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

We here show that FMDV Lb pro targets ISG15 with strong preference over ubiquitin and NEDD8 and characterize this specificity biochemically and structurally. We uncover a previously undescribed mechanism by which viruses interfere with the ubiquitin and ubiquitin-like systems. Unlike canonical deISGylases, Lb pro does not target the isopeptide bond formed Significance An understanding of the mechanisms by which viruses evade host immunity is essential to the development of antiviral drugs and viral detection strategies. Ubiquitin and ubiquitinlike modifications are crucial in cellular innate immune and infection responses and are often suppressed by viral proteins. We here identify a previously unknown mechanism of viral evasion. A viral protease, Lb pro , removes ubiquitin and the ubiquitin-like protein ISG15 incompletely from proteins. While this strategy efficiently and irreversibly shuts down these modification systems, it enables repurposing of tools and technologies developed for ubiquitin research in virus detection. Specifically, we show that foot-and-mouth disease virus infection can be detected using an anti-GlyGly antibody developed for ubiquitin mass spectrometry research. during attachment but selectively cleaves a peptide bond in the C terminus of ISG15, which results in incomplete removal of the modifier. A crystal structure of Lb pro covalently bound to a specifically designed ISG15 suicide probe reveals the molecular basis of Lb pro specificity and substrate promiscuity. Importantly, cleavage by Lb pro exposes a GlyGly epitope on substrates of the modifier, and we show that this epitope can be detected in cells during FMDV infection with relative ease. Such mechanism-and activity-based detection strategy opens avenues for distinguishing infected from vaccinated animals and may help limit the economic impact of FMDV.