USP7 interacts with K-RTA and promotes KSHV reactivation.

(A-B) HEK293T cells were co-transfected with HA-K-RTA and FLAG-USP10/CYLD (0, 0.5, 1 or 2 μg), and immunoblotting was performed at 24 h post-transfection. (C-D) SLK.iBAC-GFP cells were transduced with sh-Ctrl, sh-USP10 (C) or sh-CYLD (D), and the stable cells were induced with Dox (1 μg/ml) and sodium butyrate (0.5 mM). The expression of the indicated genes was quantified by RT-qPCR, and KSHV infectious units in the supernatants were quantified at 48 h post-induction. (E) SLK.iBAC-GFP cells were transduced with sh-Ctrl or sh-USP7, and the stable cells were induced with Dox (1 μg/ml) and sodium butyrate (0.5 mM) at 48 h post-transduction. The expression of USP7 was quantified by RT-qPCR. (F) HEK293T cells were co-transfected with FLAG-MDM2, HA-Ub (K48-only) and Myc-USP7. Denatured immunoprecipitation with anti-FLAG affinity agarose was performed, followed by immunoblotting. (G) HEK293T cells were co-expressed with FLAG-USP7 and HA-OTUD4, followed by co-immunoprecipitation and immunoblotting at 24 h post-transfection. (H) HEK293T cells were co-expressed with HA-USP7 and FLAG-OTUD4 or the indicated mutants, and WCLs were collected for immunoprecipitation with anti-FLAG affinity agarose at 24 h post-transfection. The input and precipitated samples were analyzed by immunoblotting. (I) GST or GST fusion proteins [GST-USP7 (1-208aa) or GST-USP7 (560-1102aa)] were incubated with FLAG-OTUD4 expressed in HEK293T cells. The binding fractions were analyzed by immunoblotting, and purified GST and GST fusion proteins were visualized by Coomassie Brilliant Blue staining. (J) SLK.iBAC-GFP cells stably transduced with sh-Ctrl or sh-USP7 were induced with Dox (1 μg/ml) for 48 h, and the expression of the indicated gene was quantified by RT-qPCR. (K) BCBL1-Tet-K-RTA cells transduced with sh-Ctrl or sh-USP7 were induced with Dox (1 μg/ml) for 48 h. WCLs were collected and analyzed by immunoblotting.

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