Regulation of innate antiviral defenses through a shared repressor domain in RIG-I and LGP2

Saito et al. 10.1073/pnas.0606699104.

Supporting Information

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SI Figure 5
SI Figure 6
SI Figure 7
SI Figure 8
SI Materials and Methods

SI Figure 5

Fig. 5. Genomic HCV RNA triggers the host response through RIG-I but not MDA5. (A) Huh7 cells were transfected with Flag expression constructs encoding WT RIG-I, WT MDA5, or WT LGP2, as shown. After 24 h, the cells were harvested and extracts were mixed with equal amounts of biotinylated HCV 3' NTR RNA or JFH1 HCV 2a RNA (-) or with excess nonlabeled homologous competitor RNA (+), as indicated above each lane. RNA-protein complexes were recovered by selection with avidin-agarose beads, and complexes were analyzed by SDS/PAGE followed by anti-Flag immunoblot analysis. Shown are the pull-down product (Upper) and the input material (Lower) in each lane. (B) WT, RIG-I null, or MDA5 null MEFs were cotransfected with Renilla luciferase and murine IFN-b-luciferase plasmid constructs, and 24 h later were mock transfected (no RNA) or were transfected with HCV 2a JFH1 genome RNA (JFH1). After 20 h, the cells were harvested and extracts were subjected to the dual luciferase assay. The mean relative luciferase activity (± SD) is shown. (C) Electrophoretic mobility shift assay (EMSA) demonstrates a direct interaction between RIG-I and dsRNA. Synthetic dsRNA of 25 base pairs was mixed with increasing amounts of recombinant, purified RIG-I in binding buffer and subjected to native PAGE. The gel was first stained with ethidium bromide (Center) to visualize the dsRNA, then stained with Coomassie brilliant blue (Left) to detect RIG-I protein.

SI Figure 6

Fig. 6. Signaling mechanisms of RIG-I and MDA5. (A) 293 cells were cotransfected with Renilla luciferase and PRD-II-luciferase plasmid constructs and empty vector or plasmids encoding the indicated Flag-tagged RIG-I constructs. After 16 h, the cells were infected with SenV. After an additional 18 h, the cells were harvested and subjected to dual luciferase assay. Bars show relative PRD-II-luciferase activity (± SD). (B) Huh7.5 cells were cotransfected with Renilla luciferase and IFN-b-luciferase plasmid constructs and empty vector or plasmids encoding the indicated Flag-tagged RIG-I constructs. After 16 h, the cells were infected with SenV. After an additional 18 h, the cells were harvested and subjected to dual luciferase assay. Bars show relative PRD-II-luciferase activity (± SD). (C Left) Huh7 cells were cotransfected with Myc-RIG-I 1-228 and the indicated Flag-tagged RIG-I expression construct. After 24 h, the cells were harvested and extracts were subjected to coimmunoprecipitation analysis using anti-Flag antibody for the immunoprecipitation. Immunocomplexes were resolved by SDS/PAGE and subjected to immunoblot analysis using an anti-Myc antibody to detect Myc-RIG-I 1-228 within the complex (Top) and the input material (Middle). Bottom shows the Flag-tagged RIG-I protein that was immunoprecipitated in each reaction. (C Right) WT or RIG-I null MEFs were transfected with an IFN-b promoter-luciferase plasmid and vector alone or a plasmid encoding RIG-I 1-228. After 16 h, the cells were harvested for luciferase assay. Bars show the average luciferase activity in each experiment. (D) Huh7 cells were cotransfected with vector control (white bars) or plasmid encoding Flag-RIG-I 735-925 (black bars) and Renilla luciferase construct, IFN-b-luciferase construct, and empty vector or plasmid encoding Flag-RIG-I 1-228. Cells were harvested after 16 h, and extracts were subjected to dual luciferase assay. The set of bars at right shows cells that were cotransfected with plasmid encoding the Flag-RIG-I WT construct but, after 16 h, were infected with SenV for an additional 16 h before harvesting for dual luciferase assay. Bars show relative IFN-b-luciferase activity (± SD). (E) Huh7.5 cells were cotransfected with vector control or plasmid encoding Flag-MDA5 constructs, Renilla luciferase construct, and IFN-b-luciferase construct. After 16 h, the cells were mock infected or SenV infected. After an additional 16 h, the cells were harvested and extracts were subjected to dual luciferase assay. Bars show relative IFN-b-luciferase activity and SD. (F) Huh7 cells were cotransfected with Myc-tagged expression plasmids encoding vector alone (control), WT LGP2, LGP2 476-678, and plasmid encoding Flag-tagged WT RIG-I or WT MDA5. After 24 h, the cells were infected with SenV (+) or mock infected for 20 h (-), as indicated above each lane. Cell extracts were subjected to coimmunoprecipitation analysis using anti-Flag antibody for the immunoprecipitation. Immunocomplexes were resolved by SDS/PAGE and subjected to immunoblot analysis using an anti-Myc antibody to detect RIG-I or MDA5 within the complex (Top) and the input material (Middle). Bottom shows the Flag-tagged protein that was immunoprecipitated in each reaction.

SI Figure 7

Fig. 7. LGP2 blocks RIG-I but not MDA5 signaling. Huh7.5 cells were cotransfected with Renilla luciferase and IFN-b-luciferase plasmid constructs. Left, cells were cotransfected with 20 ng of expression plasmid encoding vector alone, WT RIG-I, or RIG-I 1-228, and the indicated amount of WT LGP2 expression plasmid. Right, cells were cotransfected with 20 ng of expression plasmid encoding vector alone, WT MDA5, or MDA5 1-294, and the indicated amount of WT LGP2 expression plasmid. Cells were mock infected (white bars) or infected with SenV (black bars) and were harvested 20 h later for dual luciferase assay. Bars show relative mean luciferase activity (± SD) for each condition.

SI Figure 8

Fig. 8. Characterization of RIG-I cell lines. (A and B) Huh7, Huh7.5, Huh7-RIG-I-wt, and Huh7 RIG-I-735-925 cells were cotransfected with Renilla luciferase and IFN-b-luciferase plasmid constructs. After 16 h, the cells were infected with SenV. After a further 16 h, the cells were harvested for dual luciferase assay (A) (bars show relative IFN-b-luciferase activity ± SD) and for immunoblot analysis of ISG56, Flag-tagged protein, and GAPDH abundance (B). (C) Huh7 cells, Huh7.5, Huh7-RIG-I wt, and Huh7-RIG-I-735-925 cells were infected with vesicular stomatitis virus at a multiplicity of infection of 0.1. At 10 h after infection, supernatants were harvested and titered for infectious virus. Bars show plaque-forming units per ml and SD.

SI Materials and Methods

Antibodies. Immunoblot analysis was conducted using a mAb specific to RIG-I (provided by T.F.), FLAG (M2; Sigma), Myc (Bethyl Laboratories, Montgomery, TX) or polyclonal rabbit serum specific to RIG-I (provided by M.G.), GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA) or ISG56 (G. Sen, Cleveland Clinic Foundation, Cleveland, OH). HCV proteins were detected by using a fully characterized patient serum (obtained from W. Lee, University of Texas Southwestern Medical Center, and used with informed consent). All secondary antibodies were obtained from Jackson Immunoresearch Laboratories (West Grove, PA). Immunoreactive bands were detected by using the ECL Plus Western blotting detection system (Amersham Pharmacia Biosciences). Coimmunoprecipitation products were recovered by using anti-Flag M2 agarose beads (Sigma) in accordance with the manufacturer's protocol for protein recovery and analysis.

Analyses of RIG-I or IRF-3 multimerization/dimerization were conducted using methods described previously for determining IRF-3 dimerization [Sumpter R, Loo YM, Foy E, Li K, Yoneyama M, Fujita T, Lemon S, Gale M J, Jr (2005) J Virol 79:2689-2699], except that proteins were separated by electrophoresis through precast 7.5% Tris·HCl gels (BioRad, Hercules, CA). Proteins were transferred to nitrocellulose membrane, and membranes were probed using anti-RIG-I polyclonal antibodies.

Preparation of Cell Extracts for RNA Binding Assay. Cytoplasmic extracts were prepared from 5 ´ 10⁸ cells. Cells were collected from culture dishes, washed with PBS, and then concentrated by resuspending in five packed-cell volumes of buffer A [10 mM Hepes (pH 7.9)/1.5 mM MgCl₂/10 mM KCl/0.2 mM DTT] and incubated for 5 min on ice. After centrifugation for 10 min at 2,000 ´ g, cells were resuspended in two packed-cell volumes of buffer A and lysed by 10 up-and-down strokes in a glass Dounce homogenizer. The homogenate was centrifuged for 10 min at 2,000 ´ g. Supernatant was recovered and used for cytoplasmic extract preparation by adding an additional 10% volume of 10´ cytoplasmic extract buffer [0.3 M Hepes (pH 7.9)/0.03 M MgCl₂/1.4 M KCl]. The mixture was then centrifuged at 100,000 ´ g in a Beckman 50 Ti ultracentrifuge rotor for 60 min at 4°C. The supernatant was recovered and dialyzed against 20 mM Hepes (pH 7.8)/100 mM KCl/2 mM MgCl₂/20% glycerol/0.2 mM EDTA/0.5 mM DTT for 16 h at 4°C. The dialyzed material was then centrifuged at 55,000 ´ g for 20 min, and supernatant was collected and stored at -80°C until use in RNA binding studies. The protein concentration of each aliquot was determined by using the BioRad protein assay kit, in accordance with the manufacturer's instructions.