Effects of DDX39B and DDX39A on polymerase activity depend on ATP to recruit TREX complex subunits THOC1, THOC4 and CIP29.

(A) HEK293T cells were co-transfected with HA-DDX39B or FLAG-DDX39B, HA-DDX39A or FLAG-DDX39A, MYC-NP or empty pCAGGS vectors, at 48h after transfection, the cells were lysed treated with or without RNase A, followed by co-IP with anti-FLAG mouse Mab. (B) HEK293T cells were co-transfected with FLAG-NP and HA-DDX39B, HA-DDX39A or empty pCAGGS vectors, at 48h after transfection, the cells were lysed and treated with or without RNase A, followed by co-IP with anti-HA mouse Mab. (C) HEK293T cells were co-transfected with FLAG-NP and PB2 together with HA-DDX39B, HA-DDX39A or empty pCAGGS vectors, at 48h after transfection, the cells were lysed, followed by co-IP with anti-PB2 mouse Mab. (D) HEK293T cells were co-transfected with FLAG-NP and PB1 together with HA-DDX39B, HA-DDX39A or empty pCAGGS vectors, at 48h after transfection, the cells were lysed, followed by co-IP with anti-PB1 mouse Mab. (E) HEK293T cells were co-transfected with FlAG-NP and MYC-NP together with HA-DDX39B, HA-DDX39A or empty pCAGGS vectors, at 48h after transfection, the cells were lysed, followed by co-IP with anti-FLAG mouse Mab. (F) HEK293T cells were co-transfected with NP and HA-DDX39B or HA-DDX39A, at 24h after transfection, the cells were lysed and cell lysates were divided into 2 portions: stored at 4°C or heated at 100°C for 5 min. Reducing SDS/PAGE with 5% acrylamide in the stacking gel and 10% in the running gel was carried out at 4°C and at low current. (G) HEK293T cells were co-transfected with FlAG-DDX39B and HA-DDX39B (WT), HA-DDX39B (WT), HA-DDX39B (95A), HA-DDX39B (197A) or HA-DDX39B (199A) together with MYC-NP or empty pCAGGS vectors, at 48h after transfection, the cells were lysed, followed by co-IP with anti-FLAG mouse Mab. (H) Polymerase genes (PB2627E), poll-Luc, RL-TK and HA-DDX39B together with plasmids encoding THOC1, THOC2, THOC3, THOC4, THOC5, THOC6, THOC7, CIP29 or empty pCAGGS vectors were co-transfected into HEK293T cells, 48h after transfection, the polymerase activity was detected. (I, J and K) HEK293T cells were co-transfected with MYC-THOC1 (I), HA-THOC4 (J) or MYC-CIP29 (K) and FLAG-DDX39B or FLAG-DDX39A, at 48h after transfection, the cells were lysed, followed by co-IP with anti-FLAG mouse Mab. (L) HEK293T cells were transfected with FLAG-DDX39B (WT), FLAG-DDX39B (95A) or empty pCAGGS vectors, at 24h after transfection, the cells were infected with recombinant influenza viruses WSN-H7 (627E) (MOI = 1) and cultured for 12 h, then cells were lysed and treated with or without RNase A, followed by co-IP with anti-FLAG mouse Mab.