Fig 2

(A-B) HEK293T ZAP^-/- cells were transfected with HIV-1_WT or HIV-1_CG plasmids and plasmids encoding human ZAP-L-HA or PARP12-HA. Supernatant was harvested after 48h and the infectious virus yield was determined using MT4-R5-GFP target cells (A). Whole cell lysates were analysed by western blotting (B). IU, infectious units. (C) HEK293T ZAP^-/- cells were transfected with plasmids encoding a luciferase reporter gene that contained VSV-G wildtype (WT) or CG-enriched (VSV-G CG) sequences, or IAV-NP WT or CG-enriched sequences as 3’ UTRs, together with plasmids expressing ZAP-L, PARP12 or an empty plasmid (vector). RLU, relative light units. (D) HEK293T ZAP^-/- cells were transfected with plasmids expressing ZAP-L-3xHA or PARP12-3xHA and 24h later culture medium was supplemented with 100μM 4SU. After overnight incubation cells were irradiated with UV light, and ZAP-L/PARP12 were immunoprecipitated. RNA bound to each protein was radiolabeled 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.