Decanoyl rvkr cmk

All studies were performed based on the approved IRB protocols by the University of California (UCSD), San Diego. The human carcinoma cell lines Calu-3 and Caco-2 were purchased from ATCC and were cultured in MEM medium supplemented with 1×NEAA, 100 IU/mL penicillin–streptomycin, and 20% FBS. HEK293T (293T), A549 and Vero cells were maintained in our lab and were cultured in DMEM medium supplemented with 10% FBS. MT4 cell was cultured in RPMI medium 1640 with 10% FBS. 293T cells transduced with lentivirus encoding human ACE2 and TMPRSS2 was maintained in DMEM medium with 10% FBS and 2 ug/ml puromycin. Brefeldin A was purchased from BioLegend; kifunensine, swainsonine, decanoyl-RVKR-CMK, and NGI-1 were purchased from Cayman Chemical; Endo H and PNGase F were purchased from New England Biolabs; The DNA fragments encoding spike of SARS-CoV-2 strains USA-WA1/2020 (WT), B. 1. 351 (Beta) were inserted into pLVX-puro and C-terminal was tagged with Flag, no organelle localization signal was included in this vector . The spike plasmids of B.1.617.2 (Delta), and B.1.1.529 (Omicron) were obtained from InvivoGen. The plasmids of SARS-CoV (CUHK-W1) spike, MERS-CoV (HCoV-EMC/2012) spike, IAV H5N1 (Thailand/KAN-1/2004) HA and NA were purchased from Sino Biological. All the plasmids of viral glycoproteins above were codon-optimized.

Vero cells cultured in a 96-well plate at a cell density of 1 × 10⁵ cells/well were treated with 1% dimethyl sulfoxide (Sigma-Aldrich) or 100 µM decanoyl-RVKR-CMK (Cayman Chemical, MN, USA) and 50 µM E64d (Calbiochem, Darmstadt, Germany) or 50 µM nafamostat (Cayman Chemical), or both, for 1 h. The cells were challenged with 15 µL of pseudotype virus or 50 PFU of recombinant virus and incubated for 15 and 20 h, respectively. Luciferase activity was measured using the Luciferase Assay System (Promega) for pseudotype viruses. The infected cells were visualized by an indirect fluorescence assay using an anti-SARS-CoV-2 N protein antibody (a kind gift by Dr. Okamoto) and anti-mouse IgG antibody-CF488 conjugate (Nacalai Tesque).