SUPPLEMENTARY INFORMATION

Transcription

1 doi: /nature05732 SUPPLEMENTARY INFORMATION Supplemental Data Supplement Figure Legends Figure S1. RIG-I 2CARD undergo robust ubiquitination a, (top) At 48 h posttransfection with a GST, GST-RIG-I-2CARD (1-200 aa) or GST-MDA5 2CARD (1-220 aa) fusion construct, HEK293T cells were used for GST pulldown, and purified proteins were separated by SDS-PAGE followed by IB with anti-ubiquitin (αub). Arrows indicate the ubiquitinated bands. (bottom) WCLs were used for IB with αgst. b, At 48 h posttransfection with vector or Flag-tagged RIG-I with or without HA-tagged ubiquitin, HEK293T WCLs were used for IP with αflag followed by IB with αha or αflag. WCLs were used for IB with αha. Arrow indicates an ubiquitinated protein. c, HA-ubiquitin and actin expression. At 39 h posttransfection with Flag-RIG-I and HA-Ub, HEK293T cells were mock infected or infected with SeV at 25 HA unit/ml. At 9 h postinfection, WCL were used for IB with αha and αactin antibodies. Figure S2. Lys63 -linked ubiquitination of GST-RIG-I-2CARD and full length RIG-I. At 48 h posttransfection with GST-RIG-I-2CARD (a) or Flag-tagged full length RIG-I (b) together with HA-tagged wt, K 48 R or K 63 R Ub mutant, HEK293T WCLs were used for GST-PD (a) or IP with αflag antibody (b), followed by IB with αha (a and b), αgst (a) and αflag (b) antibodies. WCLs were used for IB with αha to show HA-Ub expression and αactin antibody for loading control. 1

2 doi: /nature05732 SUPPLEMENTARY INFORMATION Figure S3. K 172 R mutation abolishes RIG-I ubiquitination and signaling activity. a, At 48 h posttransfection with GST-RIG-I-2CARD wt or its K R mutants, HEK293T WCLs were used for GST pull down (GST-PD) followed by IB with αgst (top) or αub (bottom). Because of weak ubiquitination, the ubiquitinated GST-RIG-I-2CARD was detected by IB with αgst for a longer exposure than the unubiquitinated GST-RIG-I-2CARD. Arrows indicate the ubiquitinated bands. b, At 48 h posttransfection with GST-RIG-I-2CARD wt or its K R mutants together with IFN-β luciferase or NF-κB luciferase and constitutive β-gal-expressing pgk-β-gal, HEK293T cells were harvested and luciferase and β-galactosidase values were determined. Luciferase values were normalised to β-galactosidase activity for transfection efficiency control. IFN-β promoter or NF-κB promoter luciferase activity is presented as fold induction. Figure S4. a, TRIM25 SPRY expression suppresses the ubiquitination of endogenous RIG- I. At 24 h posttransfection with vector, V5-TRIM25 or V5-TRIM25 SPRY together with HA- Ub, HEK293T cells were treated with IFN-β (1000U/ml) for 24 h. WCLs were used for IP with αrig-i followed by IB with αha or αrig-i. WCLs were used for IB with αha, αv5 or αactin. b, TRIM25 induces the ubiquitination of GST-RIG-I-2CARD but not GST-MDA5-2CARD. At 48 h posttransfection with GST-RIG-I-2CARD or GST-MDA5-2CARD together with vector, TRIM25 and TRIM25ΔRing, HEK293T WCLs were used for GST-PD, followed by IB with αgst antibody. WCLs were used for IB with αv5 antibody to show TRIM25 and TRIM25ΔRing expression. 2

3 doi: /nature05732 SUPPLEMENTARY INFORMATION Figure S5. a, TRIM25 shrna significantly suppresses the ubiquitination of Flag-RIG-I. At 48 h posttransfection with Flag-RIG-I and HA-ubiquitin together with empty retroviral psuper vector or an increasing amount of TRIM25 shrna specific retroviral psuper vector, HEK293T WCLs were used for IP with αflag followed by IB with αha or αflag. WCLs were used for IB with αtrim25 or αactin to show their expression. Arrows indicate the ubiquitinated forms of Flag-RIG-I. b, Non-specific scrambled shrna does not affect the ubiquitination of GST-RIG-I-2CARD. At 48 h posttransfection with GST-RIG-I-2CARD together with empty retroviral psuper vector or increasing amount of non-silencing shrna retroviral psuper vector, HEK293T WCLs were used for GST-PD, followed by IB with αgst. WCL were used for IB with αtrim25 and αactin antibodies to show their expression. Figure S6. a, TRIM25 catalyses the in vitro ubiquitination of RIG-I-2CARD. MBP-T7-RIG- I-2CARD, MBP-T7-RIG-I-2CARD(170stop), and MBP-T7 were subjected to in vitro ubiquitination assay and to immunoblotting with αt7 antibody. Arrows indicate the ubiquitinated forms of MBP-T7-RIG-I-2CARD. b, TRIM25 enhances RIG-I-2CARD ability to induce NF-κB promoter activity. At 48 h posttransfection with GST or GST-RIG-I-2CARD and NF-κB luciferase together with an increasing amount of TRIM25 expression vector, HEK293T cells were harvested and luciferase values were determined. pgk-β-gal was also included for transfection efficiency control. NF-κB promoter luciferase activities are presented as fold induction. Figure S7. TRIM25 SPRY expression suppresses RIG-I 2CARD signaling ability. 3

4 doi: /nature05732 SUPPLEMENTARY INFORMATION At 48 h posttransfection with GST or GST-RIG-I-2CARD and IFN-β luciferase or NF-κB luciferase together with an increasing amount of TRIM25 SPRY expression vector, HEK293T cells were harvested and luciferase values were determined. pgk-β-gal was also included for transfection efficiency control. IFN-β promoter and NF-κB promoter luciferase activities are presented as fold induction. Figure S8. K 172 R mutation significantly decreases full-length RIG-I ubiquitination. At 48 h posttransfection with Flag-tagged full length RIG-I wt, K 172 R or K 172only mutant together with HA-tagged Ubiquitin, HEK293T WCLs were used for IP with α-flag, followed by IB with αha or αflag antibody (top two panels). WCLs were used for IB with αha and αactin antibodies. Figure S9. RIG-I K 172only mutant but not RIG-I K 172 R mutant induced IFN-β production in RIG-I-/- MEFs upon SeV infection. Vector, RIG-I wt, RIG-I K 172only or RIG-I K 172 R mutant was stably expressed in RIG-I-/- MEFs using pbabe retroviral vector and these cells were then mock infected or infected with SeV for 9 h. IFN-β production per 10 5 cells in the culture supernatants was measured by enzyme-linked immunosorbent assay. Data are representative of three independent experiments. Figure S10. Role of TRIM25-mediated ubiquitination in RIG-I antiviral activity. a, TRIM25 is critical for RIG-I-mediated activation of IFN-β promoter activity. wt, TRIM25+/-, and TRIM25-/- MEFs were transiently transfected with GST or GST-RIG-I-2CARD and IFN-β reporter luciferase. Renilla luciferase construct was included as an internal transfection control. 4

5 doi: /nature05732 SUPPLEMENTARY INFORMATION WCLs were prepared and subjected to a luciferase assay. The fold induction of IFN-β promoter activity at the y-axis is presented as the comparison between GST and GST-RIG-I 2CARD. Error bars indicate SD, and data are representative of three independent experiments. b, TRIM25 knockout MEFs support higher VSV replication than wt MEFs. wt, TRIM25+/-, and TRIM25-/- MEFs were infected with VSV-eGFP at different MOIs for 36 h. GFP expressing cells were determined by flow cytometry. c, TRIM25 knockout MEFs support higher NDV replication than wt MEFs. wt, TRIM25+/-, and TRIM25-/- MEFs were infected with NDV-GFP at MOI 1 for 48 h. Cells and GFP expression were visualised by inverted fluorescence microscopy. 5

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