Deepa Chaphekar, University of Toronto, Scarborough

Deepa Chaphekar1,2, Rubesan CJ Rajakumar1, Arvin T Persaud1,2, Claire Fernandes1,2, Aiman Farheen1,2, Christina Guzzo1,2,3. 1. Department of Biological Sciences, University of Toronto Scarborough, 1265 Military Trail, Toronto, ON M1C 1A4, Canada 2. Department of Cell and Systems Biology, University of Toronto, 25 Harbord Street, Toronto, ON M5S 3G5, Canada 3. Department of Immunology, University of Toronto, 1 King's College Circle, Toronto, Canada

Building & Escaping

Virus stocks have a ubiquitous presence of extracellular vesicles (EVs), and since these two particles (viruses and EVs) exhibit similarities in size and surface protein compositions, it can be challenging to discriminate them with currently available tools. Distinguishing EVs from virus particles is critical to better understand how EV-virus interactions can influence outcomes of viral infection. Herein, we have expanded the applications of flow virometry (FV) to discern putative EVs and HIV-1 particles within virus samples by developing a dual staining approach for simultaneously targeting surface antigens and internal virus structural proteins (HIV Gagp24; capsid). Since the HIV-1 capsid protein is known to be minimally incorporated into EVs it was utilized in this study as an EV-virus differentiating factor. Our data identified heterogeneity in antigen expression on putative EVs (Gagp24 negative) and viruses (Gagp24 positive), highlighting the importance of using multiple markers to reliably differentiate viruses and EVs. Immunocapture assays targeting putative EVs and/or viruses were used to deplete different particles from the crude virus stocks, and depletions were confirmed with our FV-based dual staining approach. Moreover, a loss of Gagp24 concentration and particle infectivity was observed in virus-depleted fractions, further supporting our discernment of putative EVs from viruses using this new dual staining approach. This study provides a novel approach to understand the breadth of particle heterogeneity present within crude virus samples. These new FV-based approaches to discern EVs and viruses can be used to better characterize the interplay of EVs and viruses in infection.