TRIM31 is recruited to mitochondria after infection with SeV.

Transcription

1 Supplementary Figure 1 TRIM31 is recruited to mitochondria after infection with SeV. (a) Confocal microscopy of TRIM31-GFP transfected into HEK293T cells for 24 h followed with SeV infection for 6 h. MitoTracker (red) were used to label mitochondria. Scale bar 10 μm. (b) Immunoblot of TRIM31 protein and TOMM20 protein in the crude mitochondrial extracts (upper) and TRIM31 protein and β-actin in the whole cell lysate (lower) prepared from peritoneal macrophages infected with SeV for indicated times. (c) Immunoblot of TRIM31 protein in mouse peritoneal macrophages infected with SeV for indicated times. (d) qpcr analysis of Trim31, Trim26, Trim30α, Ifnb1 expression in primary peritoneal macrophages transfected with mice TRIM31 sirna, TRIM26 sirna, TRIM30α sirna or control sirna for 48 h, followed with SeV infection for indicated times. mrna results are presented relative to those of untreated cells transfected with control sirna. (e) qpcr analysis of TRIM31 and IFNB1 mrna in THP-1 cells transfected with human TRIM31 sirna or control sirna for 48 h, followed with SeV infection for indicated times. mrna results are presented as in d. (f) qpcr analysis of IFNB1 mrna in Hela cells transfected with Flag-TRIM31 expression plasmid or control vector for 24 h, followed with SeV infection or transfection with poly(i:c) for the indicated times. mrna results are presented relative to those of untreated cells transfected with control plasmid. The data are representative of 3 independent experiments with three biological replicates (means ± S.D. in d-f). *p < (two-tailed Student s t-test).

2 Supplementary Figure 2 TRIM31 deficiency impairs RNA-virus-induced production of type I interferon and antiviral responses. (a) Schematic diagram of the deletions of the Trim31 deficient mice generated by transcription activation-like effector nuclease (TALEN) technology. The deletions were confirmed by DNA sequencing analysis of genomic DNA isolated from Trim31 +/+ and Trim31 / mice tails. (b) Immunoblot of TRIM31 protein in Trim31 +/+ and Trim31 / peritoneal macrophages. (c) qpcr analysis of Ifnb1, Ccl5 and Cxcl10 mrna in the macrophages prepared form Trim31 / (G) mice or Trim31 / (5) mice infected with SeV for indicated times. mrna results are presented relative to those of untreated wild-type cells. (d,e) qpcr analysis of Ifnb1, Ccl5 and Cxcl10 mrna in bone marrow-derived macrophages (BMDMs) (d) or MEFs (e) prepared from Trim31 +/+ and Trim31 / mice followed with infection of SeV and HSV-1 or stimulation of 5 ppp-rna, LPS and poly(i:c) for indicated times. mrna results are presented as in c. The data are representative of 3 independent experiments with three biological replicates (means ± S.D. in c-e). *p < 0.05 and **p < (two-tailed Student s t-test).

3 Supplementary Figure 3 Trim31-deficient mice are not impaired in their response to infection with a DNA virus. (a) qpcr analyses of Ifnb1 mrna in brains and ELISA analysis of IFN-β production in sera of Trim31 +/+ and Trim31 / mice intraperitoneally infected with HSV-1 ( PFU/mouse)for 24 h. mrna results are presented relative to those of untreated wild-type cells. (b) qpcr analysis of HSV-1 genomic DNA and plaque assays of HSV-1 titer in brains of Trim31 +/+ and Trim31 / mice as treated in (a). (c) Survival of Trim31 +/+ and Trim31 / mice (n=7) after intraperitoneally injection of HSV-1 ( pfu/mouse). The data are representative of 3 independent experiments with three biological replicates (means ± S.D. in a, b). *p < 0.01 (two-tailed Student s t-test(a,b) or two-way analysis of variance (ANOVA) (c)).

4 Supplementary Figure 4 TRIM31 targets MAVS. (a) Luciferase activity in HEK293T cells transfected with IFNB1 luciferase reporter and expression plasmids for RIG-IN, MDA5, MAVS, TBK1, IKKε and IRF3 5D together with Flag-TRIM31 expression plasmid for 24 h. (b) Luciferase activity in HEK293T cells transfected with control sirna or TRIM31 sirna and IFNB1 luciferase reporter or ISRE luciferase reporter together with expression plasmids for RIG-IN, MAVS and TBK1 for 24 h. (c) Coimmunoprecipitation analysis of Flag-TRIM31 interaction with Myc-IRF3 or Myc-STING in HEK293T cells. The data are representative of 3 independent experiments with three biological replicates (means ± S.D. in a, b). *p < (two-tailed Student s t-test).

5 Supplementary Figure 5 TRIM31 catalyzes K63-linked polyubiquitination of MAVS. (a) Coimmunoprecipitation analysis of RIG-I ubiquitination in HEK293T cells transfected with Myc-RIG-I and HA- Ubiquitin together with control plasmid, Flag-TRIM31 plasmid or Flag-TRIM25 plasmid. (b) Luciferase activity in HEK293T cells transfected with IFNB1 luciferase reporter or ISRE luciferase reporter together with Flag-TRIM31-WT plasmid or Flag-TRIM31-C53/56A plasmid for 24 h followed with SeV infection for indicated times. (c) qpcr analysis of IFNB1 mrna and VSV RNA in HEK293T cells transfected with control plasmid, Flag-TRIM31-WT plasmid or Flag- TRIM31-C53/56A plasmid for 24 h followed with VSV infection (MOI, 0.1) for indicated times. mrna results are presented relative to those of untreated cells transfected with control plasmid. (d) Immunoblot of phosphorylated and total IRF3 protein in lysates of HEK293T cells transfected with Flag-TRIM31-WT or Flag-TRIM31-C53/56A plasmid for 24 h followed with SeV infection for indicated times. The data are representative of 3 independent experiments with three biological replicates (means ± S.D. in b, c). *p < (two-tailed Student s t-test).

6 Supplementary Figure 6 TRIM31 promotes the formation of MAVS aggregates. (a) SDD-AGE analysis of MAVS aggregation in Trim31 +/+ and Trim31 / MEFs infected with SeV for indicated times (upper). SDS-PAGE analysis of MAVS protein and TRIM31 protein in the whole cell lysates prepared above (lower). (b) SDD-AGE analysis of MAVS aggregation in HEK293T cells transfected with Flag-TRIM31 expression plasmid and Myc-MAVS for 24 h. (c) Immunoblot of TRIM31 protein in Trim31 / MEFs reconstituted with mtrim31- WT or mtrim31-c52/55a. TRIM31 +/+ MEFs were used as controls. qpcr analysis of Ifnb1, Ccl5 and Cxcl10 mrna in TRIM31 +/+ or Trim31 / MEFs reconstituted with mtrim31-wt or mtrim31-c52/55a followed with SeV infection for 12 h. mrna results are presented relative to those of untreated wide-type cells transfected with control plasmid. (d) qpcr analysis of Ifnb1 mrna in Mavs / MEFs transfected with MAVS expression plasmid or control plasmid for 24 h followed with SeV infection for 12 h. mrna results are presented relative to those of untreated cells transfected with control plasmid. (e) Luciferase activity in Mavs / MEFs transfected with IFN-β or ISRE luciferase reporter and

7 expression plasmids for MAVS-WT, MAVS-K10R, MAVS-K10/311R, MAVS-K10/311/461R along with Flag-TRIM31 expression plasmid for 24 h followed with SeV infection for 12 h. (f) qpcr analysis of Ifnb1 mrna and SDD-AGE analysis of MAVS aggregation in Mavs / MEFs transfected with MAVS-WT, MAVS-K10R, MAVS-K311R and MAVS-K461R along with Flag-TRIM31 expression plasmid or control plasmid for 24 h followed with SeV infection for 12 h. (g) qpcr analysis of Ifnb1 mrna and SDD-AGE analysis of MAVS aggregation in Mavs / MEFs transfected with MAVS-WT or MAVS-W56R along with Flag-TRIM31 expression plasmid or control plasmid for 24 h followed with SeV infection for 6 h. mrna results are presented as in d. The data are representative of 3 independent experiments with three biological replicates (means ± S.D. in c-g). *p < (two-tailed Student s t-test).

8 Supplementary Table 1 Details of antibodies used. Antibody Clone Distributor Anti-HA sc-7392 Santa Cruz Biotechnology Anti-Ub sc-8017 Santa Cruz Biotechnology Anti-Actin sc Santa Cruz Biotechnology Anti-MAVS PRS4053 Sigma-Aldrich Anti-TRIM31 AV34717 Sigma-Aldrich Anti-Flag F1804 Sigma-Aldrich Anti-VSV-G V5507 Sigma-Aldrich Anti-Myc #2276 Cell Signaling Technology Anti-TBK1 #3504 Cell Signaling Technology Anti-phospho-TBK1 #5483 Cell Signaling Technology Anti-IRF3 #4302 Cell Signaling Technology Anti-phospho-IRF3 #29047 Cell Signaling Technology Anti-PCNA #13110 Cell Signaling Technology Anti-ubiquitin K63 #5621 Cell Signaling Technology Anti-ubiquitin K48 #8081 Cell Signaling Technology Anti-TOMM20 ab Abcam

9 Supplementary Table 2 Primers for RNA Quantification, Related to Experimental Procedures Gene Forward primer (5'-3') Reverse primer (5'-3') Trim31 AACAGCCTGAATAAAGAAG CACAGTGGAAAGGGTAGA Trim26 AGACGAGGAGGTTGGCTAT CTGGGACTCTGCATTGGT Trim30α GCAACTGCCCTGTATG CTTCTGCTCCTCCTCTG Ifnb1 AGTTACACTGCCTTTGCC GTTGAGGACATCTCCCAC Ccl5 CCTCACCATATGGCTCGGAC TCTTCTCTGGGTTGGCACAC Cxcl10 CCAAGTGCTGCCGTCATTTT GATAGGCTCGCAGGGATGAT Actin CCACACCCGCCACCAGTTCG TACAGCCCGGGGAGCATCGT IFNB1 CAACAAGTGTCTCCTCCAAAT TCTCCTCAGGGATGTCAAAG ACTIN GGAAATCGTGCGTGACATTAA AGGAAGGAAGGCTGGAAGAG

10 Supplementary Table 2 Primers for RNA Quantification, Related to Experimental Procedures Gene Forward primer (5'-3') Reverse primer (5'-3') Trim31 AACAGCCTGAATAAAGAAG CACAGTGGAAAGGGTAGA Trim26 AGACGAGGAGGTTGGCTAT CTGGGACTCTGCATTGGT Trim30α GCAACTGCCCTGTATG CTTCTGCTCCTCCTCTG Ifnb1 AGTTACACTGCCTTTGCC GTTGAGGACATCTCCCAC Ccl5 CCTCACCATATGGCTCGGAC TCTTCTCTGGGTTGGCACAC Cxcl10 CCAAGTGCTGCCGTCATTTT GATAGGCTCGCAGGGATGAT Actin CCACACCCGCCACCAGTTCG TACAGCCCGGGGAGCATCGT IFNB1 CAACAAGTGTCTCCTCCAAAT TCTCCTCAGGGATGTCAAAG ACTIN GGAAATCGTGCGTGACATTAA AGGAAGGAAGGCTGGAAGAG