OTUD4 bridges the association of K-RTA and USP7 to promote KSHV lytic reactivation.

(A) BCBL-1 Tet-K-RTA was induced with Dox (1 μg/ml) for 24 h to trigger lytic reactivation. Co-immunoprecipitation and immunoblotting were performed with the indicated antibodies. (B) HEK293T cells were transfected with the indicated plasmids. WCLs were collected for immunoprecipitation with anti-FLAG affinity agarose, followed by immunoblotting analysis. (C) HEK293T cells transduced with sh-Ctrl or sh-OTUD4 were transfected with the indicated plasmids, followed by immunoprecipitation and immunoblotting analysis at 24 h post-transfection. (D) HEK293T cells transduced with sh-Ctrl or sh-USP7 were co-transfected with PAN-Luc, HA-K-RTA, and FLAG-OTUD4 WT/C45A. Luciferase activities were determined at 24 h post-transfection. (E) SLK.iBAC-GFP cells transduced with sh-Ctrl or sh-USP7 were further stably transduced with vector control, OTUD4 WT or the C45A mutant. Then the stable cells were induced with Dox (1 μg/ml) and sodium butyrate (0.5 mM) to trigger lytic reactivation for 48 h. KSHV infection units in the supernatants were quantified. (F) HEK293T-control or HEK293T-shOTUD4 cells were co-transfected with FLAG-K-RTA, HA-Ub (K48-only) and Myc-USP7, and then treated with MG132 (10 μM). Denatured immunoprecipitation with anti-FLAG affinity agarose was performed, followed by immunoblotting. (G) HEK293T cells were transfected with FLAG-USP7, HA-K-RTA and HA-OTUD4 (1-425aa) (2, 4 or 6 μg). WCLs were collected for immunoprecipitation with anti-FLAG affinity agarose, followed by immunoblotting analysis. (H) HEK293T cells were co-transfected with PAN-Luc, FLAG-K-RTA, and OTUD4 WT or OTUD4 (1-425aa). Luciferase activities were determined at 24 h post-transfection. (I) OTUD4 knockdown SLK.iBAC-GFP cells were stably reconstituted with control vector, OTUD4 or OTUD4 (1-425aa). The stable cells were induced with Dox (1 μg/ml) and sodium butyrate (0.5 mM) to induce lytic reactivation. KSHV infectious units were quantified at 48 h post-induction.