Grl0617

293T-ACE2 cells were cultured and infected with SARS-CoV-2 as described above. When indicated, cells were treated with 50 μM of GRL0617 (MedChemExpress #HY-117043) at the time of infection. Cultures were rinsed with PBS and fixed with 4% paraformaldehyde (electronic microscopy grade; Alfa Aesar) in PBS for 10 min at room temperature, treated with 50 mM NH4Cl for 10 min, permeabilized with 0.5% Triton X-100 for 15 min, and blocked with 0.3% BSA for 10 min. Cells were incubated with primary and secondary antibodies for 1 h and 30 min, respectively, in a moist chamber. Nuclei were labeled with Hoechst dye (Molecular Probes). Images were acquired using a LSM700 (Zeiss) confocal microscope equipped with a 63X objective or by Airyscan LSM800 (Zeiss). Image analysis and quantification was performed using ImageJ (Fiji) v2.1.

The Smart^TM chemical library was purchased from Chemdiv (San Diego, USA), which contains 50,080 small-molecule compounds with enhanced diversity and drug-like properties. Remdesivir and its prodrug GS-441524, as well as GRL0617 were purchased from MedChemExpress (NJ, USA). Arg-Leu-Arg-Gly-Gly-AMC (RLRGG-AMC) was purchased from Bachem Bioscience (Bubendorf, Switzerland). ISG15-AMC, ubiquitin-AMC, and HA-Ub-VS were purchased from R&D systems (Minneapolis, USA). Cellular protease assay kit (Cat# 539125) was purchased from Sigma-Aldrich. CellTiter-Glo cell viability assay kit was purchased from Promega. Other chemicals were purchased from Sigma-Aldrich unless specified.