The last two decades have seen the emergence of three major corona virus(CoV) outbreaks. The SARS-CoV-2 spike glycoprotein (S) is a 1273 amino acids type I membrane protein that binds to the ACE2 receptor on the host cell. On the cytosolic side of the membrane is a short endodomain that contains a cysteine rich domain (CRD) capable of undergoing Sacylation/palmitoylation. Palmitoylation of viral proteins has been implicated in replication, viral assembly, budding, and cell fusion. This method presents therole of palmitoylation of the SARS-CoV-2 spike protein using a pseudotyped luciferase lentivirus system as well as SARS-CoV-2 virus. The researchers evaluate the role of spike palmitoylation and it is identified that DHHC9 as a PAT palmitoylating SARS-CoV-2 S protein and demonstrate its co-localization and physical interaction with the S protein in transfected cells and in SARS-CoV-2 infected cells. It also includes SARS-CoV-2-mNG (SARS-CoV-2 stably encoding mNeonGreen) and show that PAT inhibitors also reduce SARS-CoV-2 infection and syncytia formation. Together, these results establish DHHC9 as a potential target against SARS-CoV-2.
The images illustrate the effect of DHHC9 knockdown onSARS-CoV-2 infection in Caco-2 cells. Image A shows that Caco-2 cells were knocked down for DHHC5 or DHHC9 and 48 h later, infected with 36 icSARS-CoV-2-mNG ;24, 48 and 72 h post-infection, the cells were fixed, nucleus stained with DAPI and visualized under a fluorescence microscope. Image B shows that the signal from image A was quantitated, normalized to DAPI and plotted to show the effect of the respective acyltransferase knocked down on icSARS-CoV-2-mNG infection.