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

Analysis of ISG15 Specificity. Another interaction site between ISG15 and Lb pro centers on the conserved Trp123 of ISG15 ( Fig. 3A and Fig. S4A ), the main hydrophobic ISG15 surface used in other deISGylases (Fig. S4B) (18, 19, 22, 25) . Lb pro utilizes a corresponding hydrophobic ISG15 binding surface formed by Leu92, Pro99 in the α3/α4 loop, and residues, including Leu102 on the α4 helix to shape the overall structure of the binding site ( Fig. 3A and Fig. S3C ); mutations in these residues decrease Lb pro activity toward ISG15 (Fig. 3B and Fig. S4C ). Importantly, this loop is not involved in interactions used during self-processing (Fig. 2E) , and consequently and consistently, mutants in the ISG15 binding site are neither affected in their ability to self-process nor affected in their ability to cleave eIF4G (Fig. S5) . Hence, substrate and ISG15 binding sites are spatially separated. In ISG15, mutation of Trp123 to Ala leads to a significant reduction of cleavage by Lb pro (Fig. 3C) . Trp123 is also a major difference between ISG15 and ubiquitin, where the structurally equivalent residue is Arg42 (Fig. 3D) . Mutation of ubiquitin Arg42 to Trp in the context of an Met1-linked ubiquitin chain significantly reduced cleavage of this chain type by the Met1 linkage-specific DUB OTULIN (26) (Fig. S4D ) but enhanced ubiquitin chain cleavage by Lb pro (Fig. 3E and Fig. S4E ). This reveals one component of how Lb pro distinguishes modifiers.