The C-terminal PARP domain of the long ZAP isoform contributes essential effector functions for CpG-directed antiviral activity

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 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.

Zinc-finger antiviral protein-mediated inhibition of porcine epidemic diarrhea virus growth is antagonized by the coronaviral nucleocapsid protein

“…Two major isoforms of ZAP (short and long isoforms) contribute to viral restriction function. Despite sharing a common RNA-binding domain, the long isoform exerts stronger viral inhibition due to the extended C-terminal domain containing the inactive poly-(ADP-ribose) polymerase (PARP)like domain, which has been shown to aid in RNA binding, interact with its cofactor (Kmiec et al, 2021) and recruit an endonuclease protein to degrade bound RNA (Ficarelli et al, 2019). Moreover, the long isoform is constitutively expressed, while the short isoform is dependent on IFN response (Ryman et al, 2005;Wang et al, 2010;Hayakawa et al, 2011;Schwerk et al, 2019).…”

“…As products of alternative RNA splicing, the short and long isoforms of ZAP (ZAPS and ZAPL, respectively) share the N-terminal domain, which contains a highly conserved zinc finger domain. The long isoform has an additional C-terminal portion with an inactive poly-(ADPribose) polymerase (PARP)-like domain that has been shown to contribute to increased antiviral activity (Schwerk et al, 2019;Kmiec et al, 2021). In general, ZAPS is upregulated as one of the interferon (IFN)-stimulated genes (ISGs) during the type I IFN response, whereas ZAPL is constitutively expressed and less dependent on stimulation by interferon or ISGs (Schwerk et al, 2019;Kmiec et al, 2021).…”

The Roles and Pathogenesis Mechanisms of a Number of Micronutrients in the Prevention and/or Treatment of Chronic Hepatitis, COVID-19 and Type-2 Diabetes Mellitus

“…In a study, it was demonstrated that the two human zinc finger antiviral protein (hZAP) isoforms (i.e., h-ZAP S and h-ZAP L) inhibited the replication of HBV in human hepatocyte-derived cells via the viral pgRNA posttranscriptional down-regulation [77]. In a mechanistic manner, the zinc finger motif-containing N-terminus of hZAP has the responsibility of reducing HBV RNA, as well as altering the four zinc finger motifs' integrity in order to facilitate the binding of ZAP to HBV RNA and therefore fulfil the expected antiviral function [78].…”

Positive selection analyses identify a single WWE domain residue that shapes ZAP into a super restriction factor

“…At the C-terminus, there is a PARP-like domain. Despite being one of the 17 PARPs, ZAP is the only PARP with a PARP-like domain that is catalytically inactive and cannot ADP-ribosylate substrates (18,19), but confers more antiviral activity on the longer isoforms (7,15,20,21). Even though the RNA binding activity of ZAP has been extensively studied, how the other domains contribute to ZAP antiviral activity are not well characterized.…”