Viral determinants of cis- and trans-cleavage by SARS-CoV-2 Nsp3 and an on-off reporter for monitoring intracellular protease activity.

Non-structural protein 3 (nsp3), the largest multifunctional membrane-associated protein encoded by coronaviruses, contains a papain-like protease (PLpro) domain that cleaves viral polypeptides at the nsp1/nsp2, nsp2/nsp3 and nsp3/nsp4 junctions, as well as host substrates such as polyubiquitin chains and ISG15. While host substrate recognition by PLpro is well characterized, the processes by which PLpro identifies and interacts with membrane-associated viral substrates remains poorly defined. In this study, we developed full-length nsp3-containg reporter constructs to investigate PLpro-mediated cleavage in a membrane context. Our results show that differential cleavage sensitivity at nsp2/3 nsp3/4 junction in response to protein expression levels: nsp2/3 cleavage occurred even at low expression levels, whereas efficient nsp3/4 cleavage required high protein dosage, suggesting a reliance on trans-cleavage for the latter. Mutagenesis and structural analysis based on the recently resolved nsp3-4 complex structure suggest that membrane constraints on the substrate, along with positional and conformational requirements of the PLpro domain for cis-cleavage, contribute to the trans-cleavage preference at the nsp3/4 site. Membrane localization and specific protein-protein interactions further facilitate this trans-cleavage process. To assess nsp3/4 cleavage in a more physiologically relevant context, we developed a cleavage-induced degron (CID) system that mimics authentic viral polyprotein processing. This system generates a binary signal upon cleavage at the nsp3/4 junction and response to inhibition by the PLpro inhibitor GRL-0617. Our findings reveal that the domain arrangement of nsp3 dictates trans-dependence of the nsp3/4 cleavage, involving membrane association and protein interactions that may facilitate the subsequent double-membrane vesicle assembly.