key: cord-0979508-u5nad1fr authors: Kmiec, Dorota; Lista-Brotos, Maria-José; Ficarelli, Mattia; Swanson, Chad M; Neil, Stuart JD title: The C-terminal PARP domain of the long ZAP isoform contributes essential effector functions for CpG-directed antiviral activity date: 2021-06-22 journal: bioRxiv DOI: 10.1101/2021.06.22.449398 sha: 558b572857648206b83b811ed65182ee721ea838 doc_id: 979508 cord_uid: u5nad1fr The zinc finger antiviral protein (ZAP) is a broad inhibitor of virus replication. Its best-characterized function is to bind CpG dinucleotides present in viral RNA and, through the recruitment of TRIM25, KHNYN and other cellular RNA degradation machinery, target them for degradation or prevent their translation. ZAP’s activity requires the N-terminal RNA binding domain that selectively binds CpG-containing RNA. However, much less is known about the functional contribution of the remaining domains. Using ZAP-sensitive and ZAP-insensitive human immunodeficiency virus type I (HIV-1), we show that the catalytically inactive poly-ADP-ribose polymerase (PARP) domain of the long ZAP isoform (ZAP-L) is essential for CpG-specific viral restriction. Mutation of a crucial cysteine in the C-terminal CaaX box that mediates S-farnesylation and, to a lesser extent, the inactive catalytic site triad within the PARP domain, disrupted the activity of ZAP-L. Addition of the CaaX box to ZAP-S partly restored antiviral activity, explaining why ZAP-S lacks CpG-dependent antiviral activity despite conservation of the RNA-binding domain. Confocal microscopy confirmed the CaaX motif mediated localization of ZAP-L to vesicular structures and enhanced physical association with intracellular membranes. Importantly, the PARP domain and CaaX box together modulate the interaction between ZAP-L and its cofactors TRIM25 and KHNYN, implying that its proper subcellular localisation is required to establish an antiviral complex. The essential contribution of the PARP domain and CaaX box to ZAP-L’s CpG-directed antiviral activity was further confirmed by inhibition of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication. Thus, compartmentalization of ZAP-L on intracellular membranes provides an essential effector function in the ZAP-L-mediated antiviral activity. Author summary Cell-intrinsic antiviral factors, such as the zinc-finger antiviral protein (ZAP), provide a first line of defence against viral pathogens. ZAP acts by selectively binding CpG dinucleotide-rich RNAs, which are more common in some viruses than their vertebrate hosts, leading to their degradation. Here, we show that the ability to target these foreign elements is not only dependent on ZAP’s N-terminal RNA-binding domain, but additional determinants in the central and C-terminal regions also regulate this process. The PARP domain and its associated CaaX box, are crucial for ZAP’s CpG-specific activity and required for optimal binding to cofactors TRIM25 and KHNYN. Furthermore, a CaaX box, known to mediate post-translational modification by a hydrophobic S-farnesyl group, caused re-localization of ZAP from the cytoplasm and increased its association with intracellular membranes. This change in ZAP’s distribution was essential for inhibition of both a ZAP-sensitized HIV-1 and SARS-CoV-2. Our work unveils how the determinants outside the CpG RNA-binding domain assist ZAP’s antiviral activity and highlights the role of S-farnesylation and membrane association in this process. RNAs and this is likely to be due at least in part to restriction by ZAP less is known about the functional relevance of the other domains and motifs. 89 We aimed to determine the functional relevance of ZAP's domains and their 90 contribution to the mechanism of CpG-specific antiviral activity. In addition to the RBD, 91 we identified that the PARP domain and CaaX box found in ZAP-L, but not ZAP-S, are (Fig 1C) . 126 Moreover, mutation of residues that directly interact with a CpG dinucleotide, Y108A almost complete loss of CpG-specific inhibition (Fig 2A and B To determine if these residues modulate CpG-specific antiviral activity and 154 explain the apparent lack of inhibition by C-terminally truncated ZAP, we mutated 155 ZAP's residues 786, 818, 875 (canonical triad positions, pink) and 793, 804 and 805 156 (sites under positive selection, green) within the PARP domain ( Fig 2C) . Mutation of 157 the Y786, Y818 and V875 to alanine resulted in a large loss of antiviral function ( Fig 158 2D and 2E) though this was also associated with a substantial decrease in ZAP 159 expression ( Fig 2E) . Mutation of these residues to H-A-E did not alter ZAP expression 160 but led to a significant loss of antiviral activity. Meanwhile, alanine substitutions at 161 positions under positive selection did not affect the antiviral phenotype ( Fig 2D) . 162 Therefore, the residues in these positions in ZAP-L that constitute the triad motif in 163 catalytically active PARPs, but not the rapidly evolving residues within the PARP 164 domain, contribute to CpG-specific viral inhibition. However, this does fully account for 165 the loss of phenotype observed with deletion of ZAP's C-terminus (ΔPARP). While ZAP-L has been reported to be more active than ZAP-S lacking the C-terminal 169 domain ( Fig 3A) [40] [41]. In agreement with data obtained with the 171 C-terminally truncated mutant ΔPARP (Fig 2) , ZAP-S displayed no significant CpG-172 specific HIV-1 antiviral activity ( Fig 3B) . We also tested whether co-expression of both 173 isoforms could have synergistic activity and found that ZAP-S had no significant effect 174 on the CpG-high HIV-1 virus even in the presence of ZAP-L ( Fig S2) . The ZAP-L PARP domain ends with a well-conserved CVIS sequence that 176 forms a CaaX box (Fig S3B) , which mediates a C-terminal post-translational We then determined whether ZAP targeting to intracellular membranes is 237 required for its interaction with ZAP cofactors to mediate its antiviral activity against In this study, we demonstrate that in a robust knockout cell-based system ZAP-L, but 271 not ZAP-S, can efficiently inhibit both a CpG enriched HIV-1 as well as SARS CoV-2. 272 We further demonstrate that the C-terminal PARP domain, and particularly its 273 associated farnesylation motif, is essential for this differential activity. Interestingly, * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.001. Supporting information 575 Figure S1 . Effect of expressed ZAP mutants on viral protein levels. 576 Representative western blots of experiment shown in (A) Fig.1B and (B) Fig.2D . SnapShot: Antiviral Restriction Factors Sindbis Virus Translation Is 623 Inhibited by a PKR/RNase L-Independent Effector Induced by Alpha/Beta 624 Interferon Priming of Dendritic Cells Positive selection and increased 627 antiviral activity associated with the PARP-containing isoform of human zinc-628 finger antiviral protein PARP13 regulates cellular mRNA post-631 transcriptionally and functions as a pro-apoptotic factor by destabilizing 632 TRAILR4 transcript Sars-cov-2 is restricted by zinc finger antiviral protein despite preadaptation to 637 the low-cpg environment in humans Novel host restriction factors implicated in HIV-640 1 replication Inhibition of retroviral RNA production by ZAP, a 643 CCCH-type zinc finger protein Zinc-finger antiviral protein inhibits HIV-1 infection by selectively targeting 647 multiply spliced viral mRNAs for degradation Translational repression precedes and 651 is required for ZAP-mediated mRNA decay The Zinc 654 Finger Antiviral Protein Directly Binds to Specific Viral mRNAs through the 655 CCCH Zinc Finger Motifs The zinc-finger antiviral protein recruits the 658 RNA processing exosome to degrade the target mRNA TRIM25 Enhances the Antiviral Action of Zinc-Finger Antiviral 664 Protein (ZAP) TRIM25 Is Required 667 for the Antiviral Activity of Zinc Finger Antiviral Protein KHNYN is essential for the zinc finger antiviral 671 protein (ZAP) to restrict HIV-1 containing clustered CpG dinucleotides. eLife The role of ZAP and 675 OAS3/RNAseL pathways in the attenuation of an RNA virus with elevated 676 frequencies of CpG and UpA dinucleotides 680 Characterization of Novel Splice Variants of Zinc Finger Antiviral Protein 681 (ZAP) ADP-ribose) potentiates ZAP antiviral activity. bioRxiv CG dinucleotide suppression enables antiviral 688 defence targeting non-self RNA Structure of the zinc-finger antiviral protein in complex 693 with RNA reveals a mechanism for selective targeting of CG-rich viral 694 sequences Molecular 698 Mechanism of RNA Recognition by Zinc-Finger Antiviral Protein The rate of hydrolytic 701 deamination of 5-methylcytosine in double-stranded DNA Patterns of 704 evolution and host gene mimicry in influenza and other RNA viruses Increasing the CpG dinucleotide abundance in the HIV-1 genomic RNA inhibits 708 viral replication HIV-1 Replication through Zinc Finger Antiviral Protein (ZAP)-Dependent and -713 Independent Mechanisms Why is CpG suppressed in the 716 genomes of virtually all small eukaryotic viruses but not in those of large 717 eukaryotic viruses Zinc-Finger Antiviral Protein Inhibits XMRV 720 Infection HTLV-1 contains a high CG dinucleotide content 724 and is susceptible to the host antiviral protein ZAP CpG frequency in the 5' third of the env gene determines sensitivity 729 of primary HIV-1 strains to the zinc-finger antiviral protein Origin and evolution of the zinc finger antiviral protein Expression of the Zinc-Finger Antiviral Protein Inhibits Alphavirus 736 Structure of N-739 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