Transit mrna reagent

HeLa (CCL-2), HEK293T (CRL-11268), HepG2 (HB-8065) and KG-1 (CCL-246) cells from the American Type Culture Collection were maintained with DMEM (Thermo Fisher Scientific) supplemented with 10% FBS (Gibco) and 1% penicillin–streptomycin (Gibco). Cell lines were not authenticated. For routine subculture, 0.25% TrypLE (Thermo Fisher Scientific) was used for cell dissociation.
RNA delivery was achieved with TransIT-mRNA transfection, Lipofectamine transfection or NEON electroporation. Within each experiment, the molar amount of mRNA or circRNA delivered and transfection method used was the same for all samples unless otherwise indicated. For TransIT-mRNA transfections, 3 µl of TransIT-mRNA reagent (Mirus Bio) was used per microgram of RNA. Besides this change, RNA delivery was performed following the manufacturer’s instructions.

Huh7, Huh7 NTV, Huh7 RIG-I K/D, Huh7 14-3-3η K/D cells were infected with Sendai virus (SeV) at a concentration of 200 HA unit/mL in DMEM supplemented with 10% FBS at 37°C. For encephalomyocarditis virus (EMCV) infection, cells were first washed with PBS twice, then were incubated with virus with designated MOI in serum-free medium for 1 hour at 37°C. The cells were rinsed by PBS twice and incubated in DMEM supplemented with 2% FBS post virus solution absorption. For poly(I:C) stimulation, cells were transfected with high molecular weight (HMW) poly(I:C) (Invivogen) by TransIT-mRNA reagent (Mirus) according to manufacturer’s instructions.

ZIKV replicons have been previously reported (Rusanov et al., 2018 (link)). To determine whether NDUFA4 is involved in flavivirus genome replication, WT, NDUFA4^−/− and NDUFA4^ΔSNP iPSCs were transfected with the in vitro transcribed ZIKV replicon RNA. iPSCs were plated into 24-well plates at 62,500 cells per well. Transfection mixes for each well were prepared by mixing 0.1 μg of the replicon RNA with 0.2 μL each of the mRNA boost reagent and TransIT-mRNA reagent (TransIT-mRNA transfection kit; Mirus, WI). At 24 hpi, cells were lysed in 1X cell culture lysis reagent (Promega, WI) followed by measurement of the Renilla luciferase activity.

Transfection of HEK 293 T cells was performed with TransIT-mRNA (Mirus Bio) according to the manufacturer’s instructions: mRNA (0.3 µg) was combined with TransIT-mRNA Reagent (0.34 µL) and Boost Reagent (0.22 µL) in 17 µL of serum-free medium, and the complex was added to 6 × 10⁴ cells in 183 µL complete medium.

An AlexaFluor 568-conjugated probe was adhered to the 5′ end of the partial ZIKV 5′-UTR sequence: 5′-CTACTCCGCGTTTTAGCATATTGACAATCCGGAATCCTCCGG-3′. W+ and W− cells were plated at 3.5 × 10⁵ cells per well in 0.5 ml volumes in a 24-well plate and incubated for 24 h at 28°C with 5% CO₂. Per well, 1 µg of probe was diluted in 100 µl of Optimem with 1 µl of Mirus Bio mRNA boost and 1 µl of Mirus Bio mRNA TransIT reagent; we allowed 3 min for lipid complexes to assemble and added them to cells. Control transfection experiments without the DNA probe or transfection boost and reagent were conducted simultaneously. Cells were incubated at 28°C with 5% CO₂ for 24 h. Supernatant was removed, and cells were either fixed for imaging analysis or rinsed three times with PBS for plate reader analysis. For imaging, cells were fixed in 4% paraformaldehyde for 1 h at room temperature. Cells were rinsed 1 time with PBS and mounted in Prolong Gold and stained with DAPI. For plate reader analysis, cells were lysed in NP-40 lysis buffer and 100 µl of cell lysate was transferred to a clear-bottom plate. A Tecan Spark plate reader was used to determine fluorescence in the 568-nm channel.