Abstract
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.
Highlights
Recent discoveries indicate that several naked virus species can escape from intact cells via a non-lytic release mechanism involving enclosure in membranous structures that resemble extracellular vesicles (EV) [1,2,3,4]
We hypothesized that different types of EV are released by naked virus-infected cells
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
Summary
Recent discoveries indicate that several naked virus species can escape from intact cells via a non-lytic release mechanism involving enclosure in membranous structures that resemble extracellular vesicles (EV) [1,2,3,4]. The phylogenetically distinct hepatitis E virus (HEV, family Hepeviridae), which like HAV is a hepatotropic virus, has been shown to leave cells in an EV-enclosed form [8]. There is strong evidence that virus-containing EV can transfer the infection to new host cells [1,7]. The co-transfer of host molecules may influence the uptake of virus-containing EV, as has been described for phosphatidylserine (PS) lipids that contribute to cellular entry of EV-enclosed poliovirus and HAV [1,13,14]. EV-mediated release of virus or host products from infected cells can trigger the antiviral immune response in EV-targeted cells that are non-susceptible or non-permissive to infection with the naked virus [13]. The reported proand antiviral effects of virus-induced EV could be explained by experimental variation
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