(A) Schematic representation of the domain organization of human ZAP and its paralogues PARP12 and ZAP-like. (B) Diagrams of the organization of PARP12 (in yellow) and ZAP (in red) loci in vertebrate genomes generated using multiple genome browsers (NCBI, Ensembl, Genomicus). Evolutionary distance of the indicated species (presented in million years, Ma) is based on [36]. Chr, chromosome number. (C) Maximum likelihood tree (midpoint rooted) depicting phylogenetic relationships among PARP12- and ZAP-related genes in vertebrate and invertebrate species, generated with BEAST and 1000 bootstrap replicates. Shaded areas loosely clustered sequences from related species in the superclass Tetrapoda (in blue), clade Actinopterygii (Ray-finned fish, in green), the phylum Mollusca (molluscs, in yellow) and the phylum Cnidaria (in red).

(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.

(A) Open reading frames (ORFs) from several organisms belonging to the indicated phyla were collected from the NCBI nucleotide database and dinucleotide frequency ratio (observed/expected) was calculated and frequency distribution for each dinucleotide in ORFs was plotted. (B) Average dinucleotide observed/expected ratio in various animal species. Evolutionary distance (presented in million years, Ma) of indicated species was based on [36]. (C) Average dinucleotide observed/expected ratio in various portions of vertebrate mRNA transcripts vertebrates.

(A) Schematic representation of ZAP chimeras in which the RNA binding domain of mouse, bat, chicken and alligator ZAP was fused to the WWE and PARP-like domain of human ZAP. (B-C) HEK293T ZAP^-/- cells were transfected with HIV-1_WT (W) or HIV-1_CG (C) proviral plasmids and increasing amounts (0ng, 75ng, 145ng, or 290ng) of a plasmid encoding each ZAP chimera. Supernatant was harvested after 48h and the infectious virus yield was determined using MT4-R5-GFP target cells (B). Whole cell lysates corresponding to the highest amount of ZAP plasmids transfected were analysed by western blotting probing with antibodies against HIV-1 proteins and ZAP (C). -, Indicates co-transfection with an empty vector in place of a ZAP expression plasmid.

(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.

(A) Phylogenetic analysis of TRIM25 protein sequences from vertebrate species. Shaded areas indicate cluster sequences from mammals (in blue), birds and reptiles (in green) and ray-finned fish (in yellow). (B) HEK293T ZAP^-/- and TRIM25^-/- cells were co-transfected with an HIV-1_CG proviral plasmid as well as a fixed amount of a plasmid encoding human ZAP or ZAP chimeras from various vertebrate species. Animal names indicate source of the RNA-binding domains for each ZAP chimera. For each ZAP protein, cells were also co-transfected increasing amounts of a plasmid (0ng 10ng, 30ng or 90ng) expressing a TRIM25 protein from the 6 different indicated species. After 48h, infectious virus yield was determined using MT4-R5-GFP target cells.

(A-B) HEK293T ZAP^-/- and TRIM25^-/- cells were co-transfected with indicated proviral plasmids, increasing amounts (0ng, 75ng, 145ng, or 290ng) of a plasmid encoding an FLAG-tagged human ZAP or mouse, bat, chicken and alligator ZAP chimeras and a fixed amount of a plasmid encoding a cognate HA-TRIM25 protein. After 48h, infectious virus yield was determined using MT4-R5-GFP target cells (A). Whole cell lysates were analysed by western blotting probing with antibodies against HIV-1 proteins ZAP-FLAG and TRIM25-HA (B). (C) HEK293T ZAP^-/- and TRIM25^-/- cells were transfected with HIV-1 proviral plasmids, along with increasing amounts (0ng, 75ng, 145ng, or 290ng) of a plasmids expressing human ZAP or ZAP chimeras from various avian species. For cells transfected with human ZAP, a fixed amount of a human TRIM25 expression plasmid was co-transfected, while for the avian ZAP chimeras, chicken TRIM25 was used. After 48h, infectious virus yield was determined using MT4-R5-GFP target cells.

(A) HEK293T ZAP^-/- and TRIM25^-/- cells were transfected with an HIV-1_CG proviral plasmid and a plasmid expressing human ZAP or chicken ZAP chimera. Cells were treated with 4-thiouiridine prior to UV-crosslinking and CLIP analysis. Reads associated with human (top) or chicken (bottom) ZAP were mapped to the HIV-1_CG genome and the read density plotted against position in the HIV-1_CG genome. CpG-enriched region the HIV-1_CG genome is highlighted in red and dashed lines. (B) Contribution (% observed/expected) for each dinucleotide to reads derived from the HIV-1_CG genome to was calculated. Shift in the ratio for each dinucleotide contribution comparing human and chicken ZAP reads was also determined (blue line).

(A) Representation of the crystal structure of the RNA-binding NTD domain of human ZAP (PDB 6UEI, [12]). ZnF 1–4 indicate zinc fingers 1 through 4. Areas colored in green indicate α-helices 1 and 2 at the C-terminus of the NTD. (B, top) Schematic diagram of chicken ZAP (chZAP) chimera, containing the N-terminal 247 amino acids of chicken ZAP, in an otherwise human ZAP background, and chicken ZAP-X (chZAP-X) chimera, that contains the N-terminal 186 amino acids of chicken ZAP in a human ZAP background. chZAP-X contains the two human α-helices highlighted (A). (B, bottom) Sequence alignment of the α-helices 1 and 2 in human ZAP with the corresponding region in chicken ZAP. Colors indicate amino acid identity and conservation. The position numbers for the two α-helices in humanZAP are indicated in green. (C-D) HEK293T ZAP^-/- and TRIM25^-/- cells were co-transfected with HIV-1_WT and HIV-1_CG proviral plasmids, as well as plasmids encoding human ZAP (huZAP), chicken ZAP chimera (chZAP) and the chicken ZAP-X (chZAP-X) chimera and either human TRIM25 or chicken TRIM25. After 48h, infectious virus yield was determined using MT4-R5-GFP target cells (C). Whole cell lysates were analysed by western blotting probing with antibodies against HIV-1 proteins ZAP-FLAG and TRIM25-HA (D).