Abstract
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 RNAs and, through the recruitment of TRIM25, KHNYN and other cofactors, target them for degradation or prevent their translation. The long and short isoforms of ZAP (ZAP-L and ZAP-S) have different intracellular localization and it is unclear how this regulates their antiviral activity against viruses with different sites of replication. Using ZAP-sensitive and ZAP-insensitive human immunodeficiency virus type I (HIV-1), which transcribe the viral RNA in the nucleus and assemble virions at the plasma membrane, we show that the catalytically inactive poly-ADP-ribose polymerase (PARP) domain in 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 residues in place of the 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 antiviral activity for CpG-enriched HIV-1 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 jointly 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 antiviral activity was further confirmed by inhibition of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication, which replicates in double-membrane vesicles derived from the endoplasmic reticulum. Thus, compartmentalization of ZAP-L on intracellular membranes provides an essential effector function in ZAP-L-mediated antiviral activity against divergent viruses with different subcellular replication sites.
Highlights
Cell-intrinsic antiviral factors are an important line of defence against viral pathogens
In addition to residues in the RNA-binding domain (RBD) that directly interact with a CpG dinucleotide, we identified that the poly (ADP ribose) polymerase (PARP) domain and CaaX box in zinc finger antiviral protein (ZAP)-L are required for its antiviral activity against both viruses
Full-length ZAP contains an RNA binding domain consisting of four CCCH zinc finger domains, a central domain comprised of a fifth CCCH zinc finger and two WWE modules plus a C-terminal PARP domain (Fig 1A) [3,4,16]
Summary
Cell-intrinsic antiviral factors are an important line of defence against viral pathogens. The zinc finger antiviral protein (ZAP, known as PARP13 and encoded by ZC3HAV1) is a broadly active antiviral protein that is induced by both type I and II interferons and is under positive selection in primates [2,3,4,5]. It restricts RNA and DNA viruses as well as endogenous retroelements, with retroviruses and positive-strand RNA viruses being the most commonly used viral systems to study ZAP [6]. While CpG dinucleotides in viral genomes may have multiple deleterious effects on replication [31,32,33], since ZAP has been shown to bind CpG dinucleotides in viral RNA and restrict replication, it has been proposed that ZAP at least partially drives the CpG suppression observed in many vertebrate RNA viruses [18,34,35]
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