OTUD4 promotes KSHV lytic reactivation independent of its DUB activity.

(A) SLK.iBAC-GFP cells stably transduced with sh-Ctrl or sh-OTUD4 were induced with Dox (1 μg/ml) for 48 h, followed by quantification of viral gene expression by RT-qPCR. (B) Immunoblotting of SLK.iBAC-GFP cells as described in Fig 2A. (C) SLK.iBAC-GFP cells as described in Fig 2A were induced with Dox (1 μg/ml) and sodium butyrate (0.5 mM) for 48 h. The supernatants containing infectious virion were collected and used to infect HEK293T cells, and GFP expression was imaged at 48 h post-infection. Scale bars, 100 μm. (D) KSHV infectious units were calculated based on flow cytometry analysis of GFP-positive cell percentage as described in Fig 2C. (E) BCBL1-Tet-K-RTA cells transduced with sh-Ctrl or sh-OTUD4 were induced with Dox (1 μg/ml) and sodium butyrate (0.5 mM) for 48 h. KSHV genome copies in the supernatants were quantified by qPCR. (F) OTUD4 knockdown SLK.iBAC cells were stably reconstituted with control vector, OTUD4 or OTUD4-C45A, and induced with Dox (1 μg/ml) and sodium butyrate (0.5 mM). KSHV infectious units were quantified at 48 h post-induction. (G) SLK.iBAC stable cells as described in Fig 2F was induced with Dox (1 μg/ml) for 48 h, followed by immunoblotting analysis.