Poly(ADP-ribose) potentiates ZAP antiviral activity

Fig 1. Modular domain organization of ZAP. (A) Domain diagram of the ZAP primary sequence. Modules are colored according to their structural properties (zinc fingers Z1, Z2, Z3, Z4 and Z5 in blue; WWE1 and WWE2 domains in orange; PARP in green; inter-domain linkers L1, L2, L3, and L4 in gray, red, cyan and magenta). Indicated below are the two major naturally-occurring splice isoforms (ZAP-L and ZAP-S), the minimally active antiviral fragment (ZAP-N), and the central domain described in this study (ZAP-CD). (B) SDS-PAGE profiles of purified recombinant ZAP-CD proteins used in this study. (C) Differential scanning fluorimetry profile of wild type (WT) ZAP-CD shows a single transition. The apparent melting temperature (Tm) is 50.9 ± 0.1°C, determined with five independent protein preparations.

Fig 2. Structure of ZAP-CD. (A) Ribbons representation. Modules are colored according to Fig 1A (Z5 in blue, WWE1 and WWE2 in orange), as are the linkers (L2 in red, L3 in cyan, and L4 in magenta). The amino and carboxyl termini are also indicated. Black dashes denote a flexible loop in the second WWE module with very weak density and left unmodeled. (B) Orthogonal surface views colored according to electrostatic potential from red (negative) to blue (positive). The Z5, WWE1 and WWE2 modules are indicated, as are the putative isoADPr pocket (top) and the extended electropositive cleft (bottom).

Fig 3. Structural analysis of the ZAP WWE modules. (A) Structure of ZAP WWE1. (B) Structure of ZAP WWE2 in ribbons (left) and surface representation (right). Relevant features are labeled. (C) Structure of RNF146 WWE with bound isoADPr ligand (PDB 3V3L) [11]. Left shows the protein in ribbons (gray with the C-terminal extension in yellow) and the ligand in sticks (magenta). Right shows the protein in surface representation. Relevant features are labeled. (D) View of pocket residues in the same orientation as the ribbons in B and C, after backbone-guided superposition of ZAP WWE2 (orange) and RNF146 WWE (gray) with bound isoADPr (magenta). Residues with structural equivalents in ZAP and RNF146 are boxed; residues with no equivalents are unboxed. Hydrogen bonds are shown in yellow.

Fig 4. Structure of ZAP-CD in complex with ADPr. (A) Surface representation (orange) of the ZAP WWE2 pocket with the bound ADPr in stick representation (cyan), in the same orientation as Fig 3B, right panel. Blue mesh shows the 2mFo-DFc map for the ligand after refinement, contoured at 1σ. Green mesh shows the unbiased positive mFo-DFc densities prior to placement of ADPr, contoured at 2.5σ. (B) Details of the binding interaction, with ZAP residues shown in orange and the ADPr carbon atoms in cyan. Phosphate atoms are in pale green. Hydrogen bonds are in yellow.

Fig 5. ZAP-CD binds to PAR in vitro. (A) Size exclusion binding assay with purified ZAP-CD and PAR, performed at room temperature. The three top panels show individual analytical Superdex 200 size exclusion profiles of purified PAR alone (black), ZAP-CD alone (green) and mixed ZAP-CD and PAR after 20 min incubation (orange). The bottom panel shows an overlay of all three curves. Insets show SDS-PAGE analysis of fractions indicated in the bottom panel. Results are representative of two independent experiments, each done in two replicates. (B) Size exclusion assay performed at 4°C. Results are representative of two independent experiments. (C) Representative results of assays (4°C) performed with the indicated ZAP-CD mutants. Results are representative of two independent experiments.

Fig 6. PAR co-immunoprecipitates with ZAP. (A-B) HEK 293T cells were transfected with empty vector or the indicated HA-tagged ZAP-CD constructs (A) or ZAP-L constructs (B). Forty-eight hours later, whole cell lysates (WCL) were subjected to pull-down with anti-HA antibody followed by immunoblotting with the indicated antibodies. Actin was used as loading control. Results are representative of three (ZAP-CD) or two (ZAP-L) independent experiments. (C) Comparison of ZAP-CD and ZAP-L.

Fig 7. ZAP redistributes with PAR in cytoplasmic puncta. HeLa cells were transiently transfected with (A) vector encoding HA-tagged ZAP-CD, (B) ZAP-CD Q668R mutant, or (C) ZAP-L. Twenty-four hours later, cells were treated with sodium arsenite or mock-treated, fixed, immunostained with anti-HA and anti-PAR primary antibodies followed by dye-conjugated secondary antibodies, and imaged by using fluorescence microscopy. Results are representative of three (ZAP-CD) or two (ZAP-L) independent experiments. Scale bars, 10 μm.

Fig 8. Antiviral potency of ZAP-L is reduced by the Q668R mutation. (A) HEK 293T ZAP-/- TRIM25-/- cells were transfected with a provirus of either HIV-1 (NL4.3 WT) control or a CpG-enriched mutant (NL4.3 CG-High), together with a plasmid encoding TRIM25 and increasing concentrations of a plasmid encoding WT ZAP-L or the ZAP-L Q668R mutant. After 48 hours, produced virus was harvested, filtered and titered. (B) Immunoblots of whole cell lysates showing expression levels of HIV-1 proteins (gp160 and gp120) and ZAP-L. Tubulin was used as loading control. Results are representative of two independent experiments. (C) The same experiment as in A was performed with MLV. (D) Immunoblots of whole cell lysates showing ZAP-L expression levels from plasmids co-transfected with MLV. Actin was used as loading control. *, p<0.05; ***, p<0.001; ns, not significant.