Irreversible inactivation of ISG15 by a viral leader protease enables alternative infection detection strategies

Swatek, Kirby N.; Aumayr, Martina; Pruneda, Jonathan N.; Visser, Linda J.; Berryman, Stephen; Kueck, Anja F.; Geurink, Paul P.; Ovaa, Huib; van Kuppeveld, Frank J. M.; Tuthill, Tobias J.; Skern, Tim; Komander, David

Crystallography. The Lb pro ∼ISG15 CTD -ΔC complex was purified by anion chromatography (Resource Q) and dialyzed into 50 mM Tris, pH 8.0, 50 mM NaCl, 1 mM EDTA, 5 mM DTT, and 5% glycerol. After dialysis, the complex was concentrated to 4 mg/mL and set up at a 1:1 protein:precipitant ratio in a sitting drop vapor diffusion format. Crystals grew in 2.0 M ammonium sulfate, 0.1 M Na citrate, pH 5.6, and 0.2 M K/Na tartrate and were cryoprotected in mother liquor containing 25% glycerol. Crystallographic data were collected at the Diamond Light Source synchrotron (Table S1 ). Additional descriptions of the methods are listed in SI Methods. Model for Lb pro action against ISG15 and ubiquitin. Lb pro preferentially targets ISG15 over ubiquitin, which results from an optimized hydrophobic ISG15 binding site. The acidic groove coordinates the C terminus of ISG15 into the active site of Lb pro and enables cleavage between Arg and GlyGly of the modifiers. This has two consequences: it renders ISG15 or ubiquitin incapable of (re-) conjugation and leaves substrates modified with a GlyGly remnant on their Lys residues. GlyGly-modified Lys remnants can be detected using available antibodies, enabling infection detection strategies. NTD, N-terminal ubiquitin-like domain.