Fig 5. TRIM25 binds the ZAP NTD independently of RNA.

(A) HEK293T ZAP^-/- and TRIM25 ^-/- cells were co-transfected with plasmids expressing human ZAP-L-FLAG and full-length untagged human TRIM25 or one of two human TRIM25 truncations (corresponding to the N-terminal 371 or 410 amino acids of TRIM25), that lack the SPRY domain [21]. Proteins were immunoprecipitated from cell lysates with an anti-FLAG antibody and subjected to western blot analysis. IP, immunoprecipitation. IB, immunoblot. (B) HEK293T ZAP^-/- TRIM25 ^-/- cells were co-transfected with proviral plasmids and plasmids encoding human ZAP and full-length TRIM25 or the indicated truncated TRIM25 proteins. Infectious virus yield was determined using MT4-R5-GFP target cells. (C) HEK293T ZAP^-/- cells were transfected with an empty vector or plasmids encoding human ZAP-L-HA, a truncated ZAP lacking the RNA binding domain (ΔZnF ZAP-L) or a truncated ZAP comprising the N-terminal 254 amino acids (ZAP 254). Proteins were immunoprecipitated from cell lysates with an anti-HA antibody and analysed by western blotting to detect overexpressed ZAP-L-HA and endogenous TRIM25. (D) Antiviral activity of ΔZnF ZAP-L was assessed by co-transfecting HEK293T ZAP^-/- cells with indicated proviral plasmids and plasmids encoding full length (WT) or truncated (ΔZnF) human ZAP. Infectious virus yield was determined using MT4-R5-GFP target cells. (E) HEK293T ZAP^-/- cells were transfected with plasmids encoding human ZAP-L (WT) or an RNA-binding mutant of ZAP (RNA^null, R74A, R75A, K76A). After overnight incubation cells were irradiated with UV light, and ZAP proteins were immunoprecipitated. RNA bound to each protein was radiolabelled and protein-RNA adducts were resolved by SDS-PAGE, transferred to a nitrocellulose membrane and exposed to autoradiographic film. In parallel, a western blot of immunoprecipitated proteins was done using anti-HA antibody. IP, immunoprecipitation. IB, immunoblot. (F) HEK293T ZAP^-/- cells were transfected with plasmids encoding HA-tagged human ZAP-L (WT), ΔZnF ZAP-L or ZAP (RNA^null). Cell lysates were treated with RNAse A or a mixture of RNAse A and T1. ZAP protein complexes immunoprecipitated and subjected to western blot analysis to detect overexpressed ZAP-L-HA and endogenous TRIM25. (G) HEK293T ZAP^-/- and TRIM25 ^-/- cells were transfected with plasmids encoding HA-tagged ZAP-L (WT), ΔZnF ZAP-L or ZAP (RNA^null), along with untagged TRIM25 or a TRIM25 RNA binding mutant (TRIM25 7KA). ZAP-HA protein complexes immunoprecipitated and subjected to western blot analysis to detect overexpressed ZAP and TRIM25 proteins.