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.

Immune stimuli shape the small non-coding transcriptome of extracellular vesicles released by dendritic cells.

The release and uptake of nano-sized extracellular vesicles (EV) is a highly conserved means of intercellular communication. The molecular composition of EV, and thereby their signaling function to target cells, is regulated by cellular activation and differentiation stimuli. EV are regarded as snapshots of cells and are, therefore, in the limelight as biomarkers for disease. Although research on EV-associated RNA has predominantly focused on microRNAs, the transcriptome of EV consists of multiple classes of small non-coding RNAs with potential gene-regulatory functions. It is not known whether environmental cues imposed on cells induce specific changes in a broad range of EV-associated RNA classes. Here, we investigated whether immune-activating or -suppressing stimuli imposed on primary dendritic cells affected the release of various small non-coding RNAs via EV. The small RNA transcriptomes of highly pure EV populations free from ribonucleoprotein particles were analyzed by RNA sequencing and RT-qPCR. Immune stimulus-specific changes were found in the miRNA, snoRNA, and Y-RNA content of EV from dendritic cells, whereas tRNA and snRNA levels were much less affected. Only part of the changes in EV-RNA content reflected changes in cellular RNA, which urges caution in interpreting EV as snapshots of cells. By comprehensive analysis of RNA obtained from highly purified EV, we demonstrate that multiple RNA classes contribute to genetic messages conveyed via EV. The identification of multiple RNA classes that display cell stimulation-dependent association with EV is the prelude to unraveling the function and biomarker potential of these EV-RNAs.

The encephalomyocarditis virus Leader promotes the release of virions inside extracellular vesicles via the induction of secretory autophagy.

Naked viruses can escape host cells before the induction of lysis via release in extracellular vesicles (EVs). These nanosized EVs cloak the secreted virus particles in a host-derived membrane, which alters virus-host interactions that affect infection efficiency and antiviral immunity. Currently, little is known about the viral and host factors regulating this form of virus release. Here, we assessed the role of the encephalomyocarditis virus (EMCV) Leader protein, a 'viral security protein' that subverts the host antiviral response. EV release upon infection with wildtype virus or a Leader-deficient mutant was characterized at the single particle level using high-resolution flow cytometry. Inactivation of the Leader abolished EV induction during infection and strongly reduced EV-enclosed virus release. We demonstrate that the Leader promotes the release of virions within EVs by stimulating a secretory arm of autophagy. This newly discovered role of the EMCV Leader adds to the variety of mechanisms via which this protein affects virus-host interactions. Moreover, these data provide first evidence for a crucial role of a non-structural viral protein in the non-lytic release of picornaviruses via packaging in EVs.

Inhibition of the integrated stress response by viral proteins that block p-eIF2-eIF2B association.

Eukaryotic cells, when exposed to environmental or internal stress, activate the integrated stress response (ISR) to restore homeostasis and promote cell survival. Specific stress stimuli prompt dedicated stress kinases to phosphorylate eukaryotic initiation factor 2 (eIF2). Phosphorylated eIF2 (p-eIF2) in turn sequesters the eIF2-specific guanine exchange factor eIF2B to block eIF2 recycling, thereby halting translation initiation and reducing global protein synthesis. To circumvent stress-induced translational shutdown, viruses encode ISR antagonists. Those identified so far prevent or reverse eIF2 phosphorylation. We now describe two viral proteins-one from a coronavirus and the other from a picornavirus-that have independently acquired the ability to counteract the ISR at its very core by acting as a competitive inhibitor of p-eIF2-eIF2B interaction. This allows continued formation of the eIF2-GTP-Met-tRNAi ternary complex and unabated global translation at high p-eIF2 levels that would otherwise cause translational arrest. We conclude that eIF2 and p-eIF2 differ in their interaction with eIF2B to such effect that p-eIF2-eIF2B association can be selectively inhibited.

Intricate relationships between naked viruses and extracellular vesicles in the crosstalk between pathogen and host.

It is a long-standing paradigm in the field of virology that naked viruses cause lysis of infected cells to release progeny virus. However, recent data indicate that naked virus types of the Picornaviridae and Hepeviridae families can also leave cells via an alternative route involving enclosure in fully host-derived lipid bilayers. The resulting particles resemble extracellular vesicles (EV), which are 50 nm-1 µm vesicles released by all cells. These EV contain lipids, proteins, and RNA, and generally serve as vehicles for intercellular communication in various (patho)physiological processes. EV can act as carriers of naked viruses and as invisibility cloaks to evade immune attacks. However, the exact combination of virions and host-derived molecules determines how these virus-containing EV affect spread of infection and/or triggering of antiviral immune responses. An underexposed aspect in this research area is that infected cells likely release multiple types of virus-induced and constitutively released EV with unique molecular composition and function. In this review, we identify virus-, cell-, and environment-specific factors that shape the EV population released by naked virus-infected cells. In addition, current findings on the formation and molecular composition of EV induced by different virus types will be compared and placed in the context of the widely proven heterogeneity of EV populations and biases caused by different EV isolation methodologies. Close interactions between the fields of EV biology and virology will help to further delineate the intricate relationship between EV and naked viruses and its relevance for viral life cycles and outcomes of viral infections.

"Small Talk" in the Innate Immune System via RNA-Containing Extracellular Vesicles.

A newly uncovered means of communication between cells involves intercellular transfer of nano-sized extracellular vesicles (EV), composed of lipids, proteins, and genetic material. EV released by cells of the immune system can play a regulatory role in the induction and suppression of immune responses. These functions may be mediated not only by the bioactive lipids and proteins present in EV but also by EV-associated RNAs. The RNA in EV mainly consists of microRNAs and a large range of other small non-coding RNA species. Since many of these small RNAs have the potential to regulate gene expression, intercellular transfer of these RNAs via EV may cause long-term changes in the function of EV-targeted cells. Several types of innate immune cells release EV that affect innate immune responses and other (patho)physiological processes. Additionally, the innate immune system is influenced by EV released by non-immune cells and EV found in body fluids. In this review, we focus on how EV-associated RNAs contribute to these immune regulatory processes.