Cell culture and viral infection
HEK293T cells, HeLa cells and MEFs were cultured in standard conditions. TRIM25-/- MEFs were kindly provided by Dr. J.U. Jung (Department of Molecular Microbiology and Immunology, University of Southern California, Los Angeles, CA, USA). Sendaï virus (SeV) H4 and WT strains as well as infection protocol were described previously [7, 8], and the Multiplicity of infection (MOI) was 40.
Reagents
Proteasome inhibitors: MG132 (Calbiochem, Merck Chemicals Ltd. Nottingham, UK)) and Lactacystin (Calbiochem) were used at 10 μM and 25 μM, respectively. Proteases inhibitors: z-VAD-fmk (Calbiochem), qVD-fmk (Calbiochem), Leupeptin Hemisulfate (mpbio, Santa Ana, CA, USA) and Pepstatin A (Sigma-Aldrich, St. Louis, MO, USA) were used at 50 μM. Staurosporine (Sigma) was used at 2 μM. Interferons α and β (R&D Systems, Minneapolis, MN, USA) were used at 3,000 U/ml and 3,200 U/ml, respectively. HMW Poly(I:C) (Invivogen, San Diego, CA, USA) was transfected at 1 or 2 μg/ml. Lambda protein phosphatase (λ-PPase) was provided from New England Biolabs (NEB, Ipswich, MA, USA) (P0753S). Neutralizing anti-IFNAR1 (a gift of Dr. P. Eid) was used at 50 μg/ml.
Protein extraction and immunoblot analysis
Cells were lysed in buffer-A (20 mM Tris-HCl (pH 7.4), 137 mM NaCl, 2 mM EDTA, 1% Triton X-100, 2 mM sodium pyrophosphate, 10% Glycerol, 25 mM β-glycerophosphate, 1 mM sodium orthovanadate) supplemented with the protease inhibitor mixture Complete (Roche Molecular Biochemicals, Meylan, France)). After incubation on ice for 20 minutes, a soluble extract was collected after centrifugation at 11,000 g for 10 minutes at 4°C. The lysate (20 μg) was boiled in SDS sample buffer and resolved by SDS-polyacrilamide gel electrophoresis. Immunoblot analysis was performed with specific antibodies and the Ag-Ab complexes were visualized by chemiluminescence (Immobilon Western, Merck Millipore, Billerica, MA, USA). For total cell extracts, cell were lysed in buffer-A supplemented with 3% SDS.
Antibodies
The primary antibodies used in immunoblotting were as follows: mouse monoclonal anti-RIG-I (Alexis Biochemicals, San Diego, CA, USA, clone Alme-1{) (1:2,000 dilution), mouse monoclonal anti-Cardif/MAVS (Alexis Biochemicals, clone Adri-1) (1:4,000), rabbit polyclonal anti-rodent MAVS (Cell Signaling Technology, Danvers, MA, USA) (1:4,000), mouse monoclonal anti-actin (Sigma-Aldrich, St. Louis, MO, USA, clone AC-40) (1:5,000), rabbit polyclonal anti-MDA-5 (Alexis Biochemicals) (1:2,000), rabbit monoclonal anti-phospho-IRF3 (Cell Signaling Technology, clone 4D4G) (1:1,000), rabbit polyclonal anti-IRF3 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) (1:1,000), rabbit monoclonal anti-IRF3 (Cell Signaling Technology) (1:2,000), mouse monoclonal anti-phospho-IκBα (Cell Signaling Technology, clone 5A5) (1:2,000), rabbit polyclonal anti-IκBα (Santa Cruz Biotechnology, C-21) (1:2,000), mouse monoclonal anti-IKKι/IKKε/TBK1 (Imgenex, San Diego, CA, USA, clone 72B587) (1:1,000), mouse monoclonal anti-PARP (BD, Franklin Lakes, NJ, USA, clone C2-10) (1:4,000), rabbit polyclonal anti-Stat1 (Upstate Biotechnology, Merck Millipore, Billerica, MA, USA) (1:1,000), rabbit polyclonal anti-phospho-Stat1 (Cell Signaling Technology, clone Tyr701) (1:1,000), rabbit polyclonal anti-TRAF3 (Santa Cruz Biotechnology, H-122) (1:500), mouse monoclonal anti-VDAC (Calbiochem, clone 89-173/025) (1:4,000), rabbit polyclonal anti-V5 (Sigma-Aldrich) (1:5,000), mouse monoclonal anti-HA (Sigma-Aldrich) (1:5,000). The antibody used in immunoprecipitation of endogenous MAVS was rabbit polyclonal anti-Cardif/MAVS (Alexis Biochemicals, clone AT107) and rabbit polyclonal anti-Myc (Sigma-Aldrich) for the immunoprecipitation of Myc-MAVS. The primary antibodies used for immunofluorescence microscopy were rabbit polyclonal anti-IRF3 (Santa Cruz Biotechnology) (1:500), polyclonal anti-TOM20 (Santa Cruz Biotechnology) (1:800) and mouse monoclonal anti-TBK1 (ProSci Incorporated, Poway, CA, USA, Clone 108A429) (1:400).
Transfections and plasmids
Transfection of HEK293T cells was performed using the calcium phosphate precipitation method. Transfection of HeLa cells by DNA and poly(I:C) was performed using Lipofectamine 2000 (Invitrogen, Life Technologies, Grand Island, NY, USA {) and Oligofectamine (Invitrogen) was used for transfecting siRNAs. The plasmid for the expression of TRIM25-V5 was provided by Dr. J.U. Jung.
Luciferase assays
Cells were plated in 24-well plates. On the second day, cells were co-transfected with 50 ng of firefly luciferase constructs under the control of the IFN-β promoter or driven by three copies of an NF-κB enhancer, and 10 ng of the renilla luciferase pRL-TK plasmid (Promega). The next day, cells were either infected by SeV or transfected with poly(I:C) for a few hours. Transfected cells were collected and luciferase activity was assessed using the Dual-luciferase reporter assay (Promega) on a Fluorostar Optima (BMG Labtech, Ortenberg, Germany). Each experiment was carried out in triplicates. For each sample, to obtain relative fluorescence units (RLU), firefly luciferase fluorescence units were normalized to renilla luciferase fluorescence units.
Immunoprecipitation
Cell lysates were prepared in lysis buffer-B (50 mM Tris-HCl (pH 7.5), 140 mM NaCl, 5 mM EDTA, 5% glycerol, 1% Triton X-100, and 1% Nonidet P-40) supplemented with the protease inhibitor mixture Complete, on ice for 20 minutes. Soluble proteins (500 μg) were subjected to immunoprecipitation with an anti-MAVS (2.5 μg/ml), or rabbit anti-IgG antibody as a control or an anti-Myc antibody. An aliquot of the total lysates was included as a control. After one hour, 20 μl of equilibrated protein G-magnetic beads (Ademtech SA, Pessac, France) was added. Immunoprecipitation was carried out for one hour. The beads were then washed three times with buffer-B. Immune complexes were resolved by SDS-PAGE and immunoblotted.
Lambda phosphatase test
Following immunoprecipitation, the G-magnetic beads were washed twice with the lysis buffer-B, and then twice with the lysis buffer B without EDTA and without the protease inhibitor mixture. Then, each sample was incubated with the reaction mixture (2.5 μl of reaction buffer provided with the λ Phosphatase kit (NEB), 2.5 μl MnCl2 (provided with the kit), 10 μl lysis buffer-B without EDTA/inhibitors and 10 μl λ-Phosphatase (NEB) or lysis buffer-B only for control) for 30 minutes at 30°C. Finally, Immunoblot and phosphorylation of MAVS were resolved by SDS-PAGE.
Immunofluorescence microscopy
Cells grown in LabTek (Fisher Scientific, Illkirch, France) chambers were fixed for 10 minutes in 4% paraformaldehyde, followed by permeabilization with 0.15% Triton X-100 in PBS for 15 minutes. The cells were then incubated for one hour in blocking buffer (2% BSA in PBS) followed by incubation overnight with primary antibodies. Next, cells were washed three times for 10 minutes each in PBS, then incubated for 1 hr with Alexa Fluor secondary antibodies. Images were acquired using a Leica SP6 confocal microscope (Leica Microsystems, Wetzlar, Germany) through a 63x oil fluorescence objective.
Signal intensities from each channel were reconstructed by plotting pixel values of each channel along lines drawn through optical sections. Multichannel images were separated into single channels and exported to the ImageJ software (National Institute of Health, Bethesda, MD, USA). Measurements of the pixel intensities were made along the lines shown in the respective picture.
Enzyme-Linked Immunosorbent Assays (ELISA)
MEFs were plated in 24-well plates at a cell density of 2.105 cells per well. Eight hours later, cells were infected with SeV or transfected with poly(I:C). Supernatants of cells were collected and ELISA assay was performed following the manufacter's protocol (PBL Biomedical Laboratories, Piscataway Township, NJ, USA, Mouse Interferon Beta ELISA Kit v.1.4 and R&D Systems, Mouse IL-6 immunoassay).
Small interfering RNA (siRNA)
For down-regulation of proteins, siRNA oligos directed against MAVS, TRIM25, RIG-I and MDA-5 at a final concentration of 20 nM were transfected into cells for 72 hr. For HeLa cells, oligofectamine was used according to the manufacturer's instructions whereas for HEK293T cells, siRNA transfection was performed using the calcium phosphate precipitation method. siRNAs were purchased from Ambion (Life Technologies, Grand Island, NY, US). The sequence of the siRNA oligos is as follows (only sense strands are shown):
MAVS siRNA1: CCGUUUGCUGAAGACAAGAtt
MAVS siRNA2: CCACCUUGAUGCCUGUGAAtt
TRIM25 siRNAa: CCAUAGACCUCAAAAACGAtt
TRIM25 siRNAb: CAACAAGAAUACACGGAAAtt
RIG-I siRNA: GGAAGAGGUGCAGUAUAUUtt
MDA-5 siRNA: GUUCAGGAGUUAUCGAACAtt
Mass spectrometry
After SeV H4 infection for four hours, a purified MAVS complex was analyzed by means of one-dimensional gel electrophoresis in combination with the nano liquid-chromatography tandem using 10 segment GelC/MS and spectral counting by mass spectrometry. Mass spectrometry was performed by Nextgensciences (Ann Arbor, MI, USA).
Cellular fractionation
Isolation of mitochondrial and cytosolic fraction: HeLa cells were harvested in isotonic buffer-C (210 mM mannitol, 70 mM sucrose, 1 mM EDTA and 10 mM HEPES (pH 7.5)), supplemented with the protease inhibitor mixture Complete (Roche Molecular Biochemicals). Cells were broken by 15 passages through a 25-gauge needle fitted onto a 5 ml syringe, and the suspension was then centrifuged at 2,000 g at 4°C for 5 minutes to remove nuclei and unbroken cells. This procedure was repeated until nearly all of the cells were broken. Heavy membrane fractions enriched in mitochondria were obtained by centrifugation at 10,000 g at 4°C for 10 minutes, and supernatant was centrifuged at 25,000 g for 30 minutes and supernatant was kept as the "cytosolic fraction". The heavy membrane fraction was resuspended in buffer-C and layered on top of a discontinuous sucrose gradient consisting of 1.2 M sucrose in 10 mM Hepes (pH 7.5), 1 mM EDTA, and 0.1% BSA on top of 1.6 M sucrose in 10 mM Hepes, (pH 7.5), 1 mM EDTA, and 0.1% BSA. Then, samples are centrifuged at 30,000 g for 2 hr at 4°C. Mitochondria are recovered at the 1.6 to 1.2 M sucrose interface, washed in buffer C and centrifuged at 13,000 g at 4°C for 10 minutes, and resuspended in buffer C. The mitochondrial pellet was lysed and used for immunoblot analyses.
Densitometric image analysis
To measure the relative expression level of proteins in cell extracts, acquired images were densitometrically analyzed by using the ImageJ software.
Statistical analyses
Data were compared using Student's t-test. Differences were considered to be significant if P < 0.05. ***P < 0.001, **0.001 < P < 0.01, *0.01 < P < 0.05. NS, not significant.