Characterization of extracellular vesicle and virus-like particles by single vesicle tetraspanin analysis

ABSTRACT

Extracellular vesicles (EVs) are nano-sized membranous particles secreted by cells. EVs have been classified into subpopulations according to their presumed biogenesis pathway, but their detailed biogenesis mechanisms still need to be fully elucidated. Enveloped viruses are another type of cell-derived nano-vesicles, and their biogenesis processes are much better known than that of EVs. Recently, studies on the similarity between enveloped viruses and EVs have been increasingly reported. The biogenesis of EVs could be better understood if these similarities are adequately investigated. In this study, we utilized a single vesicle imaging technique to visualize the protein expressions of individual nano-sized vesicles and analyzed expression patterns within single vesicles. Using this technique, we identified unique tetraspanin expression patterns in single EVs and that these patterns were closely related to their subcellular origins. The expression of CD9 or CD81 in EVs implied that they originated from the plasma membrane, and the expression of CD63 in EVs implied that they originated from endosomal organelles. We further analyzed the tetraspanin expressions of two different types of virus-like particles (VLPs) and demonstrated that the HIV-Gag-induced VLPs were more similar to EVs than SARS-CoV-2-NP/M/E-induced VLPs. In addition, HIV-Gag-GFP-expressing VLPs were highly colocalized with CD9, CD63, and CD81 signals, whereas SARS-CoV-NP-GFP-expressing VLPs were not. Based on these observations, we could assume that tetraspanin-expressing EVs might be produced through a similar process by which HIV is produced. © 2023