Figure 4.

Detection of VLP entry of target cells with confocal and electron microscopy.A, schematic of the GFP-VLP entry assay. GFP-VLPs were produced in HEK293 cells and used to infect target cells. After spinoculation and 2-h incubation, cells were fixed, stained with plasma membrane (WGA-Alexa647) and nuclear (Hoechst 3342) stains, and imaged on the confocal microscope. B, APEX2-VLPs were produced in HEK293 cells and used to infect target cells. After spinoculation and 2-h incubation, cells were fixed, processed, and imaged with TEM. APEX reaction was performed on coverslips and blocks sectioned en face to be able to image stained VLPs at the cell periphery (B, and magnified image of the area within white box in A, shown in C and F). At this stage, endosomes filled with stained VLPs are observed entering the cell (indicated with a white box in C and at higher magnification in D). E, at higher magnification, these stained structures are seen to have a darker stained periphery as expected of VLPs with stained S protein (D, red arrow and magnified image of the same in E). F, magnified image of the stained VLPs at the periphery of the cell shows a size distribution that falls within the expected range reported for SARS-CoV-1 and SARS-CoV-2 virus.