Date Published: February 19, 2019
Publisher: Public Library of Science
Author(s): Susanne G. van der Grein, Kyra A. Y. Defourny, Huib H. Rabouw, Chenna R. Galiveti, Martijn A. Langereis, Marca H. M. Wauben, Ger J. A. Arkesteijn, Frank J. M. van Kuppeveld, Esther N. M. Nolte-‘t Hoen, Carolyn B Coyne.
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.
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) [1–4]. EV are submicron-sized (50–300 nm) lipid bilayer-enclosed particles containing proteins and RNA, and are increasingly recognized as an important means of intercellular communication employed by all cells (reviewed in ). EV can play a role in maintaining homeostasis as well as in various pathologies via the delivery of cargo molecules that trigger a response in distant or neighboring recipient cells (reviewed in ). Enclosure of naked virus particles in EV has been predominantly observed for viruses belonging to the Picornaviridae family, a group of small RNA viruses implicated in many human and veterinary diseases. Examples include members of the genus Enterovirus, such as poliovirus, coxsackievirus B3 (CVB3), and enterovirus 71 (EV71), as well as hepatitis A virus (HAV, genus Hepatovirus) [1,2,4,7]. Furthermore, 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 . EV-enclosed naked viruses have also been observed in vivo, for example in serum of HEV and HAV-infected individuals [4,9,10]. Enclosure of virus particles in EV can benefit virus infections in several ways. First, the EV can shield the virus from immune recognition [4,7,11,12]. In addition, virus exit from host cells without inducing cell rupture limits tissue damage and consequent alarming of the immune system. Moreover, 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]. Furthermore, the enclosure of multiple virions per EV could benefit infection by promoting cooperativity between genetic quasi-species . On the contrary, EV-mediated release of virus or host products from infected cells can also trigger the antiviral immune response in EV-targeted cells that are non-susceptible or non-permissive to infection with the naked virus . The reported pro- and antiviral effects of virus-induced EV could be explained by experimental variation between studies, e.g. with regard to virus strains, producer and recipient cell types, and EV isolation methods . Alternatively, multiple EV populations with variable composition and function could be released by infected cells.
To our knowledge, our data provide first evidence that a picornavirus from the Cardiovirus genus, like other members of the Picornaviridae family, can exit cells via enclosure in EV. Using advanced single EV-based analysis and isolation methods, we demonstrated that host cells respond to EMCV infection by releasing a complex mixture of EV that are heterogeneous in their molecular composition and their capacity to transfer viral infection. In addition, we demonstrate differences in the release kinetics of these distinct EV subpopulations during the pre-lytic phase of infection.