S-farnesylation is essential for antiviral activity of the long ZAP isoform against RNA viruses with diverse replication strategies

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.

Regulation of KHNYN antiviral activity by the extended di-KH domain and nucleo-cytoplasmic trafficking

“…Immunoprecipitation of KHNYN-GFP pulled down ZAP-L but no detectable ZAP-S (Figure 7A). Supporting this observation, we recently showed that immunoprecipitation of ZAP-L preferentially pulls down KHNYN compared to ZAP-S and mutation of the S-farnesylation motif decreases this interaction [35]. KHNYN∆NES-GFP immunoprecipitated less ZAP-L than wild-type KHNYN (Figure 7A), which suggests that nuclear export of KHNYN is required for it to bind ZAP-L that is localized to the cytoplasmic endomembrane system.…”

“…ZAP subcellular localization appears to regulate its antiviral activity against several viruses in that ZAP-L with an intact S-farnesylation motif mediates more potent restriction than ZAP-S [32][33][34][35]. This correlates with preferential KHNYN and TRIM25 binding to ZAP-L compared to ZAP-S, even though the binding sites for KHNYN and TRIM25 are present in both isoforms [9,12,35,60]. KHNYN subcellular localization also appears to be important in that the CRM1 nuclear export signal is required for full antiviral activity.…”

“…Because we did not observe ZAP re-localization to the nucleus upon CRM1 inhibition by leptomycin B treatment or re-localization of KHNYN to the nucleus (Figure 4D, Figure 5 and Figure 6D), we hypothesized that the KHNYN NES could be required for antiviral activity by allowing it to interact with ZAP in the cytoplasm. ZAP-L has greater antiviral activity than ZAP-S against several viruses, including HIV-1 CpG [32][33][34][35]. This is at least in part because it contains an S-farnesylation motif that localizes it to the endomembrane system and is required for its antiviral activity [33][34][35][36].…”

“…ZAP-L has greater antiviral activity than ZAP-S against several viruses, including HIV-1 CpG [32][33][34][35]. This is at least in part because it contains an S-farnesylation motif that localizes it to the endomembrane system and is required for its antiviral activity [33][34][35][36]. To determine if the nuclear export signal in KHNYN is required for it to interact with ZAP, we performed co-immunoprecipitation experiments in the KHNYN-GFP and KHNYN∆NES-GFP cell lines.…”

“…There are two predominant isoforms for ZAP, ZAP-L and ZAP-S [31,32]. ZAP-L contains a Cterminal S-farnesylation modification that localizes it to the cytoplasmic endomembrane system and it has greater antiviral activity than ZAP-S against some viruses, including CpG-enriched HIV-1 [32][33][34][35][36]. Importantly, KHNYN physically interacts with ZAP and is required for ZAP to restrict retroviruses, including CpGenriched HIV-1 [12,29].…”