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

Revealing Lb pro Activity During Viral Infection. To assess the impact of Lb pro activity during viral infection, we first exploited a chimera viral infection model (29) . A mengovirus (a strain of encephalomyocarditis, a picornavirus that is closely related to FMDV) system was engineered, in which the Leader protein was inactivated by mutations and functionally replaced by WT Lb pro or catalytically inactive Lb pro Cys51Ala as a leader protease. We monitored Lb pro activity in a time course with anti-ISG15, antiubiquitin, and anti-GlyGly antibodies (Fig. 4B and Fig. S6D ). With inactive Lb pro , ISG15 modifications were visibly increased 6-8 h postinfection, whereas no visible changes to total ubiquitin and no signal in the anti-GlyGly Western blot were apparent. In contrast, mengovirus with active Lb pro resulted in a decrease in ISG15 signals, a slight decrease in ubiquitin signals, and importantly, the appearance of GlyGly-modified proteins at 6 and especially, 8 h postinfection (Fig. 4B and Fig. S6D ). This indicates that, indeed, production of active Lb pro by the virus leads to incomplete hydrolysis of ISG15 and ubiquitin from substrates, which can be visualized using available ubiquitin proteomics antibodies.