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A determinant in the ZAP NTD contributes to the species-specific cognate TRIM25 requirement.(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-1WT and HIV-1CG 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).
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