Selective Isolation of Retroviruses from Extracellular Vesicles by Intact Virion Immunoprecipitation

Abstract

There exists a wide variety of techniques to isolate and purify viral particles from cell culture supernatants. However, these techniques vary greatly in ease of use, purity, yield and impact on viral structural integrity. Most importantly, it is becoming evident that secreted extracellular vesicles (EVs) co-purify with retroviruses using nearly all purification methods due to nearly indistinguishable biophysical characteristics such as size, buoyant density and nucleic acid content. Recently, our group has illustrated a means of isolating intact and highly enriched retroviral virions from EV-containing cell supernatants using an immunoprecipitation approach targeting the viral envelope glycoprotein of the Moloney Murine Leukemia Virus ( Renner et al., 2018 ). This technique, that we call intact virion immunoprecipitation (IVIP), enabled us to characterize the accessibility of epitopes on the surface of these retroviruses and assess the orientation of the virus-encoded integral membrane protein Glycogag (gPr80) in the viral envelope. Proper implementation of this protocol enables fast, simple and reproducible preparations of intact and highly purified retroviral particles devoid of detectable EV contaminants.

Keywords: Exosomes; Extracellular vesicles; Flow virometry; Glycogag; HIV; IVIP; Immunoprecipitation; Intact virion immunoprecipitation; MLV; Nanoscale flow cytometry; Retrovirus purification; gPr80.