Renilla substrate

LIPS assay (Figure S1) was performed according to a protocol previously described by Burbelo et al. [19 (link)] with modification. Renilla antigen was prepared by transfecting 293FT cells with Renilla luciferase-orf3b expression plasmid, followed by lysis at 48 h post-transfection. Heat-inactivated patient sera were 1:100 diluted and incubated with 1×10⁷ light units of Renilla antigen on a rotary shaker. 1.5 µL of Protein A/G resin (Thermo Fisher Scientific, USA) diluted in PBS was then added to each sample and incubated to capture the antibody–antigen complex. Following three washes, the beads were transferred to an opaque 96-well plate. Renilla substrate (Promega, USA) was then added and luciferase signal was measured using the Wallac Victor3 Multilabel Counter (PerkinElmer, USA).

Rex specifically binds to the Rex-responsive element (RxRE) in the 3′-UTR of HTLV-1 mRNA and mediates CRM1-dependent nuclear export. To measure the functional activity of Rex, we inserted the RxRE sequence downstream of the β-globin coding region of Renilla-WT (Renilla-WT-RxRE). HEK293FT cells were seeded at 5 × 10⁵ cells/mL and 10 mL/10 cm culture dish one day before transfection. At 24 h after seeding, HEK293FT cells were transfected with 10 μg of shNONO#1, shNONO#2, or shLuc-CS-RfA-EvBsd by PEI. After 48 h, three types of shRNA-expressing HEK293FT cells were seeded onto 48-well plates at 2 × 10⁴ cells/well, 6 wells each, and after 24 h, Renilla-WT-RxRE was transfected at 200 ng per well by PEI. After 24 h, the activity of Renilla luciferase was measured with addition of Renilla substrate (Promega, Corp., Madison, WI, USA) and on Centro LB 960 luminometer (Berthold Technologies GmbH & Co KG, Bad Wildbad, Germany). The protein level of the cell lysate in each well was measured, and the corresponding Renilla activity was normalized by the protein level (mg protein).