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.

For measurement of IFN-β (IFNB1) and IFN-λ (IFNL3, IL-28), 0.5 µg of total RNA was reverse transcribed using High-Capacity cDNA Reverse Transcription kit with RNase Inhibitor (Applied Biosystems). Diluted cDNA was analyzed by quantitative real-time PCR using SYBR Green PCR Master Mix on a 7500 Real Time PCR System (Applied Biosystems). The primer sequences used were as follows: IFNB1 forward primer 5′-CAG GAG AGC AAT TTG GAG GA-3′, IFNB1 reverse primer 5′-CTT TCG AAG CCT TTG CTC TG-3′, IFNL3 forward primer 5′-TCC TTC AGC AGA AGC GAC TC-3′, IFNL3 reverse primer 5′-GCC ACA TAG CCC AGT TCA AG-3′, ACTB forward primer: 5′-GCA CAG AGC CTC GCC TT-3′, ACTB reverse primer 5′-GTT GTC GAC GAC GAG CG-3′. IFNB1 or IFNL3 transcripts were first normalized to β-actin expression, and then shown as fold-changes relative to 0 h for each siRNA (uninfected cells). lentivirus particle production Specific lentiviral transfer vectors (all in pLenti-III-UbC/ mCherry) were constructed as described in the Plasmids and molecular cloning section. VSV-G-pseudotyped lentivirus particles were generated by transient co-transfection into 293T cells of the specific transfer vector together with the packaging plasmids pCMV delta R8.2 (HIV-1 GAG/ POL, Tat, and Rev expressing plasmid, Addgene #12263) and pCMV VSV-G (VSV-G envelope expressing plasmid; Addgene #8454; Stewart et al., 2003) using calcium phosphate precipitation (Kingston et al., 2003) . In brief, 13 million 293T cells were seeded in poly-l-lysine (Sigma-Aldrich) coated Cell Culture Treated TripleFlasks (Nunc). When cells reached 95% confluency, 250 µg of specific transfer vector, 125 µg pCMV delta R8.2, and 42 µg of pCMV VSV-G were precipitated with calcium phosphate, mixed with 100 ml of complete IMDM, and added to the cells. DNA precipitates were washed out 12 h after transfection and cell supernatants were collected daily for 3 d (stored at 4°C), filtered through 0.22 µm pore-size filter (GE), concentrated by centrifugation at 18,000× g for 3 h at 4°C, and resuspended in Opti-MEM I reduced serum media (Gibco). Lentivirus preparations were stored at −80°C until use.