Ifn 1

HeLa and HEK293T cells were seeded in 6-well plates and transfected at a confluence of 70%. HeLa cells were transfected using TransIT-LT1 (Mirus) according to the manufacturer’s instructions at a ratio of 3 μl TransIT-LT1 to 1 μg DNA. HEK293T cells were transfected according to the manufacturer’s instructions using PEI (1 mg/ml; Sigma-Aldrich) at a ratio of 4 μl PEI to 1 μg DNA. For each transfection, 0.5 μg pHIV-1 and 0.5 μg pGFP, pGFP-FLAG, pKHNYN-1, pVSVG, pHA-ZAP-S, pHA-ZAP-L, or pHA-Renilla was used, for a total of 1 μg DNA. The transfection medium was replaced with fresh medium 6 h (HEK293T) or 24 h (HeLa) posttransfection. The cells were lysed 48 h posttransfection, and the medium was recovered. In experiments performed with type I interferon, 1,000 U/ml of IFN-I (Universal Type I Interferon; PBL Assay Science) was added to the cells upon medium change 6 h posttransfection. The medium was filtered through a 0.45-μm filter, and the virions were pelleted for 2 h at 20,000 × g through a 20% sucrose cushion in phosphate-buffered saline (PBS) solution.

Bone marrow cells from C57BL/6 mice were cultured in DMEM (Corning) with 10% FBS, 10 mM L-glutamine (Gibco, ThermoFisher), and 20% L929 supernatants. After 7 days 2 × 10⁶ BMDMs were cultured in 48-wells plate in 300 μl of medium without FBS. The BMDM were stimulated with 1:5 of red blood cells (RBC) or infected RBCs (iRBC), 40 ng/ml of IFNγ (Sigma), and 500 U/ml of IFN-I (Pbl Assay Science), as indicated in the figure legends. Between 16 and 24 h of in vitro stimulation, BMDMs were lysed with RIPA buffer (Sigma) solution. Culture supernatants were collected, and the soluble proteins were precipitated using chloroform (25% of supernatant volumes) and methanol (same supernatant volumes). After centrifugation at 20,000 × g for 10 min at 4 °C, the proteins pellet was dried in 56 °C and suspended in sample buffer.