FIG 1.

Ectopic expression of TRIM56 inhibits multiplication of YFV, DENV2, and HCoV-OC43 but not that of EMCV. (A) (Left side) Immunoblot analysis of the expression of N-terminally HA-tagged TRIM56 (using anti-HA antibody) and YFV NS3 (using anti-YFV NS3 antibody) in 293-Flp-In T-REx-TRIM56 cells (293-FIT-T56) in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of tetracycline (Tet) and mock infected (lanes 1 and 2) or infected with YFV-17D for 24 h (MOI = 0.1; lanes 3 and 4). Where indicated, cells were treated with Tet (2 μg/ml) 48 h before and during viral infection. Actin served as a loading control demonstrating equal sample loading. (Right side) Progeny virus production determined by TCID₅₀ assay in culture supernatants of 293-FIT-T56 cells with or without Tet treatment at 24 h after infection with YFV-17D (MOI = 0.1). An asterisk indicates that statistical differences exist between −Tet and +Tet cells with a P value of <0.05. (B) Tet-inducible expression of HA-TRIM56 in HeLa-Flp-In-T-REx-hACE2-TRIM56 cells (HeLa-FitA2-T56) inhibited NS3 protein expression at 72 h after infection with YFV-17D (MOI = 0.5). (C, E, and F) (Left side) Immunoblot analysis of HA-TRIM56 and viral protein expression in mock-infected (lanes 1 and 2) or virus-infected (lanes 3 and 4) 293-FIT-T56 cells with (lanes 2 and 4) or without (lanes 1 and 3) Tet treatment. (Right side) Progeny viral titers in cell-free supernatants of the infected cells in the absence or presence of Tet treatment, under experimental settings similar to those for panel A except that the cells were infected with DENV2-16681 (MOI = 0.1) for 24 h for immunoblot analysis and 72 h for TCID₅₀ assay for panel C, with HCoV-OC43 (MOI = 0.1) for 24 h for panel E, or with EMCV (MOI = 0.01) for 18 h for panel F. The viral protein antibodies used for immunoblotting included anti-DENV2 envelope (E) in panel C, anti-HCoV-OC43 nucleocapsid (N) protein in panel E, and anti-EMCV 3D polymerase (Pol) in panel F. Note that in panel E, densitometry analysis of the HCoV-OC43 N protein bands (which were invariably detected as double bands) showed that there was a 4-fold decrease in N protein abundance in HA-TRIM56-expressing cells (lane 4) compared to nonexpressing cells (lane 3). (D) Tet-inducible expression of HA-TRIM56 in HeLa-FitA2-T56 cells inhibited prM protein expression at 36 h after infection with DENV2 (MOI = 0.1).