Donghyuk Shin^1,2,3, Rukmini Mukherjee^1,2, Diana Grewe², Denisa Bojkova⁴, Kheewoong Baek⁵, Anshu Bhattacharya^1,2, Laura Schulz⁶, Marek Widera⁴, Ahmad Reza Mehdipour⁶, Georg Tascher¹,  Paul P. Geurink⁷, Alexander Wilhelm^4,11, Gerbrand J. van der Heden van Noort⁷, Huib Ovaa^7,13, Stefan Müller¹, Klaus-Peter Knobeloch⁸, Krishnaraj Rajalingam⁹, Brenda A. Schulman⁵, Jindrich Cinatl⁴, Gerhard Hummer^6,10, Sandra Ciesek^4,11,12 & Ivan Dikic^1,2,3,12,*

¹Institute of Biochemistry II, Faculty of Medicine, Goethe University, Theodor-Stern-Kai 7, 60590, Frankfurt am Main, Germany. ²Buchmann Institute for Molecular Life Sciences, Goethe University, Max-von-Laue-Str. 15, 60438, Frankfurt am Main, Germany. ³Max Planck Institute of Biophysics, Max-von-Laue-Str. 3, 60438, Frankfurt am Main, Germany. ⁴Institute of Medical Virology, University Hospital Frankfurt, Frankfurt am Main, Germany. ⁵Department of Molecular Machines and Signaling, Max Planck Institute of Biochemistry, Martinsried, Germany. ⁶Department of Theoretical Biophysics, Max Planck Institute of Biophysics, Max-von-Laue-Str. 3, 60438, Frankfurt am Main, Germany. ⁷Oncode Institute and Department of Chemical Immunology, Leiden University Medical Centre, Einthovenweg 20, 2333 ZC, Leiden, The Netherlands. ⁸Institute of Neuropathology, Faculty of Medicine, University of Freiburg, Freiburg, Germany. ⁹Cell Biology Unit, University Medical Center of the Johannes Gutenberg University Mainz, 55131, Mainz, Germany. ¹⁰Institute of Biophysics, Goethe University Frankfurt, Frankfurt am Main, Germany. ¹¹Institute of Pharmaceutical Biology, Goethe-University, Frankfurt/Main, Germany. ¹²Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Branch Translational Medicine and Pharmacology, Frankfurt, Germany. ¹³Deceased.

^*Corresponding author

The papain-like protease PLpro is an essential coronavirus enzyme required for processing viral polyproteins to generate a functional replicase complex and enable viral spread1,2. PLpro is also implicated in cleaving proteinaceous post-translational modifications on host proteins as an evasion mechanism against host anti-viral immune responses3–5. Here, we provide biochemical, structural and functional characterization of the SARS-CoV-2 PLpro (SCoV2-PLpro) and outline differences to SARS-CoV PLpro (SCoV-PLpro) in controlling host interferon (IFN) and NF-κB pathways. While SCoV2-PLpro and SCoV-PLpro share 83% sequence identity, they exhibit different host substrate preferences. In particular, SCoV2-PLpro preferentially cleaves the ubiquitin-like protein ISG15, whereas SCoV-PLpro predominantly targets ubiquitin chains. The crystal structure of SCoV2-PLpro in complex with ISG15 reveals distinctive interactions with the amino-terminal ubiquitin-like domain of ISG15, highlighting this high affinity and specificity. Furthermore, upon infection, SCoV2-PLpro contributes to the cleavage of ISG15 from interferon responsive factor 3 (IRF3) and attenuates type I interferon responses. Importantly, inhibition of SCoV2-PLpro with GRL-0617 impairs the virus-induced cytopathogenic effect, fosters the anti-viral interferon pathway and reduces viral replication in infected cells. These results highlight a dual therapeutic strategy in which targeting of SCoV2-PLpro can suppress SARS-CoV-2 infection and promote anti-viral immunity.