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.

Firefly luciferase plasmid driven by the human IFN-β promoter (IFNB-pGL3; Promega; Lin et al., 2000) and the constitutively expressed Renilla luciferase reporter plasmid (pRL-TK; Promega) were gifts from Y. He (National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD). The firefly luciferase plasmids driven by the IFN-stimulated response element (pGL4.33-luc2P/ISRE/ Hygro; Promega) and the NF-κB response element luciferase (pGL4.32-luc2P/NF-κB-RE/Hygro; Promega) were modified to express GFP instead of the hygromycin resistance cassette. In brief, pGL4.33-luc2P/ISRE/Hygro and pGL4.32-luc2P/NF-κB-RE/Hygro plasmids were digested with BamHI and NotI (New England Biolabs) and gelpurified (QIAquick Gel Extraction kit; QIA GEN) to remove the hygromycin resistance cDNA. The coding sequence of EGFP was PCR-amplified from pcDNA3-EGFP (Addgene #13031) using AccuPrime Pfx SuperMix (Invitrogen) and subcloned into the linearized vector backbones using the In-Fusion HD Cloning kit (Clontech), to generate pGL4.33-luc2P/ISRE/EGFP and pGL4.32-luc2P/NF-κB-RE/EGFP. pCL20c MSCV-GFP-T2A is a modified version of the lentiviral transfer vector pCL20c MSCV-GFP (Hanawa et al., 2004) . In brief, a self-cleavage T2A peptide sequence plus additional restriction sites were added in-frame to the 3′ end of the GFP cDNA from pCL20c MSCV-GFP. This was accomplished by PCR amplification of pCL20c MSCV-GFP using AccuPrime Pfx SuperMix (Invitrogen) with forward primer 5′-CTA GGC GCC GGA ATT ACC GGT GGC CGG CCG CGG GCC ACC ATG GTG AGC AAG GGC GAG GAG-3′ and reverse primer 5′-GGC ATC GAT GCG GCC GCA TGC TCA CCT GCA GGG GCC GGG GTT CTC CTC CAC GTC GCC GCA GGT CAG CAG GCT GCC CCG GCC CTC CTT GTA CAG CTC GTC CAT GCC GAG AGT GAT CC-3′. The PCR-amplified product was resolved by agarose gel electrophoresis, gel-purified (QIAquick Gel Extraction kit; QIA GEN), and ligated using the In-Fusion HD Cloning kit (Clontech) into the EcoRI-and NotI-digested (New England Biolabs) and gel-purified vector backbone of pCL20c MSCV-GFP (digested and purified to remove the original GFP and multiple cloning site).