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.

Human IFIH1 sequence-verified cDNA was purchased from Open Biosystems (clone ID: 40008600) and subcloned into pcDNA3.1 (Invitrogen), pCL20 MSCV GFP T2A (immediately 3′ to T2A sequence), and pLenti-III-UbC/ mCherry (under the ubiquitin C promoter) mammalian expression plasmids using the In-Fusion HD Cloning kit (Clontech). Site-directed mutagenesis was used to generate constructs encoding the MDA5 mutants K365E, gain-offunction mutants R337G and R779H, and 38 other MDA5 constructs containing point mutations found in ExAC database with MAF ranging from 0.01 to 9.3%. In brief, wildtype MDA5-expressing plasmids were PCR-amplified using AccuPrime Pfx SuperMix (Invitrogen) and primers containing appropriate point mutations (Table S3 ). This was followed by Dpn1 digestion (New England Biolabs) and transformation into TOP10 competent cells (Invitrogen). Constructs encoding MDA5, skipping either Exon 8 or Exon 14 because of splicing mutations, were generated using In-Fusion HD Cloning kit. Plasmid DNA was purified using the PureLink HiPure Filter Plasmid kit (Invitrogen), and the introduced mutations were confirmed by Sanger dideoxy sequencing.