Supporting Methods

Cell Culture. Human embryonic kidney (HEK) 293 and hepatoma Huh7 cells were cultured by using standard protocols. Huh7 2-3 cells, which contain genome-length replicating HCV RNA; their IFN-a 2b-cured progeny, 2-3c cells; and the osteosarcoma cell line UNS3-4A-24, which conditionally expresses NS3/4A under tetracycline regulation (D. Moradpour, University of Freiburg, Freiburg, Germany), were cultured as described in refs. 1 and 2. HeLa SL1 cells (a kind gift from C. Seeger, Fox Chase Cancer Center, Cheltenham, PA) (3) contain a replicating subgenomic HCV RNA; their IFN-cured progeny, SL1c cells, were produced by using a the protocol described in ref. 2.Where indicated, cells were infected with 100 hemagglutinin units/ml of Sendai virus (Charles River) and harvested 16 h later for luciferase reporter assays or at the times indicated for immunoblot analysis, as described in ref. 4. SCH6 was kindly provided by Drs. S. Bogen and B. Malcolm of the Schering-Plough Research Institute.

Plasmids. TRIF and TLR3 cDNAs from MRC-5 cells were cloned into pCDNA6 V5-His B (Invitrogen). pCDNA6-TRIF-His contains TRIF fused to a C-terminal 6XHis tag; pCDNA6-mycTRIF-His includes an additional N-terminal myc tag. We generated pET21d-TRIF-His for expression of TRIF in Escherichia coli. pCDNA6-NS3/4A and pRcCMV-NS5AB were constructed from HCV-N sequence. pCMV-NS3/4A-S139A was provided by C. Johnson (University of Texas Southwestern Medical Center). pCMVb -gal or pEGFP-C1 (CLONTECH) were used to normalize transfection efficiencies. pPRDII-luc is described in ref. 4. pIFN-b -luc and pRANTES-Luc were provided by R. Lin (Lady Davis Institute for Medical Research, Montreal). pCDNA3-Flag-TBK1 and pCDNA3-Flag-IKKe were gifts from K. Fitzgerald (University of Massachusetts Medical School, Worcester, MA) and T. Maniatis (Harvard University, Cambridge, MA); pMALc-NN-scNS3 (pYB57) was provided by I. Benhar (Tel Aviv University, Tel Aviv). We generated C192R, C326R, C372R, D N372, and D C340 TRIF mutants and C1125A mutant scNS3 by QuikChange site-directed mutagenesis (Stratagene). Cells were transfected with the indicated plasmids by using FuGene 6 transfection reagents according to the manufacturer’s protocol.

Protein Expression and Purification. Linearized plasmids served as template for in vitro transcription of TLR3, TBK1, IKKe , and TRIF RNA with T7 polymerase. The RNA transcripts (300 ng) were used to program translation in 10 m l of Flexi-Rabbit reticulocyte lysate (Promega) containing [35S]-Met. Recombinant His-tagged NS3p, scNS3, and TRIF were expressed in E. coli and isolated from bacterial inclusion bodies. TRIF was purified by affinity (Ni-NTA; Qiagen, Valencia, CA) and gel filtration (Amersham Biosciences) chromatography. NS3p and scNS3 were purified by Ni-NTA chromatography followed by removal of the His tag and further purification by Ni-NTA and SP Sepharose (Amersham Biosciences) chromatography. MB-scNS3, a fusion of scNS3 to the C terminus of E. coli maltose-binding protein, was purified on an amylose resin column (New England Biolabs). Rabbit antisera to TRIF were obtained by immunization of rabbits with purified recombinant full-length TRIF protein.

Immunoblots. Cells were harvested and extracts were prepared and subjected to immunoblot analysis as described in ref. 4 by using the following mAbs or polyclonal antibodies (pAbs): anti-Flag M2 and anti-actin mAbs (Sigma), anti-myc mAb (Santa Cruz), anti-GFP mAb (Roche), anti-HCV NS3 mAb (Novacastra), anti-IRF-3 pAb (from M. David, University of California at San Diego, La Jolla, CA), anti-TRIF pAb, anti-ISG15 pAb (from A. Haas, Medical College of Wisconsin, Milwaukee), anti-Sendai virus pAb (from I. Julkunen, National Public Health Institute, Helsinki), and anti-ISG56 pAb (from G. Sen, Lerner Research Institute, Cleveland). Immunostaining of NS5A antigen was conducted as described by using mAb anti-NS5A (Maine Biotech).

EMSA. Nuclear extracts were prepared from UNS3-4A-24 cells with and without NS3/4A expression after polyI:C treatment at the indicated times. EMSA was carried out by using a 32P-labeled double-stranded oligonucleotide probe corresponding to the PRDII element of human IFN-b promoter (5).

Metabolic Labeling and Immunoprecipitation. Twenty-four hours posttransfection, cell culture medium was replaced with DMEM (without methionine or cysteine) supplemented with 2% dialysed FBS. One hour later, 200 m Ci (1 Ci = 37 GBq) of trans-35S-label (ICN) was added. The cells were placed at 37°C for 30 min, washed, and refed with DMEM containing10% FBS supplemented with 2 mM methionine and cysteine. Cells were incubated at 37°C as indicated before lysis and immunoprecipitation with anti-myc or anti-Flag M2. To identify the C-terminal TRIF cleavage fragment, the products of an in vitro cleavage reaction containing MB-scNS3 and 35S-labeled in vitro-translated TRIF-His were subjected to immunoprecipitation with rabbit anti-His (Cell Signaling).

RNA Interference. Cells were transfected with 80 nM short interfering RNA (siRNA) targeting TLR3, TRIF, RIG-I, or a negative control siRNA (Ambion) by using Oligofectamine (Invitrogen) according to the manufacturer’s recommended procedures. siRNA (Dharmacon and Ambion) target sequences were as follows: TLR3,GGTATAGCCAGCTAACTAG; TRIF, GACCAGACGCCACTCCAAC (6) or GAAGATACCACCTCTCCAA; RIG-I, GGAAGAGGTGCAGTATATT. For promoter assays, cells were cotransfected with reporter plasmids 24 h after transfection of the siRNA. After an additional 24 h, cells were treated or mock-treated with polyI:C and subsequently harvested as described above. Silencing of specific mRNAs was confirmed by semiquantitative RT-PCR. Primer sequences are available upon request.

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