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

Substrate Cleavage in Cells. Given the high level of Lb pro activity against ISG15 in vitro ( Fig. 1 A and B) , we were curious whether this activity and noncanonical cleavage were detectable in a biological context. Transfection of FLAG-tagged ISG15 and the ISG15 assembly machinery (UBE1L, UBE2L6, and HERC5) led to robust ISGylation in HeLa cells. Treatment of these cell lysates with Lb pro collapsed the ISGylated proteins, while free ISG15 remained seemingly unchanged ( Fig. 4A and Fig. S6C ). Serendipitously, we observed that ISG15 cross-reacted with a polyclonal antiubiquitin antibody (Fig. S6 A and B) . Treatment with Lb pro specifically removed this cross-reaction, indicating that a fraction of the polyclonal antibody detects an epitope spanning the identical C terminus of both modifiers (Fig. S6 A and B) . Antibody cross-reactivity provided us with a tool to monitor ISG15 cleavage by Lb pro in cell extracts. Indeed, when transfection cell lysates were probed with the polyclonal antiubiquitin antibody, the ISG15 cross-reactive band was no longer detected after Lb pro treatment, indicating that Lb pro had hydrolyzed the ISG15 C terminus (Fig. 4A, red box) . The antiubiquitin blot also showed that ubiquitin modifications in Lb pro -treated samples were only partially processed, confirming ISG15 preference in complex samples (Fig. 4A) . This was in stark contrast to the CCHFV vOTU domain, which cleaves ubiquitin and ISG15 similarly (18, 19) and collapsed both types of signals to the same extent (Fig. 4A) .