viruses Review Recent Progress on Exosomes in RNA Virus Infection Liying Zhang, Yichen Ju, Si Chen and Linzhu Ren * College of Animal Sciences, Key Lab for Zoonoses Research, Ministry of Education, Jilin University, Changchun 130062, China; [email protected] (L.Z.); [email protected] (Y.J.); [email protected] (S.C.) * Correspondence: [email protected] Abstract: Recent research indicates that most tissue and cell types can secrete and release membrane- enclosed small vesicles, known as exosomes, whose content reflects the physiological/pathological state of the cells from which they originate. These exosomes participate in the communication and cell-to-cell transfer of biologically active proteins, lipids, and nucleic acids. Studies of RNA viruses have demonstrated that exosomes release regulatory factors from infected cells and deliver other functional host genetic elements to neighboring cells, and these functions are involved in the infection process and modulate the cellular responses. This review provides an overview of the biogenesis, composition, and some of the most striking functions of exosome secretion and identifies physiological/pathological areas in need of further research. While initial indications suggest that exosome-mediated pathways operate in vivo, the exosome mechanisms involved in the related effects still need to be clarified. The current review focuses on the role of exosomes in RNA virus infections, with an emphasis on the potential contributions of exosomes to pathogenesis. Keywords: exosome; RNA virus; infection 1. Introduction Citation: Zhang, L.; Ju, Y.; Chen, S.; Extracellular vesicles (EVs) are small lipid bilayer-enclosed nanoparticles that are Ren, L. Recent Progress on Exosomes released from multiple types of cells [1–3]. The content, size, and membrane composition in RNA Virus Infection. Viruses 2021, of EVs are highly heterogeneous and dynamic and are affected by the cellular source, state, 13, 256. https://doi.org/10.3390/ and environmental conditions [4]. In recent decades, several EVs, including microvesicles, v13020256 apoptotic bodies, and exosomes, have been found to play important roles in various physiological and pathological processes [3,4]. Academic Editor: Philippe Gallay Exosomes were first identified by Harding et al. in 1983 and confirmed by Johnstone Received: 20 January 2021 et al. in 1987 [5,6]. As reported, multivesicular bodies (MVBs) are formed by endosome Accepted: 4 February 2021 budding in the cytoplasm [3], while exosomes are released into the extracellular fluids by Published: 8 February 2021 fusion of MVBs with the cell surface, resulting in secretion [7]. The size of exosomes ranges from 30–120 nm, and exosomes contain a variety of proteins, lipids, and nucleic acids. Publisher’s Note: MDPI stays neutral Exosomes are important intercellular communicators because of their roles in transferring with regard to jurisdictional claims in signals and materials. Depending on the cell type, many cellular functions have been published maps and institutional affil- attributed to exosomes [7]. Importantly, increasing evidence indicates that exosomes play iations. complex roles in viral diseases [8,9]. For example, exosomes can enhance viral infection by spreading viral components, such as proteins and nucleic acids, and inhibit the immune response or promote immune escape [7,10–12]. They can also inhibit infection by triggering innate or acquired immunity and eliminating pathogens via induction of apoptosis or Copyright: © 2021 by the authors. other signaling pathways [7,10–12]. In addition, the viral replication cycle from virus entry Licensee MDPI, Basel, Switzerland. to assembly and release also involves the exosomal pathway [7,10,11]. At present, there This article is an open access article are still no effective preventative, therapeutic, or even diagnostic methods for many viral distributed under the terms and diseases, including human immunodeficiency virus (HIV) infection, viral hepatitis, and conditions of the Creative Commons Attribution (CC BY) license (https:// carcinoma. Exosomes may be used as markers for disease diagnosis, vehicles for treatment, creativecommons.org/licenses/by/ or targets for treatment or vaccine research. Moreover, further research on the relationships 4.0/). between viruses and exosomes is warranted. Viruses 2021, 13, 256. https://doi.org/10.3390/v13020256 https://www.mdpi.com/journal/viruses Viruses 2021, 13, 256 2 of 12 2. Exosome Biogenesis Johnstone first coined the term exosome to describe the process that underlies the transformation from a reticulocyte to a mature erythrocyte. Because the vesicular secretion process was similar to “reverse endocytosis,” small vesicles were termed “exosomes” [6]. Exosomes are formed within the endosomal network, and early endosomes have three fates: recycling, degradation, and exocytosis. The remainder becomes late endosomes, also called MVBs, because of the observation that an MVB contains many small lysosomes to target related proteins and lipids for degradation; the second fate is fusion with the plasma membrane to release internal vesicles into the extracellular milieu, which then are considered exosomes, and the last fate is to remain in the cytoplasm and storage compart- ments [10]. Although the exact mechanism of how this process is regulated is still unclear, scientists have established many theories of exosome biogenesis, including endosomal sorting complex required for transport (ESCRT)-dependent [13] and ESCRT-independent pathways [14], which include the oligomerization of tetraspanin complexes [15], the sph- ingomyelinase pathway that catalyzes ceramide synthesis [16], or phospholipase D2 and adenosine diphosphate (ADP) ribosylation factor-6-mediated intraluminal vesicle (ILV) budding [17]. ESCRTs consist of approximately twenty proteins that assemble into four complexes (ESCRT-0, ESCRT-I, ESCRT-II, and ESCRT-III) with the associated proteins vacuolar protein sorting 4 (VPS4), vesicle trafficking 1 (VTA1), and ALG-2 interacting protein X (ALIX) (Figure1). The ESCRT-0 complex recognizes and sequesters ubiquitylated proteins in the endosomal membrane, whereas the ESCRT-I and ESCRT-II complexes appear to be respon- sible for inward membrane budding with sequestered cargo, and ESCRT-III components subsequently drive vesicle scission. ESCRT-0 comprises hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), which recognizes monoubiquitylated cargo proteins and associates in a complex with signal-transducing adaptor molecule (STAM), epidermal growth factor receptor pathway substrate 15 (Eps15), and clathrin. HRS recruits tumor susceptibility 101 (TSG101) of the ESCRT-I complex, and ESCRT-I is then involved in the recruitment of ESCRT-III through ESCRT-II or ALIX, an ESCRT-accessory protein. Finally, the dissociation and recycling of the ESCRT machinery require an interaction with the AAA-ATPase VPS4 [18,19]. The ESCRT-independent pathway includes ceramide-dependent manner, cluster of differentiation 63 (CD-63)-dependent mechanism, and ESCRT-independent exosomal nanovesicle formation in human embryonic kidney cells (HEK293) (Figure1)[ 20]. For example, oligodendrocytes generate exosomes via the production of ceramide [16], while other cell types rely on the oligomerization of tetraspanin complexes [15,21]. Moreover, Rab GTPases, a family of conserved proteins that regulate vesicular traffick- ing and membrane fusion events, are also involved in exosome formation. Each member of the Rab GTPase family is differentially enriched in each subcompartment of an exosomal pathway: Rab5 is enriched in EVs; Rab7 and Rab 27 in MVBs; and Rab11, Rab25, Rab4, and Rab35 in the slow and rapid recycling routes. Rab9 is present in vesicles intended for the trans-Golgi network (TGN). Many viruses use these Rabs in various phases of the viral life cycle, although whether this usage influences exosomal release has not been investigated [11]. Viruses 2021, 13, 256 3 of 12 Viruses 2021, 13, x FOR PEER REVIEW 3 of 12 Figure 1. ExosomeExosome biogenesis and composition. ExosomesExosomes areare formedformed within within the the endosomal endosomal network network via via ESCRT-dependent ESCRT-depend- entand and ESCRT-independent ESCRT-independent pathways pathways and and fuse fuse with with the plasmathe plasma membrane membrane for releasefor release into into the extracellularthe extracellular space. space. Moreover, More- over, Rab GTPases are also involved in the trafficking and membrane fusion steps of exosome formation. Exosomes are Rab GTPases are also involved in the trafficking and membrane fusion steps of exosome formation. Exosomes are rich in a rich in a wide variety of proteins, lipids, and nucleic acids, which depend on the type of cell and physiological or patho- wide variety of proteins, lipids, and nucleic acids, which depend on the type of cell and physiological or pathological state. logical state. Abbreviations: ADP-ribosylation factor (Arf), ALG-2 interacting protein X (ALIX), charged multivesicular bodyAbbreviations: protein (CHMP), ADP-ribosylation cluster of differentiation factor (Arf), ALG-2 (CD), endoso interactingmal sorting protein complex X (ALIX), required charged for multivesicular transport (ESCRT), body proteinepider- mal(CHMP), growth cluster factor of receptor differentiation pathway (CD), substrate endosomal 15 (Eps15), sorting glyceraldehyde-3-phosphate complex required for transport dehydrogenase (ESCRT), epidermal (GAPDH), growth heat shockfactor protein receptor (HSP),