNA3.1-HA, pcDNA3.1-HA-NSP6, or pcDNA3.1-HA-ORF7a. At 48 h after transfection, IP was performed by anti-HA antibody. The immunocomplex was analyzed by Western blotting with the indicated antibodies.

Fig 5: Anti-miR-146a rescues TRAF6, IRAK1, IRAK2 genes. CHME3 cells were transfected with 100 pmol anti-miR-146a. Cy3-labeled negative control and mock transfected cells were used as control. After 24 h of transfection, JEV infection was given (MOI 5). For better comparison, we also used a JEV-infected group untransfected with miR-146a mimic harvested after 24 h post infection. (A) Western blots showing upregulation of TRAF6, IRAK1, and IRAK2 genes upon silencing miR-146a. Anti-miR-146a also upregulated TRAF6, IRAK1, and IRAK2 genes after JEV infection. (B) Densitometry plot showing upregulation of TRAF6, IRAK1, and IRAK2 genes by anti-miR-146a w.r.t. JEV infection. The image density was normalized by β-tubulin. For statistical analysis, the protein levels of JEV-infected samples were compared to anti-miR-146a and anti-miR-146a + JEV samples. The fold change is significant where *denotes P < 0.05, **denotes P < 0.005, ***denotes P < 0.001. (C) Taqman RT-PCR analysis of anti-miR-146a-transfected samples to confirm silencing of miR-146a. No silencing was observed in Cy3-labeled negative control. Cy3-labeled control was used as control for t-test. (D) Graph bars showing reduced luciferase activity upon miR-146a overexpression. JEV infection after 24 h post miR-146a mimic transfection further reduced NF-κB activity. β-galactosidase activity was used to normalize luciferase activity. miR-146a was overexpressed, JEV was infected, and miR-146a + JEV samples were compared to scrambled control for statistical analysis. (E) Graph showing increased luciferase activity of NF-κB luciferase vector upon silencing of miR-146a by anti-miR-146a. Anti-miR-146a treatment 24 h prior to JEV infection also increased NF-κB luciferase activity. JEV-infected group was used for comparison for statistical analysis. All experiments were repeated thrice and are represented as mean ± SE. The fold change is significant where *denotes P < 0.05, **denotes P < 0.005, and ***denotes P < 0.001.