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KHNYN is a manganese-dependent endoribonuclease required for ZAP-mediated antiviral restriction

Rebecca L Youle1,2, Maria Jose Lista2, Emma L Brudenell1,3

  • 1The Francis Crick Institute, Macromolecular Structure Laboratory, 1 Midland Road, London NW1 1AT, UK.

Nucleic Acids Research
|December 17, 2025
PubMed

Insights

The Zinc finger antiviral protein (ZAP) pathway uses KHNYN protein to inhibit viral replication. KHNYN

Area of Science:

  • Virology
  • Immunology
  • Structural Biology

Background:

  • Zinc finger antiviral protein (ZAP) is crucial for innate immunity against viral infections.
  • KHNYN protein binds ZAP and is essential for restricting CpG-rich viral genomes.
  • KHNYN possesses diKH, PIN nuclease, and CUE-like domains, all vital for its antiviral function.

Purpose of the Study:

  • To investigate the structural, enzymological, and virological properties of KHNYN's PIN nuclease domain.
  • To elucidate the role of the extended PIN domain (ex-PIN) in ZAP-mediated antiviral activity.

Main Methods:

  • X-ray crystallography to determine the structure of the KHNYN ex-PIN domain.
  • Enzymatic assays to characterize the nuclease activity of KHNYN.
  • Virological studies to assess the impact of KHNYN on viral replication.

Main Results:

  • The crystal structure revealed an extended PIN domain (ex-PIN) with a stabilizing N-terminal arm and an active site tetra-Asp motif.
  • The KHNYN ex-PIN domain exhibits high Mn2+-dependent single-stranded RNA endonuclease activity.
  • Cleavage preference was observed for ApC, ApA, and UpA dinucleotides.

Conclusions:

  • The structure and activity of the KHNYN ex-PIN domain are critical for ZAP-mediated antiviral defense.
  • KHNYN acts as a potent RNA endonuclease, suggesting a novel mechanism in host antiviral response.
  • Manganese ion activation plays a significant, previously unrecognized role in the ZAP-KHNYN antiviral pathway.

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