Applying intravirion staining to discern extracellular vesicles from viruses with flow virometry

Discriminating virus particles from extracellular vesicles (EVs) is critical for studying their distinct roles in infection. However, it remains a longstanding challenge to discriminate viruses from EVs because both particle types can exhibit similarities in size and protein compositions. Here, we used flow virometry (FV) with a dual staining strategy that combines surface antibody labeling of host-derived antigens with intravirion staining of the viral capsid protein, HIV Gag_p24. This approach revealed substantial heterogeneity in surface proteins present on EVs (Gag_p24 negative particles) and viruses (Gag_p24 positive particles) present within HIV-1 virus stocks. The dual staining approach was also able to successfully confirm immunodepletions of putative EVs and viruses that were targeted for their prominent surface antigens. Reduced Gag_p24 concentration and decreased infectivity from the virus-depleted supernatants further validated the discernment of putative viruses from EVs using the dual staining approach. Furthermore, the persistence of particles within immunodepleted supernatants indicated the presence of EV and virus subsets bearing distinct repertoires of surface antigens. To our knowledge, this is the first demonstration of staining an antigen inside intact HIV-1 particles using FV. These FV-based dual staining approaches provide a powerful new tool to better differentiate HIV-1 particles from EVs, and could be useful in future studies aimed at characterizing the interplay of EVs and viruses in infection.