King's College London

Research portal

A Nuclear Export Signal in KHNYN Required for Its Antiviral Activity Evolved as ZAP Emerged in Tetrapods

Research output: Contribution to journalArticlepeer-review

Original languageEnglish
JournalJournal of virology
Issue number1
Early online date12 Jan 2023
Accepted/In press21 Dec 2022
E-pub ahead of print12 Jan 2023
PublishedJan 2023

Bibliographical note

Funding Information: We thank members of the Neil and Swanson laboratories, as well as Michael Malim for helpful discussions. The following reagents were obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: TZM-bl from John C. Kappes, Xiaoyun Wu, and Tranzyme Inc; HIV-1 p24 Hybridoma (183-H12-5C) from Bruce Chesebro. The Antiserum to HIV-1 gp120 #20 (ARP421) was obtained from the NIBSC Centre for AIDS Reagents. These studies were funded by Medical Research Council grants MR/S000844/1 and MR/W018519/1 to S.J.D.N. and C.M.S., a Deutsche Forschungsgemeinschaft (German Research Foundation) fellowship to D.K. (Project number: KM 5/1-1), a Wellcome Trust Senior Research Fellowship (WT098049AIA) to S.J.D.N., a Royal Society/Wellcome Trust Sir Henry Dale Fellowship (206200/Z/17/Z) to C.O. and The Francis Crick Institute (IAT and RLY), which receives its core funding from Cancer Research UK (FC001178), the UK Medical Research Council (FC001178) and the Wellcome Trust (FC001178). M.F. was supported by the UK Medical Research Council (MR/R50225X/1) and was a King’s College London member of the MRC Doctoral Training Partnership in Biomedical Sciences. R.L.Y. is a joint King’s College London-The Francis Crick Institute PhD student. Funding Information: This work was supported by the Department of Health via a National Institute for Health Research Comprehensive Biomedical Research Centre award to Guy’s and St. Thomas’ NHS Foundation Trust in partnership with King’s College London and King’s College Hospital NHS Foundation Trust. We declare no competing interests. Publisher Copyright: Copyright © 2023 Lista et al.


King's Authors


The zinc finger antiviral protein (ZAP) inhibits viral replication by directly binding CpG dinucleotides in cytoplasmic viral RNA to inhibit protein synthesis and target the RNA for degradation. ZAP evolved in tetrapods and there are clear orthologs in reptiles, birds, and mammals. When ZAP emerged, other proteins may have evolved to become cofactors for its antiviral activity. KHNYN is a putative endoribonuclease that is required for ZAP to restrict retroviruses. To determine its evolutionary path after ZAP emerged, we compared KHNYN orthologs in mammals and reptiles to those in fish, which do not encode ZAP. This identified residues in KHNYN that are highly conserved in species that encode ZAP, including several in the CUBAN domain. The CUBAN domain interacts with NEDD8 and Cullin-RING E3 ubiquitin ligases. Deletion of the CUBAN domain decreased KHNYN antiviral activity, increased protein expression and increased nuclear localization. However, mutation of residues required for the CUBAN domain-NEDD8 interaction increased KHNYN abundance but did not affect its antiviral activity or cytoplasmic localization, indicating that Cullin-mediated degradation may control its homeostasis and regulation of protein turnover is separable from its antiviral activity. By contrast, the C-terminal residues in the CUBAN domain form a CRM1-dependent nuclear export signal (NES) that is required for its antiviral activity. Deletion or mutation of the NES increased KHNYN nuclear localization and decreased its interaction with ZAP. The final 2 positions of this NES are not present in fish KHNYN orthologs and we hypothesize their evolution allowed KHNYN to act as a ZAP cofactor. IMPORTANCE The interferon system is part of the innate immune response that inhibits viruses and other pathogens. This system emerged approximately 500 million years ago in early vertebrates. Since then, some genes have evolved to become antiviral interferon-stimulated genes (ISGs) while others evolved so their encoded protein could interact with proteins encoded by ISGs and contribute to their activity. However, this remains poorly characterized. ZAP is an ISG that arose during tetrapod evolution and inhibits viral replication. Because KHNYN interacts with ZAP and is required for its antiviral activity against retroviruses, we conducted an evolutionary analysis to determine how specific amino acids in KHNYN evolved after ZAP emerged. This identified a nuclear export signal that evolved in tetrapods and is required for KHNYN to traffic in the cell and interact with ZAP. Overall, specific residues in KHNYN evolved to allow it to act as a cofactor for ZAP antiviral activity.

Download statistics

No data available

View graph of relations

© 2020 King's College London | Strand | London WC2R 2LS | England | United Kingdom | Tel +44 (0)20 7836 5454