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Poly(ADP-ribose) potentiates ZAP antiviral activity

['Guangai Xue', 'Department Of Molecular Physiology', 'Biological Physics', 'University Of Virginia', 'Charlottesville', 'Virginia', 'United States Of America', 'Klaudia Braczyk', 'Daniel Gonçalves-Carneiro', 'Laboratory Of Retrovirology']

Date: 2022-04

Zinc-finger antiviral protein (ZAP), also known as poly(ADP-ribose) polymerase 13 (PARP13), is an antiviral factor that selectively targets viral RNA for degradation. ZAP is active against both DNA and RNA viruses, including important human pathogens such as hepatitis B virus and type 1 human immunodeficiency virus (HIV-1). ZAP selectively binds CpG dinucleotides through its N-terminal RNA-binding domain, which consists of four zinc fingers. ZAP also contains a central region that consists of a fifth zinc finger and two WWE domains. Through structural and biochemical studies, we found that the fifth zinc finger and tandem WWEs of ZAP combine into a single integrated domain that binds to poly(ADP-ribose) (PAR), a cellular polynucleotide. PAR binding is mediated by the second WWE module of ZAP and likely involves specific recognition of an adenosine diphosphate-containing unit of PAR. Mutation of the PAR binding site in ZAP abrogates the interaction in vitro and diminishes ZAP activity against a CpG-rich HIV-1 reporter virus and murine leukemia virus. In cells, PAR facilitates formation of non-membranous sub-cellular compartments such as DNA repair foci, spindle poles and cytosolic RNA stress granules. Our results suggest that ZAP-mediated viral mRNA degradation is facilitated by PAR, and provides a biophysical rationale for the reported association of ZAP with RNA stress granules.

Zinc-finger antiviral protein (ZAP), also known as poly(ADP-ribose) polymerase 13 (PARP13), functions as a host defense mechanism against viruses, including important human pathogens such as hepatitis B virus and type 1 human immunodeficiency virus (HIV-1). ZAP recognizes and binds viral RNA by virtue of their nucleotide composition and directs selective degradation of these viral RNA. Here, we report the X-ray crystal structures of ZAP’s central domain, which we found to bind poly(ADP-ribose) (PAR), a cellular polynucleotide. In cells, PAR is associated with macromolecular assemblages that are implicated in virus inhibition and antiviral signaling. We confirm through biochemical experiments that ZAP indeed binds PAR, both in vitro and in cells. However, the PAR-binding activity of ZAP is not essential to its antiviral function. Instead, we find that PAR binding is an ancillary activity that contributes to the potency of ZAP-mediated virus inhibition.

Funding: This study was funded by the following grants from US National Institutes of Health (NIH), National Institute of Allergy and Infectious Diseases (NIAID): R01-AI150479 (OP), U54-AI150470 (OP, BKGP, PDB) and R01-AI150111 (PDB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2022 Xue et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

In both ZAP-L and ZAP-S, the RBD is connected by a long linker segment to a fifth zinc finger (Z5) and two WWE domains (WWE1 and WWE2) ( Fig 1A ). These additional ZAP domains have unknown function, but WWE domains in other proteins are reported to have a general role in binding to poly(ADP-ribose) (PAR) [ 10 , 11 ]. PAR is a cellular polynucleotide that has been shown to function as a scaffold or collective docking site for multiple protein partners, thereby allowing for sustained co-localization of the components of cellular pathways [ 12 , 13 ]. Here, we show that Z5, WWE1 and WWE2 are sub-domains or modules that integrate into a composite fold, which we term the ZAP central domain (ZAP-CD). Structural and biochemical analyses revealed that ZAP-CD binds to PAR through the second WWE module. Both ZAP [ 14 – 18 ] and PAR [ 14 , 19 ] have been previously reported to localize to so-called RNA stress granules, which constitute a type of non-membranous cytoplasmic compartment that facilitates RNA turnover and antiviral responses [ 20 , 21 ]. Our studies suggest that PAR may coordinate the stable association of ZAP and its co-factors and thereby facilitate efficient recognition and/or degradation of ZAP-bound RNA.

(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 (T m ) is 50.9 ± 0.1°C, determined with five independent protein preparations.

ZAP has a modular organization and is expressed as two major isoforms called ZAP-L and ZAP-S, that arise from alternative splicing and are distinguished by the presence of a C-terminal PARP or poly(ADP-ribose) polymerase-like domain ( Fig 1A ). Both isoforms contain an N-terminal RNA-binding domain (RBD) with four zinc fingers (here termed Z1 to Z4) that bind to CpG dinucleotides in RNA [ 5 , 6 ]. Vertebrate genomes are depleted of CpG content, and it is the relative scarcity of this dinucleotide in cellular RNA compared to susceptible viral RNA that explains selective ZAP-mediated degradation [ 7 ]. A truncated ZAP fragment (here called ZAP-N; Fig 1A ) that essentially consists of only the RBD is both necessary and sufficient for directing viral RNA degradation [ 1 ]. However, there are indications that other ZAP domains are also important for its antiviral function. For example, the PARP-like domain in ZAP-L shows signatures of positive selection, as is found in a host protein that is locked in an antagonistic “arms race” with viruses [ 8 ]. The absence of the PARP-like domain and mutagenesis of its vestigial catalytic site are also reported to negatively affect ZAP antiviral activity [ 8 , 9 ].

Cells encode a variety of nucleic acid sensors that detect the presence of viral RNA or DNA by virtue of non-self features or inappropriate localization. The zinc-finger antiviral protein ZAP (also known as poly(ADP-ribose) polymerase 13 or PARP13) is one such sensor and selectively binds to viral messenger RNA or viral RNA genomes [ 1 , 2 ]. The ZAP-bound RNA molecules are subjected to degradation or translational inhibition, which consequently decreases production of viral proteins and suppresses virus replication [ 1 , 3 , 4 ]. Depending on the virus, the action of ZAP can selectively suppress viral protein expression by up to 30-fold, while cellular protein expression levels remain largely unaffected [ 1 ].

Results

ZAP-CD binds PAR in vitro To directly test whether ZAP interacts with PAR, we enzymatically synthesized and purified PAR polymers in vitro [24,25] (S3A Fig). We then used analytical size exclusion chromatography to test for a binding interaction (Fig 5). In this experiment, a positive interaction can be generally expected to manifest in one of two ways. High-affinity binding can result in formation of a stable complex that elutes with an apparent size (strictly speaking, hydrodynamic radius) greater than the early-eluting component. Less stable complexes can dissociate and exchange with the unbound components during the chromatography run to generate an elution profile in which the late-eluting component peak is smeared towards earlier volumes. For these experiments, we used a PAR fraction whose elution volume (S3B Fig) allowed us to distinguish the unbound PAR from both the bound complexes and unbound ZAP-CD. Note that the PAR polymers in this fraction run as an apparent single band on agarose gel electrophoresis (S3C Fig), but are heterogeneous in length and may even constitute both linear and branched forms. PPT PowerPoint slide

PNG larger image

TIFF original image Download: 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. https://doi.org/10.1371/journal.ppat.1009202.g005 In control experiments, ZAP-CD alone eluted as a single peak from an analytical Superdex 200 column with an elution volume of ~18 mL (Fig 5A, green curve). PAR eluted at an earlier volume of ~14.5 mL from the same column (Fig 5A, black curve). When the two components were mixed prior to sample injection, both types of elution behavior described above were observed. The profile consisted of a peak with an elution volume of ~13.5 mL, which is earlier than either PAR or ZAP-CD alone and indicative of a stable complex; in addition, the trailing ZAP-CD peak was also smeared towards earlier elution volumes (Fig 5A, orange curve). SDS-PAGE confirmed the presence of protein in all the relevant fractions (insets in Fig 5A). These results show that ZAP-CD indeed binds PAR in vitro, and that furthermore, two types of ZAP-CD/PAR interactions can be discerned from the exchange behavior of the complexes during size exclusion chromatography. The above experiments were performed at room temperature, and so we repeated them at 4°C to test if lower temperature would promote more stable binding. Most of the protein shifted to the early-eluting peak when mixed with PAR prior to injection (Fig 5B). This result indicates that the lower temperature indeed disfavored dissociation of the complexes during the chromatography run. As described above, the putative PAR-binding pocket in ZAP-CD contains a buried glutamine residue, Q668, surrounded by hydrophobic sidechains including W611 (Fig 3D). To confirm the importance of this pocket for PAR binding, we purified and tested ZAP-CD proteins harboring W611A, Q668A or Q668R mutations (Fig 1B). Chromatography was performed at 4°C; more efficient binding was observed with the wild type (WT) protein at this temperature and hence is a more stringent test for loss of binding. The mutant proteins did not bind PAR as evidenced by the elution profiles of the mixed samples, which were simple sums of the profiles of the individual components (Fig 5C). These results confirm that the shifts in elution volume arise from specific interactions between ZAP-CD and PAR, and that these interactions involve binding of an ADP-containing unit of PAR to the WWE pocket.

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[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1009202

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