Recurrent rhinovirus infections in a child with inherited MDA5 deficiency

Lamborn, Ian T.; Jing, Huie; Zhang, Yu; Drutman, Scott B.; Abbott, Jordan K.; Munir, Shirin; Bade, Sangeeta; Murdock, Heardley M.; Santos, Celia P.; Brock, Linda G.; Masutani, Evan; Fordjour, Emmanuel Y.; McElwee, Joshua J.; Hughes, Jason D.; Nichols, Dave P.; Belkadi, Aziz; Oler, Andrew J.; Happel, Corinne S.; Matthews, Helen F.; Abel, Laurent; Collins, Peter L.; Subbarao, Kanta; Gelfand, Erwin W.; Ciancanelli, Michael J.; Casanova, Jean-Laurent; Su, Helen C.

Affinity precipitations 500,000 293T cells were seeded per well in tissue culture-treated 6-well plates (Corning). 12-16 h later, cells were transfected with 4 µg of pCL20c MSCV GFP-T2A-WT MDA5, pCL20c MSCV GFP-T2A-K365E MDA5, or pmaxGFP (Lonza), complexed with Lipofectamine 2000 (Invitrogen) according to the manufacturer's recommendations. 48 h later, cells were lysed in 0.05% Nonidet P-40 (Calbiochem), 20 mM Hepes, 1.5 mM magnesium chloride (both from Quality Biological), 150 mM sodium chloride, 0.038% of 2-ME (both from Sigma-Aldrich), and 1x cOmplete EDTA-free protease inhibitor cocktail (Roche). After incubating on ice for 20 min, cell lysates were mechanically disrupted by passing through a 25-gauge needle 10 times. Lysates were centrifuged at 13,200 g for 15 min at 4°C, before collecting supernatants. Proteins contained within the supernatants were quantified by BCA (Thermo Fisher Scientific). β,γ-methyleneadenosine 5′-triphosphate (ADP CP; Sigma-Aldrich) was added to 2 mg of protein to a final concentration of 2 mM before adding 1 µg of biotin-labeled high molecular weight (HMW) poly(I:C) (InvivoGen). After incubating at 37°C for 10 min, the mixture was added to M-270 hydrophilic streptavidin Dynabeads (Invitrogen) that had been preblocked for 20 min at 4°C with 600 µg to 1 mg of lysate from pmaxGFP-transfected (Lonza) control 293T cells. After incubation at 4°C for 3 min, the beads were washed three times with lysis buffer containing 2 mM ADP CP. MDA5 protein was eluted by incubating at 95°C for 5 min in 2X SDS buffer protein gel loading solution (Quality Biological) supplemented with 0.3 M sodium chloride and 5% vol/vol 2-ME. Proteins were separated by SDS-PAGE, and immunoblotting for MDA5 was performed as described in the Immunoblotting section. luciferase reporter gene assays 50-100,000 293T cells were seeded per well in 24-well tissue culture plates. After culture for 18-24 h to ∼70-80% confluency, Lipofectamine 2000 (Invitrogen) was used for co-transfections of cells with pcDNA3.1 mammalian expression plasmids expressing wild-type MDA5 and/or mutants (20 ng or 100 ng), a firefly luciferase reporter plasmid (200 ng) driven by human IFN-β promoter, and a constitutively expressed Renilla luciferase reporter plasmid (20 ng). For some experiments, firefly luciferase reporter plasmids driven by the interferon-stimulated response element (ISRE) or NF-κB response element were instead used. In dominant interference experiments, 20 ng pcDNA3.1 plasmid expressing wild-type MDA5, 100 ng pCL20 MSCV plasmid expressing GFP-T2A-K365E MDA5, and/or 100 ng pCL20 MSCV plasmid expressing GFP-T2A (empty vector) were transfected, along with luciferase reporter plasmids. 6 h later, cells were stimulated with a mixture of 1.2 µg high molecular weight poly(I:C) (InvivoGen) complexed with 1.5 µl Lipofectamine 2000 in 100 µl Opti-MEM I reduced serum medium (Gibco), added to cells for an additional 18-24 h before lysis. The Dual Luciferase Reporter assay (Promega) was used on a Fluostar Omega plate reader (BMG Labtech) to measure luciferase activities contained in cell lysates. Firefly luciferase activity was normalized to Renilla luciferase activity. Fold-increase in the normalized activity in MDA5-transfected cells was reported relative to normalized activity in untransfected cells.