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. 2011 Oct;85(20):10899–10904. doi: 10.1128/JVI.00690-11

Fig. 2.

Fig. 2.

Reduction of RIG-I ubiquitination induced by KSHV ORF64. (A) HEK293T cells were transfected with vector (Vec), RIG-I–2CARD, or MAVS together with vector or KSHV ORF64. IFN-β, NF-κB, and ISRE promoter activities were assessed by dual-luciferase assays. (B) Vector, ORF64, RTA, or vIRF2 was transfected together with RIG-I–2CARD and reporter plasmids. IFN-β promoter activity was assessed by a dual-luciferase assay. (C) GST-RIG-I–2CARD was transfected with vector, ORF64 (WT), or vIRF2 into HEK293T cells. RIG-I–2CARD ubiquitination was analyzed by GST pulldown (GST-PD), followed by immunoblotting (IB) with antibodies as indicated. Whole-cell lysates (WCL) were analyzed by IB to show expression levels. aUB, anti-Ub. (D) GST–RIG-I–2CARD was transfected with vector, ORF64 (WT), or the ORF64-C29G mutant (C29G) into HEK293T cells, followed by treatment with dimethyl sulfoxide (DMSO) or MG132 (20 μM) for 4 h before harvest. RIG-I–2CARD ubiquitination was analyzed as described for panel C. (E) GST–RIG-I–2CARD and Flag-MAVS-CARD were transfected with vector, ORF64 (WT), or the ORF64-C29G mutant (C29G) into HEK293T cells as indicated. Cell lysates were subjected to GST-PD, followed by IB with antibodies as indicated. (F) GST–RIG-I–2CARD was transfected with vector or ORF64. TRIM25 (WT) or the TRIM25-RINGCS mutant (RINGCS) was transfected together as indicated. RIG-I–2CARD ubiquitination was analyzed as described for panel C. HA, hemagglutinin. (G) The effect of TRIM25 on ORF64 was analyzed by a luciferase assay. Plasmids were transfected as indicated. The experimental procedures were as described for panel A.