Picornavirus infection induces temporal release of multiple extracellular vesicle subsets that differ in molecular composition and infectious potential

Several naked virus species, including members of the Picornaviridae family, have recently been described to escape their host cells and spread infection via enclosure in extracellular vesicles (EV). EV are 50-300 nm sized lipid membrane-enclosed particles produced by all cells that are broadly recognized for playing regulatory roles in numerous (patho)physiological processes, including viral infection. Both pro- and antiviral functions have been ascribed to EV released by virus-infected cells. It is currently not known whether this reported functional diversity is a result of the release of multiple virus-containing and non-virus containing EV subpopulations that differ in composition and function. Using encephalomyocarditis virus infection (EMCV, Picornaviridae family), we here provide evidence that EV populations released by infected cells are highly heterogeneous. Virus was contained in two distinct EV populations that differed in physical characteristics, such as sedimentation properties, and in enrichment for proteins indicative of different EV biogenesis pathways, such as the plasma membrane resident proteins Flotillin-1 and CD9, and the autophagy regulatory protein LC3. Additional levels of EV heterogeneity were identified using high-resolution flow cytometric analysis of single EV. Importantly, we demonstrate that EV subsets released during EMCV infection varied largely in potency of transferring virus infection and in their kinetics of release from infected cells. These data support the notion that heterogeneous EV populations released by virus-infected cells can exert diverse functions at distinct time points during infection. Unraveling the compositional, temporal and functional heterogeneity of these EV populations using single EV analysis technologies, as employed in this study, is vital to understanding the role of EV in virus dissemination and antiviral host responses. Author summary Picornaviruses constitute a family of viruses that cause many human and animal diseases. These naked' viruses are known to spread by causing infected cell to burst open. However, challenging recent data indicate that virions also escape intact cells enclosed in small lipid membrane-enclosed particles, called extracellular vesicles' (EV). Inside these cell-derived EV, virions can stay under the radar of the host's immune system, but there is also evidence that EV can facilitate anti-viral immune responses. We therefore hypothesized that different types of EV are released by naked virus-infected cells. Testing this hypothesis required advanced methods with which the very small EV (50-300 nm) can be individually characterized. We investigated the pool of EV released by cells infected with encephalomyocarditis virus (EMCV) using an in-house developed method for analysis and isolation of individual EV. We demonstrate that EMCV infection induces the release of several EV subpopulations that differ in the molecules they carry, the timing of their release from the infected cell, and in potency to transfer virus infection. Our discovery that EV released by naked virus-infected cells vary in composition and function is a critical step in further unraveling the role of EV in virus spreading and virus-host interactions.