Functional anatomy of zinc finger antiviral protein complexes

Figures

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  Fig. 1. Proteins required for ZAP antiviral activity. a Single-cycle HIV-1WT (black) and HIV-1CG (red), infection of HeLa control cells (transduced with empty CRISPR-no guide- vector) multiplicity of infection (MOI) for input virus is plotted on the x-axis and subsequent virus yield from those infected cells (infectious units/mL) is plotted on the y-axis. Data were reported as mean ± standard error of the mean (sem) of three biological replicates. b–f Single-cycle infection of ZAP−/− (b) TRIM25−/− (c) KHNYN−/− (d) N4BP1−/− (e) KHNYN−/− N4BP1−/− Hela cells, as in (a). g, h Single-cycle infection of KHNYN−/− N4BP1−/− Hela cells as in (a) following reconstitution with a doxycycline-inducible KHNYN-FLAG (g) or N4BP1-3xHA (h) expression vector. Expression of KHNYN-FLAG or N4BP1-3xHA in the presence or absence of Dox is shown on the right. i EV71WT (dark gray bars) vs EV71CG (red bars) replication in the HeLa cell lines as in (a–f) above. Luminescence (relative light units) generated by the NanoLuc reporter EV71 viruses measured at 48 h after infection is shown and error bars represent six biological replicates. j Cartoon schematic representation of HEK293T ZAP−/−/TRIM25−/−/KHNYN−/−/N4BP1−/−…

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  Fig. 2. ZAP domain requirement for CG-specific antiviral activity. a Schematic depicting ZAP domain organization and truncated proteins used for reconstitution experiments, noting C-terminal domain boundaries. ZAP has an N-terminal RNA-binding domain (RBD, red) containing four zinc fingers (ZnFs), a putatively disordered domain (green), a fifth ZnF, WWE (blue), and PARP (purple) domains are at the C-terminus. b Reconstitution experiments in which increasing amounts of plasmids (ng) expressing the truncated ZAP expression plasmids shown in (a) were co-transfected with a constant amount (75 ng) of KHNYN expression plasmid in 293T ZAP−/− TRIM25−/− KHNYN−/− N4BP1−/− cells. Effects on HIV-1WT and HIV-1CG virus yield was measured as infectious units (IU/ml). Data were reported as mean ± sem of n = 4 biological replicates. c Same as (b) except that 75 ng of a TRIM25 expression plasmid was included in each transfection. d Co-immunoprecipitation of C-terminally 3xHA-tagged ZAP proteins (shown in (a)) transiently transfected in 293 T ZAP−/− TRIM25−/− KHNYN−/− N4BP1−/− cells, with or without doxycycline-induced expression of ZAP-N-FLAG or ZAP-L-FLAG. Proteins were immunoprecipitated from cell…

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  Fig. 3. KHNYN domain requirements for complex assembly and antiviral activity. a Schematic showing KHNYN domain organization with an N-terminal KH domain (blue), a NYN domain (orange), and putative endonuclease catalytic site and a C-terminal domain (CTD. green) with homology to formerly annotated CUBAN domains. b Reconstitution experiments in which increasing amounts of KHNYN expression plasmids (ng) were co-transfected with constant amounts of TRIM25 (75 ng) and ZAP-L (150 ng) expression plasmids. Effects on HIV-1WT and HIV-1CG virus yield was measured as infectious units (IU/ml). Data were reported as mean ± sem of n = 5 biological replicates. c Same as (b) except that the TRIM25 expression plasmid was omitted. d Co-immunoprecipitation of TRIM25 with KHNYN-FLAG following transfection of 293T ZAP−/− TRIM25−/− KHNYN−/− N4BP1−/− cells with plasmids expressing the KHNYN-FLAG proteins described in (a, b). Proteins were immunoprecipitated from cell lysates with an anti-FLAG antibody and subjected to western blot analyses. e Co-immunoprecipitation of ZAP-L-3xHA with KHNYN-FLAG following transfection of 293T ZAP−/− TRIM25−/− KHNYN−/− N4BP1−/− cells with plasmids expressing the KHNYN-FLAG…

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  Fig. 4. KHNYN nuclease activity. a Schematic representation of RNA substrates for KHNYN nuclease assays. Positions of CpG dinucleotides are in red on the CG-5 58-mer (right panel), with corresponding ApA substitutions in white on the CG-0 56-mer (left panel). b, c In vitro nuclease assay using KHNYNWT-FLAG (b) and KHNYNcat-FLAG (D565A/D566A) (c) purified by FLAG affinity chromatography from lysates of 293T ZAP−/− TRIM25−/− KHNYN−/− N4BP1−/− cells Nuclease activity was measured for each protein against the single-stranded CG-0 substrate over a 4-h time course. Activity showed vs a mock reaction with no protein added for comparison.

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  Fig. 5. Structure of ZAPX–KHNYNCTD complex. a Formation of a ZAPX-KHNYNCTD complex. Size-exclusion chromatography profiles of ZAPX (dotted trace), KHNYNCTD (dashed trace) and a 1:2 mixture of ZAPX and KHNYNCTD (solid trace) illustrate the larger complex eluting in the protein mixture, consistent with the SDS-PAGE analysis of fractions from the ZAPX-KHNYNCTD mixture shown below the chromatography profile. Red lines mark the fractions containing the ZAPX-KHNYNCTD complex. One experiment was performed. b Overall structure of the ZAPX-KHNYNCTD complex. ZAPX is shown in blue and KHNYNCTD is shown in purple. Zinc ions are shown as gray spheres. c Details of the interaction between ZAPX (blue) and KHNYNCTD (purple) highlighting the three interacting interfaces. Hydrogen bonds are shown in dashed lines. The zinc (gray sphere) binding site formed by H17 and E29 on ZAPX and H692 on KHNYNCTD is shown with dashed lines. A water molecule bound to zinc is shown as a red sphere. d Reconstitution experiments in which increasing amounts of plasmids (ng) expressing WT and point mutant ZAP proteins informed by the crystal structure were co-transfected with a constant amount (75 ng) of KHNYN expression…

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  Fig. 6. Molecular determinants of TRIM25 function. a Schematic representation of TRIM25 highlighting the N-terminal RING domain (orange), two B-box domains (brown), a coiled-coil domain (yellow) that drives inherent dimerization, and a C-terminal SPRY domain (green). b Structure of the TRIM25 RING-domain dimer (from PDB 5EYA) with each subunit displayed in a different gray shade and hydrophobic amino acids at the dimer interface (Leu63, Leu69, and Val72) colored red and green on the respective subunits. c Reconstitution experiments in which increasing quantities of plasmids (ng) expressing wildtype and point mutant TRIM25 were co-transfected with a constant amount of plasmids expressing KHNYN (75 ng) and ZAP-L (150 ng). Wild-type TRIM25 was compared with a deletion of the RING domain, point mutants at hydrophobic residues in the RING domain (L63A, L69A, V72A, and V68A/L69A), a TRIM25 SPRY and proximal 7-Lys (7 K) deletion mutants were tested as well as a 7KA substitution mutant. Effects on HIV-1WT (black) and HIV-1CG (red) virus yield were measured as infectious units (IU/ml). Data were reported as mean ± sem of n = 3 biological replicates. The right panel shows a summary of the relative…

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  Fig. 7. Reconstitution of antiviral activity with designed chimeric antiviral proteins. a Schematic representation of a ZAP-KHNYN chimera constructed by fusing the ZAP RNA binding domain (RBD) to the catalytic NYN domain from KHNYN. b Reconstitution experiments in which 293T ZAP−/− TRIM25−/− KHNYN−/− N4BP1−/− cells were transfected with increasing amounts of plasmids (ng) expressing the ZAPRBD-NYN chimera in the absence of KHNYN or ZAP, in the presence or absence of a TRIM25 expression plasmid (75 ng). Effects on HIV-1WT (black) and HIV-1CG (red) virus yield was measured as infectious units (IU/ml). Data were reported as mean ± sem of n = 4 biological replicates. c Lysates from 293T ZAP−/− TRIM25−/− KHNYN−/− N4BP1−/− cells, transfected as in Fig. 7b were analyzed by western blotting probing with antibodies against RBD-NYN-FLAG, HIV-1 gp120, and GAPDH. d Schematic representation of a TRIM25/ZAP chimera constructed by fusing the N-terminus of TRIM25 (RING-B-box-Coiled-Coil) with the ZAP RBD. e Reconstitution experiments in which 293T ZAP−/− TRIM25−/− KHNYN−/− N4BP1−/− cells were transfected with increasing amounts of plasmids (ng) expressing the TRIM25-ZAPRBD chimera plus KHNYN in the…

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  Fig. 8. Model for mechanism of action of ZAP, KHNYN, and TRIM25 in CpG-specific antiviral activity. a Model for the assembly of ZAP, KHNYN (or N4BP1), TRIM25 and a target RNA that enables the nuclease to specifically deplete CpG RNAs that are recognized through multivalent interaction with ZAP-containing complexes whose multimerization is driven by ZAP:ZAP, ZAP:TRIM25, and TRIM25:TRIM25 interactions. b Details of the interactions between the core functional ZAP NTD a CpG dinucleotide containing RNA and the KHNYN CTD that enable monovalent recruitment of a KHNYN nuclease to a target RNA, (c) Details of the TRIM25 RING-RING interaction that contribute to the formation of ZAP containing complexes that recognize CpG-rich RNA in a multivalent manner.