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

Lb pro Substrate Specificity and Cleavage. We used ubiquitin and ubiquitin-like model substrates (17) to characterize Lb pro DUB activity in vitro (Fig. S1A ) and surprisingly found that while the enzyme lacked robust DUB activity, it targeted ISG15 with high activity and specificity (Fig. 1A) . Ubiquitin cleavage required 1,000fold higher enzyme concentration (Fig. S1B ). Kinetic analysis revealed deISGylase activity comparable with previously studied viral (18, 19) and human enzymes (20) (Fig. 1B and Fig. S1C ). However, a number of standard measurements for deISGylation activity were negative. ISG15-derived suicide probes that covalently modify ISG15-reactive Cys proteases (21, 22) were unable to modify Lb pro ( Fig. 1C and Fig. S1D ). Hydrolysis of ISG15 7-amido-4-methylcoumarin (AMC) by deISGylases, such as the viral ovarian tumor (vOTU) domain of CCHFV, leads to fluorescence, yet Lb pro did not generate a fluorescent signal in this assay ( Fig. S1 E and F) . In contrast, slightly extended precursor forms of full-length proISG15 (amino acids 1-165; featuring an additional eight residues at the C terminus) or proISG15 CTD (amino acids 79-165) were rapidly hydrolyzed by Lb pro (Fig. 1D) . Strikingly, mass spectrometry (MS) analysis of Lb pro -treated mature ISG15 CTD (amino acids 79-157) revealed a truncated ISG15 molecule; Lb pro had cleaved ISG15 between Arg155 and the C-terminal Gly156-Gly157 sequence ( Fig. 1 E and F) .