castanospermine  (MedChemExpress)

Journal: Journal of Virology

Article Title: Disruption of spike protein N-glycosylation induces its endoplasmic reticulum retention and attenuates SARS-CoV-2 infectivity

doi: 10.1128/jvi.00270-26

Figure Lengend Snippet: Suppression of host N-glycosylation diminishes the infectivity of SARS-CoV-2. ( A ) Schematic diagram of the SARS-CoV-2 spike (S) glycoprotein. N-linked glycosylation sites are depicted as branched structures. The following protein domains are labeled: the N-terminal domain (NTD), receptor binding domain (RBD), fusion peptide (FP), heptad repeat 1 (HR1), heptad repeat 2 (HR2), central helix (CH), transmembrane domain (TM), and C-terminal domain (CTD). ( B–D ) Infectivity of SARS-CoV-2 pseudoviruses. SARS-CoV-2 pseudoviruses were produced in HEK293T cells treated with 1 mM NB-DNJ, 1 mM castanospermine, or 1 μg/mL tunicamycin. The viruses were then used to infect HEK293T-ACE2 ( B ), Caco-2 ( C ), and Calu-3 ( D ) cells. Luciferase activity was analyzed as the fold change relative to that of the WT group ( n = 3). ( E and F ) Infectivity of SARS-CoV and MERS-CoV pseudoviruses. ( G ) Infectivity of SARS-CoV-2 variants of concern (VOCs) in Caco-2 cells. Pseudoviruses were produced in HEK293T cells with or without 1 mM NB-DNJ treatment. Caco-2 cells were used as targets, and luciferase activity was analyzed as the fold change relative to that of the WT group ( n = 3). ( H ) Replication of SARS-CoV-2 △N-GFP (MOI = 0.01) in Caco-2-N cells in the presence of N-glycosylation inhibitors (1 mM NB-DNJ, 1 mM castanospermine, or 1 μg/mL tunicamycin). GFP fluorescence was observed 24 h post-infection via fluorescence microscopy. ( I ) Quantitation of SARS-CoV-2 △N-GFP (MOI = 0.01)-infected Caco-2-N cells via flow cytometry analysis. The data represent three biological replicates. The gating of the analysis is shown in . ( J and K ) Replication of authentic SARS-CoV-2. A549-ACE2 cells were infected with SARS-CoV-2 SH01 strain (MOI = 0.01), in the presence or absence of 1 mM NB-DNJ. Cells were harvested, and viral N gene mRNA levels were determined by RT-PCR ( J ). Viral S protein cleavage was analyzed by WB ( K ). ( L ) Cell-cell fusion mediated by the SARS-CoV-2 S protein. HEK293T cells were transfected with wild-type S and GFP plasmids. Twenty-four hours post-transfection, the spike-expressing cells were cocultured with HEK293T-ACE2 cells, and the GFP fluorescence was visualized via a fluorescence microscope after 8 h of coculture. ( M ) Western blot analysis of S protein cleavage and S1 subunit shedding induced by sACE2 in wild-type spike-expressing cells. sACE2 was added 4 h before cell collection. The supernatants were subjected to S1 immunoprecipitation via anti-FLAG M2 beads, with heavy and light chains serving as loading controls for supernatant IP. ( N ) Western blot analysis was performed to assess the effects of sACE2 (with or without NB-DNJ) on WT pseudovirus packaging, S protein cleavage, and S1 subunit shedding. The cells and supernatants were collected 4 h after sACE2 addition, and virions were purified from the supernatants via ultracentrifugation ( n = 3). ( O ) Schematic diagram of the experimental design for SARS-CoV-2 infection in K18-ACE2 mouse model ( n = 5). ( P–R ) Effect of NB-DNJ treatment on SARS-CoV-2 infection in a mouse model. K18-ACE2 mice treated with PBS or NB-DNJ were infected with BA.5.2. Infectious progeny viruses in lung tissue were determined by focus-forming units (FFUs) ( P ). Total viral N gene mRNA levels in lungs were measured by RT-PCR ( Q ). Lung tissues were analyzed by H&E staining and Immunofluorescence ( R ).

Article Snippet: Reagents, including Miglustat (NB-DNJ) (Cat# HY-17020), Castanospermine (Cat# HY-N2022), tunicamycin (Cat# HY-A0098), furin (Cat# HY- P78747 ), and Camostat mesylate (Cat# HY-13512), were purchased from MedChemExpress (MCE).

Techniques: Glycoproteomics, Infection, Labeling, Binding Assay, Produced, Luciferase, Activity Assay, Fluorescence, Microscopy, Quantitation Assay, Flow Cytometry, Reverse Transcription Polymerase Chain Reaction, Transfection, Expressing, Western Blot, Immunoprecipitation, Purification, Staining, Immunofluorescence