Single Particle Tracking Assay to Study Coronavirus Membrane Fusion

Single particle tracking (SPT) of individual virion fusion with host cell membranes using total internal reflection microscopy (TIRFM) is a powerful technique for quantitatively characterizing virus–host interactions. One significant limitation of this assay to its wider use across many types of enveloped viruses, such as coronavirus, has been incorporating non-lipid receptors (proteins) into the supported lipid bilayers (SLBs) used to monitor membrane fusion. Here, we describe a method for incorporating a proteinaceous viral receptor, feline aminopeptidase N (fAPN), into SLBs using cell blebbing of mammalian cells expressing fAPN in the plasma membrane. This receptor binds feline coronavirus (FECV 1683). We describe how to carry out single particle tracking of FECV fusion in this SLB platform to obtain fusion kinetics.