Extracellular Vesicles Exploit Viral Entry Routes for ... · deliver viral genomes into recipient...

Extracellular Vesicles Exploit Viral Entry Routes for Cargo Delivery

Helena M. van Dongen,a Niala Masoumi,a Kenneth W. Witwer,b,c D. Michiel Pegtela

Department of Pathology, Exosomes Research Group, Cancer Center Amsterdam, VU University Medical Center, Amsterdam, The Netherlandsa; Department of Molecularand Comparative Pathobiologyb and Department of Neurology,c The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .369INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .370

Clarification of terminology and abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .371VIRUS AND VIRAL GENOME DELIVERY VIA EVs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .371

Common Transport Mechanisms of EVs and Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .371HCV, molecular hitchhiker . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .371Do other enveloped viruses shuttle genomes in EVs? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .372No envelope? No problem . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .372eHAV, HIV, and exosome biogenesis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .372EVs: vehicle or trap? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .372

Target Cell Selectivity of EVs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .372Challenges in EV targeting experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .372

Virus Attachment Molecules Are Exposed on the EV Surface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .373Adhesion molecules on EV surfaces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .373Tetraspanins: a role in EV target cell selection? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .373

MECHANISM OF VIRAL FUSION WITH THE PLASMA MEMBRANE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .374Direct Fusion of Viruses: a Model for EV Uptake? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .374

Common mechanism of fusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .374Human immunodeficiency virus. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .374Epstein-Barr virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .374

A Molecular Viral Toolbox for EV Fusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .374Fusion is utilized as an entry mechanism for subtypes of EVs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .375Endogenous retroviruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .375

THE ENDOCYTIC PATHWAY AS ENTRY MECHANISM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .375Clathrin-Mediated Endocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .376

CME of viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .376CME of EVs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .376

HCV Cell Entry as a Model To Study EV Uptake Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .377Hepatitis C virus. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .377The complex process of HCV cell entry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .377

MACROPINOCYTOSIS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .378SHARED RECEPTORS FOR VIRUS AND EV ENTRY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .378

Lectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .378Heparan Sulfate Proteoglycans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .379Apoptotic Mimicry by EVs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .379

PERSPECTIVES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .380ACKNOWLEDGMENTS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .380REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .380AUTHOR BIOS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .386

SUMMARY

Extracellular vesicles (EVs) have emerged as crucial mediatorsof intercellular communication, being involved in a wide arrayof key biological processes. Eukaryotic cells, and also bacteria,actively release heterogeneous subtypes of EVs into the extra-cellular space, where their contents reflect their (sub)cellularorigin and the physiologic state of the parent cell. Within thepast 20 years, presumed subtypes of EVs have been given arather confusing diversity of names, including exosomes, mi-crovesicles, ectosomes, microparticles, virosomes, virus-likeparticles, and oncosomes, and these names are variously de-fined by biogenesis, physical characteristics, or function. Thelatter category, functions, in particular the transmission of bi-ological signals between cells in vivo and how EVs controlbiological processes, has garnered much interest. EVs have

pathophysiological properties in cancer, neurodegenerativedisorders, infectious disease, and cardiovascular disease, high-lighting possibilities not only for minimally invasive diagnosticapplications but also for therapeutic interventions, like macro-molecular drug delivery. Yet, in order to pursue therapies in-volving EVs and delivering their cargo, a better grasp of EV

Published 2 March 2016

Citation van Dongen HM, Masoumi N, Witwer KW, Pegtel DM. 2016. Extracellularvesicles exploit viral entry routes for cargo delivery. Microbiol Mol Biol Rev80:369-386. doi:10.1128/MMBR.00063-15.

Address correspondence to Kenneth W. Witwer, [email protected], or D. MichielPegtel, [email protected].

Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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targeting is needed. Here, we review recent progress in under-standing the molecular mechanisms underpinning EV uptakeby receptor-ligand interactions with recipient cells, highlight-ing once again the overlap of EVs and viruses. Despite theirhighly heterogeneous nature, EVs require common viral entrypathways, and an unanticipated specificity for cargo delivery isbeing revealed. We discuss the challenges ahead in delineatingspecific roles for EV-associated ligands and cellular receptors.

INTRODUCTION

Viruses and extracellular vesicles (EVs) are heterogeneous,mostly submicron-sized biological particles produced by liv-

ing, metazoan cells; they are capable of intercellular transfer ofbiological materials and genetic information. While the machin-eries that produce viruses and EVs in mammalian cells have manycommonalities (1), viruses have been presumed to be unique intheir ability to replicate a genome in host cells. Enveloped virusescover their capsid structure in a surrounding, host-derived mem-brane, while envelope proteins on the surface coordinate cellulartropism (2). Nonenveloped viruses have an outer protein coat thatis resistant to harsh conditions, such as dryness and extreme pH ortemperature. These viruses are often virulent, causing host celllysis upon virion release.

Recent observations have challenged the traditional classifica-tion of enveloped and nonenveloped viruses. It appears that bothenveloped and nonenveloped viruses have evolved with ingeniouscell entry mechanisms, hijacking host cellular membranes for ge-nome delivery into selected permissive cells. Thus, the distinctionsbetween viruses and certain types of EVs are blurring. These recentinsights fit with the increasing realization that viruses exploit EVsfor several purposes: (i) to enter host cells, (ii) to promote viralspread, and (iii) to avoid immune responses. The contribution ofEVs to viral infections may have broad implications for futurevaccine development (3, 4). A compelling argument for EVs hav-ing a role in viral infections is that EVs produced by virus-infectedcells have altered physiological properties (5), for example, inter-fering with, rather than triggering, immunological responses (6).Apart from incorporating viral proteins, recent studies have indi-cated that EVs can contain virus-derived nucleic acids, includingfunctional, noncoding microRNAs (miRNAs) (7, 8).

Despite major advances in EV research concerning molec-ular characterization, EV cell entry mechanisms have receivedless attention, and it has remained unclear if selective cell tar-geting is achieved and how, for example, EV RNA content isdelivered. Recent in vivo studies have begun to address a por-tion of these questions by looking at biodistribution upon in-travenous administration of “purified” EV populations, butthese studies have so far not revealed highly specific targetingmechanisms (9). One explanation could be that such studieshave typically relied on administering nonphysiological amounts ofpurified EV preparations. Despite limitations, selectivity in EVtargeting in vivo has been revealed between astrocytes and micro-glia (10) in mice, in experiments with purified astrocyte-derivedEVs, although appropriate control EVs from a different cell type,such as neurons or completely unrelated immune cell exosomes,were not used. In a more recent study, it was shown that specificintegrin expression patterns on EVs may cause specific target cellselection (11). EV targeting in vivo was also demonstrated withgenetic mouse models that made use of the Cre-lox system withdefined donor and recipient cells (12–14). This innovative strat-

egy, successfully employed by independent groups in differentmouse models, has demonstrated that functional cell-cell RNAtransfer in vivo can occur via EVs, providing new opportunities tostudy EV targeting and entry mechanisms in a physiological con-text. Nevertheless, many molecular details await elucidation.

In contrast, viral entry mechanisms that are mediated byspecific receptors have been broadly studied both in vitro and invivo, and research in this area has culminated in successfulclinical applications (2). Increased knowledge of the molecularmechanisms by which EVs deliver their cargo into target cellsmay ultimately improve virus-based vaccination, drug deliv-ery, and gene therapy strategies and expand basic understand-ing of oncogenesis (15). Relative to their size, viruses display acomplex “life” cycle, coevolving with their hosts presumablyover long periods of time. EVs, however, are far from uniformand exist in many shapes, sizes, and forms, all presumably withmultiple physiological functions and properties, although as-signing specific tasks to EV subtypes has proven challenging.The same holds true for (sub)viral particles that share similar-ities with EVs or perhaps are better classified as virus-modifiedEVs. Arguably, exosomes are the most recognized EV subtypethat is exploited by viruses (1). Exosomes are commonly de-fined as budding into multivesicular bodies (MVBs), a special-ized type of late endosomes, as intraluminal vesicles, and theyare secreted when the delimiting MVB membrane fuses withthe plasma membrane (PM) (16). Direct mechanistic evidencefor the phenomenon of MVB-PM fusion remains under-whelming, and it is currently unknown if MVB-PM fusion andexosome exocytosis are a regulated or constitutive process (orboth) (17). Exosomes probably also come in subtypes thatcarry distinct cargo molecules, depending on the subcellularlocation and membrane domain from which they were formed(18). We have referred above to several excellent reviews thathave documented our recent understanding in exosome bio-genesis.

Exosomes have been shown to serve two prominent biolog-ical purposes during viral infections: one is the ability to trans-port complete viral genomes into target cells, and the second isto facilitate infection by changing the physiology of target cells.Exosomes that promote viral infection, for example, have beenshown to blunt immune responses (19). There also two mainmechanisms by which exosomes may exert specific physiolog-ical functions on target cells, apart from a purely cellular wastedisposal function, as indicated by early evidence (20, 21). First,binding of exosomes to the target cell surface may be mediatedby specific ligand-receptor recognition, triggering downstreamsignaling events. Second, exosomes could deliver cargo eitherby rapid fusion with the target cell, leading to “direct” deliveryof exosomal content into the cytoplasm, or upon an entry pro-cess generally referred to as receptor-mediated endocytosis.Internalized exosomes may subsequently fuse with the limitingmembrane of the endosome, leading to the release of exosomalcontents into the cytoplasm. It must be noted, however, thatthese processes are highly interrelated (22). In recent years,ample evidence has been provided that exosomes carry anddeliver viral genomes into recipient cells in vitro, as shown forhepatitis C virus (HCV), hepatitis A virus (HAV), and humanherpesvirus 6 (HHV-6) (3, 23, 24). Encapsulation of viral(RNA) genomes in host membranes may be a conserved strat-egy to protect from antibody-mediated neutralization. There is

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some evidence that the human immunodeficiency virus (HIV)can utilize dendritic cell-to-T cell vesicle transfer as an alterna-tive route for productive infection, a mechanism called trans-infection (25), which may also be utilized by certain types ofEVs (26). Combined, these studies seem to confirm the premiseof the Trojan exosome hypothesis that was proposed over adecade ago by Gould et al. (27) and was recently adapted (28).Apart from EVs packaging viral genomes, EVs can also incor-porate selective sets of virus-encoded proteins and/or nucleicacids that could trigger immune responses. Several publishedreviews have dealt with the immunological properties of suchEVs during viral infection and have categorized their compo-sition and molecular contents to a large extent (5, 6, 19, 29).Unfortunately, the exact mechanisms and specific moleculesthat are required for EV-mediated cargo delivery are poorlyunderstood. To effectively combat pathogenic viruses that ex-ploit EVs for their transmission, a detailed understanding oftheir entry mechanisms is required.

In this communication, we review the current understandingof mechanisms of EV entry and propose how infection strategiesof viruses may provide clues into the mechanisms involved. Wefurther address how EVs convey physiologic effects, how they aretransmitted between cells, and how specificity of cellular targetingmight be achieved. For example, it is difficult to imagine that allEVs mediate functional transfer of nucleic acids. In circulation, ithas been estimated that 1 ml of plasma contains 1 to 10 billion EVs(30). As may be expected, the copy number of any given nucleicacid, for example, miRNAs, in this EV fraction is much smaller(31). Due to the vast heterogeneity of EVs and our lack in under-standing their specific production and release pathways, it re-mains difficult to study, or even predict, what the physiologicaleffects of EVs are in vivo and how to discern the role of viruses inEV biology and vice versa. It seems reasonable to conjecture that,considering the large overlap in molecular compositions andphysical properties of EVs and viruses, EVs probably “choose”their target cell and deliver their cargo in a similar fashion as de-scribed for viruses. Because EVs are complex— highly diverse insize, molecular composition, and presumably function—thestudy of the underpinnings of their cell entry mechanism is diffi-cult. Indeed, results from in vitro uptake studies using purified EVpopulations need to be interpreted with caution and may be inap-plicable to the in vivo situation, for which new tools must be de-veloped.

Clarification of terminology and abbreviations. Extracellularvesicles (EVs): a heterogeneous group of generally submicron-sized particles, including exosomes. Exosomes: EVs of 50 to 100nm in diameter, secreted by many cell types; they are producedwithin a multivesicular body as intraluminal vesicles and secretedwhen the membrane of the multivesicular body fuses with theplasma membrane. Multivesicular body (MVB): a specialized typeof late endosome, presumably the origin of exosomes. When it wasclear in a reviewed paper that the described vesicles were derivedfrom MVBs, they are referred to here as exosomes. In all otherinstances when the origin was not defined, the term extracellu-lar vesicles, the official nomenclature used by the InternationalSociety for Extracellular Vesicles (ISEV), has been used. Wewould also like to clarify upfront that in our reviewed papers,the distinction between binding and fusion, as a molecularevent, was challenging.

VIRUS AND VIRAL GENOME DELIVERY VIA EVs

Because of the difficulties that arise from studying the heteroge-neous population of EVs, it is reasonable to study overlap betweentargeting pathways of EVs and viruses. Just as viruses might useEVs to deliver their genomes, EVs might use entry receptors andfusion machinery similar to those used by viruses.

Common Transport Mechanisms of EVs and Viruses

It has been argued provocatively that viruses may have adoptedexisting EV-mediated communication pathways for their infec-tion strategies (32). A notable example is the recent realizationthat many enveloped viruses utilize phosphatidylserine receptorsas part of their targeting and entry strategies (2, 33). The exposureof phosphatidylserine groups on the surfaces of exosomes hasbeen observed in many studies (10, 34, 35) and may thus be one ofthe shared targeting mechanisms of exosomes and viruses. SinceEVs participate in viral pathogenesis, an understanding of themechanisms behind EV targeting and transmission may have im-portant implications for the design of next-generation antiviralvaccines and therapeutics. To provide a perspective on thesemechanisms, we first examined the evidence for virus or viral ge-nome delivery by EVs (Table 1).

HCV, molecular hitchhiker. In 2013, Ramakrishnaiah and col-leagues reported that productive hepatitis C virus infection of cul-tured human hepatoma cells could be established by virion-freeexosomes (36) purified from infected cells. To be sure, it had beenknown for some time that HCV protein can be incorporated intononvirion EVs (37). Also, electron micrographs of HCV prepara-tions from cell cultures showed the presence of exosome-like par-ticles that were larger than canonical HCV virions (38), although

TABLE 1 List of exosome-utilizing viruses

Viruses that may utilizeexosomes Reference(s)

Enveloped DNA virusesEBV Pegtel et al. (7), Vallhov et al. (98), Ruiss

et al. (100)HHV-6 Mori et al. (55), Ota et al. (24)

Nonenveloped DNA virusesAdenoviruses 2 and 5 Gastaldelli et al. (120)Reovirus Stewart et al. (125)KSHV Veettil et al. (126)

Enveloped RNA virusesDengue virus Chahar et al. (167), Van Der Schaar et al.

(122), Meertens et al. (170)HCV Masciopinto et al. (37), Ramakrishnaiah

et al. (36), Longatti et al. (42), Bukonget al. (43)

Influenza A virus Rust et al. (123)HIV Puryear et al. (145), Akiyama et al. (146),

Cladera et al. (154)GBV-C Maidana Giret and Kallas (44)SFV Taylor et al. (121), Gastaldelli et al. (120)

Nonenveloped RNA virusesPicornavirus Feng et al. (23, 59)Coxsackievirus Robinson et al. (51), Klionsky et al. (53),

Paloheimo et al. (52)

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these particles were eliminated by affinity purification (39). Fi-nally, a provocative article in 2012 showed that HCV RNA couldbe transferred to nonpermissive plasmacytoid dendritic cells(pDCs), triggering innate immune responses (40). However, theelaboration that replication-competent RNA could be transmit-ted by EVs blurred the distinction between host and viral vesicles,attracting broad attention to the topic (3, 41). Full-length RNAwas transmitted by exosomes not only from cells infected withHCV clone Jc1 but also from cells containing a subgenomic rep-licon that did not encode viral structural proteins E1, E2, and core(36). This suggested that at least some viral proteins were dispens-able for the “quasi-infectivity” of genome-containing EVs. Fur-ther evidence for virion-independent genome shuttling was pro-vided recently in a study that employed a set of cells with resistanceto different antibiotics to distinguish between true EV-mediatedRNA transfer and cell-cell fusion (42). Szabo’s group, meanwhile,provided an important step toward understanding the in vivo rel-evance of the phenomenon, finding infectious exosomes in bloodof HCV patients (43). Those authors also reported that viral RNAin exosomes was complexed with Argonaute 2 (Ago2) and thereplication-enhancing host miR-122 (43).

Do other enveloped viruses shuttle genomes in EVs? We con-tinue to await evidence that the EV-based alternative infectionstrategy of HCV is in general use by enveloped viruses. The ge-nome of GB virus C (GBV-C, also known as human pegivirus, orhepatitis G virus), a virus associated in some studies with reducedmortality and morbidity of HIV-1-coinfected individuals (44),appears to be infectious when incorporated into serum EVs (45).To date, though, there is no convincing indication that retrovi-ruses use this method. Various HIV-1 RNAs and proteins havebeen detected in EVs, and effects have been imputed to some ofthese (8, 46–49). In one report, several unspliced HIV RNA se-quences were found in association with exosomes, consistent withfull-length genome incorporation (46). Spliced RNAs were notdetected. In another study, short HIV-1 transactivation response(TAR) element transcripts were found in exosomes released frominfected cells, along with smaller RNAs processed from the TARelement (8). However, retroviral RNA conveyed in exosomes hasnot been found to be infectious (8, 46). Similarly, exosomes fromhuman T-lymphotropic virus type 1 (HTLV-1)-infected cells havenot been found to be infectious (50). Even if full-length retroviralRNA could enter cells via exosomal delivery, in contrast with, e.g.,HCV, it would be unlikely to replicate without a functional viralreverse transcriptase. However, it remains formally possible, ifsomewhat implausible, that different EVs could deliver requiredcomponents to a recipient cell in trans.

No envelope? No problem. Several nonenveloped viruses arenow known to have alternative infection capabilities via EV path-ways (Table 1). Coxsackievirus B3 is one nonenveloped virus thatsubverts host EVs. Spreading between cells via shed microvesicles,the virus avoids neutralizing antibodies that are already present(51, 52). Coxsackievirus-bearing microvesicles display not onlyautophagosomal marker LC3 II (53) but also flotillin-1, which isthought to be a general exosome marker (54). Ota et al. showedthat T cells infected by HHV-6 evaded the host immune responseby shedding major histocompatibility complex class I moleculesvia viral particles and exosomes (24). Another study reported thatHHV-6-infected cells even increase MVB and exosome formationto enhance viral glycoprotein release through the exosomal releasepathway (55). Another example of a virus that uses the EV ma-

chinery of the host cell is the nonenveloped picornavirus hepatitisA virus. It appears that there are two distinct virion fractions ofHAV (23): the classical nonenveloped form and a second form, inwhich virus-like particles are encapsulated in host-derived mem-brane structures that closely resemble exosomes (eHAV). Whencirculating in patient blood, these eHAVs are fully infectious andfully mimic enveloped viruses (23).

eHAV, HIV, and exosome biogenesis. Mechanistically, manyviruses and virus-like EVs rely on the same biogenetic pathways.For example, eHAVs involve the ESCRT pathway, the same sys-tem that supports exosome biogenesis into MVBs (23). However,ESCRT components may also be involved in domain buddingfrom the plasma membrane through ARRDC1 molecules (56) andare recruited to PM sites of HIV budding by HIV Gag (57). In fact,the mechanisms of budding at PM and MVB have many similar-ities, as recently highlighted (58). In the case of HAV envelopeacquisition, knockdown of VPS4B and ALIX, two ESCRT-associ-ated proteins, reduced release of eHAV significantly (23).

EVs: vehicle or trap? By borrowing a host envelope, presumablywith no or fewer exposed viral proteins, viruses or viral genomesare less likely to be susceptible to neutralizing antibodies in circu-lation. Indeed, eHAV is less detectable by the adaptive immunesystem, consistent with the observation that detection is delayed inHAV infection by 3 to 4 weeks before specific antibodies areformed (23). Similarly, HCV RNA-containing EVs were recog-nized, albeit significantly less readily, by neutralizing antibodies(36). Interestingly, however, the enveloped form of HAV is moreprone to capture by pDCs (59). Thus, while eluding neutralizingantibodies, eHAV may be more prone to recognition by innateimmune sensors (59). This finding supports the notion that hu-man pDCs have adapted to detect potentially pathogenic virusinfections by internalizing exosome-like vesicles carrying infec-tious HCV genomes (36, 40, 43). Apart from HCV and HAV, wehave evidence that latent herpesvirus-infected cells are also recog-nized in this manner (60), suggesting a general role for exosomesin pDC-mediated sensing of RNA and DNA viruses. Questionsremain about how EVs incorporate, transport, target, and deliverviral genomes into permissive and/or sensory target cells andwhether dedicated receptors are involved.

Target Cell Selectivity of EVs

Most published studies of the physiological properties of EVs havebeen performed in vitro and were pioneered by Morelli and col-leagues (61). We were among the first to show that viral miRNAscould be transferred from B cells via exosomes. In our model, weexploited latently Epstein-Barr virus (EBV)-infected cells to pre-vent viral DNA transmission from obscuring transfer experiments(7). While these studies unequivocally showed that EV popula-tions in coculture carry and deliver virus-derived nucleic acids,i.e., noncoding miRNAs into target cells, we did not study themechanisms by which delivery was mediated. Upon release byproducing cells, viruses must find their target cells via cell surfacereceptors. Receptor usage is a key factor in defining the tropism ofmany viruses (2).

Challenges in EV targeting experiments. In our laboratory, weregularly stain purified EVs from latently EBV-infected cells with alipid dye and incubate these with various recipient cells in culture.Most cell types, sooner or later, internalize at least a proportion ofstained exosomes, seemingly regardless of the cells of origin (Fig.1). This also reflects the heterogeneous nature of most EV popu-

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lations (18). Because the EVs from EBV-infected cells carry viralRNA and viral proteins, including on their surface, it is possiblethese types of vesicles are prone for attachment and internaliza-tion. On the other hand, infectious virions are relatively uniformin size and composition; therefore, they may be much more selec-tive in targeting experiments. Again, this is almost certainly nottrue for EVs, making it difficult to predict how and which EVs orsubpopulations thereof target what cells, since there are as yet noestablished methods to separate EVs into biologically meaningfulsubpopulations, or to study them individually. While advances arebeing made to achieve this goal, for example, via powerful flowcytometry approaches (62), one can be sure that all EVs mustattach to the surface of target cells in order to have any physiolog-ical function.

Virus Attachment Molecules Are Exposed on the EV Surface

Adhesion molecules on EV surfaces. A scan of the EV proteomicdatabases (ExoCarta and EVPedia) (63–65) reveals the presence ofmany adhesion molecules. These include integrins and cell adhe-sion molecules, both members of the immunoglobulin-like super-family. ICAM-1 and VCAM-1 from the immunoglobulin G su-perfamily are enriched in many EVs, a feature that seemsindependent of the producing cell type. The immunoglobulin su-perfamily of proteins is characterized as having domains of be-tween 7 and 9 �-strands arranged in two antiparallel sheets thatform a sandwich structure, stabilized by a conserved disulfidebridge. While VLA-4 seems enriched on the surface of reticulo-cytes and B cell exosomes (66, 67), �M integrin (ITG) and ITG�2are present on dendritic cell exosomes (68, 69). Attachment of EVsonto the target cell can be facilitated by ITG�1, ITG�1, ICAM-1,and VCAM-1, which are expressed on the target cells, although

EVs may also bind to the extracellular matrix (ECM) in an ITG�1

(ITG�4) cation-dependent matter (70, 71). Uptake of DC-derivedexosomes has been defined more clearly. DC-derived, ICAM-1-bearing exosomes bind to activated T cells via lymphocyte-associ-ated antigen 1 (LFA-1) and to DCs via LFA-1, ITG�v, and ITG�3

(61, 72, 73). Intriguingly, recent data suggested that integrins havea role in cell-specific targeting of breast cancer exosomes. Hoshinoand colleagues recently defined specific repertoires of integrinsexpressed on tumor-derived exosomes that seemed distinct fromthe tumor cells (11). They revealed that integrins might dictateexosome adhesion to specific cell types and ECM molecules inparticular organs. Notably, exosomes expressing ITG�v�5 seem tobind specifically to Kupffer cells, mediating liver tropism, whereasexosomal ITG�6�4 and ITG�6�1 bind lung-resident fibroblastsand epithelial cells, governing lung tropism. Finally, exosomesthat seem to lack a specific integrin repertoire home to bone tissue.It will be interesting to learn more about the extent to which in-tegrins are indeed dominant exosomal targeting factors in addi-tional contexts. For example, would the same integrin repertoireon exosomes from a lymphoma have a similar organ tropism tothat described? Quantitative data on lymphoblastoid cell line exo-somes compared to primary B cell exosomes suggest major differ-ences in ITG composition between the exosome populations (60).

In comparison to EVs, viruses also use various receptors fortheir multistep attachment and cellular entry process and ofteninduce changes on the target plasma membrane, activating signal-ing pathways (74). Selectivity of EV binding may also be reliant onexogenous cues. Under inflammatory conditions, surface adhe-sion molecules are activated in fibroblasts that could then tetherEVs more firmly (71). This happens, e.g., in an ITG�2- andITG�1-dependent manner. ITG�2 couples with ICAM-1, andITG�1 still binds to ECM, with possible additional binding toVCAM-1. Moreover, ITG�v�6 is actively packaged into exosomesisolated from prostate cancer cell lines. Apart from having a po-tential adhesion function, �v�6 is also efficiently transferred viaexosomes from a donor cell to an �v�6-negative recipient cell andlocalizes to the cell surface (75). Many adhesion molecules, such asICAMs and integrins, are also inserted into so-called tetraspanin-enriched microdomains (TEMs) (76, 77).

Tetraspanins: a role in EV target cell selection? Tetraspanins(TSPANs) have been proposed to have a role not only in EVformation but also in exosomal fusion with the target cell.TSPANs consist of four transmembrane regions, with intracel-lular amino- and carboxy-terminal tails. According to severalstudies, tetraspanins CD9, CD63, CD81, CD82, CD151,Tspan8 (D6.1A), CD37, and CD53 are enriched in exosomes(78–82). The exact composition of the tetraspanin web on exo-somes may have implications for target cell selection (83).Most TSPANs execute their function in cooperation with in-tegrins. For example, Tspan8 is known to form complexes withintegrins. Tspan8-CD49 complexes on exosomes seemed to in-duce uptake of exosomes by endothelial cells (84). However, atidy separation of EV subsets by assessing tetraspanin incorpo-ration is complicated by heterogeneity at the biological andtechnical levels (85). Although substantial work has been donein this area (82), many questions remain. Which tetraspaninsare enriched in exosomes versus other vesicles? Is there indeeda “tetraspanin code” to separate subclasses of EVs? To whatextent is tetraspanin enrichment dependent on cell type, cellactivation states, and experimental treatments? If certain tet-

FIG 1 Exosomes enter internal compartments of dendritic cells. Green fluo-rescent exosomes from EBV-infected B cells internalized by primary, activatedmonocyte-derived dendritic cells (MoDCs) are shown. The actin cytoskeletonis shown in red (phalloidin staining) and this highlights protrusions of theactivated MoDCs.



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raspanins clearly define EV subsets, perhaps only these EVs canintroduce proteins on the plasma membrane and deliver ge-netic material into the cytosol of target cells. Apart from suchlingering questions, it is difficult to distinguish between a func-tion mediated by attachment alone or through the actual fusionprocess. Nevertheless, it is clear that tetraspanins are involvedin membrane fusion (86) and participate in viral entry. Indeed,as we now review, both HIV and HCV exploit tetraspanins intheir life cycles.

MECHANISM OF VIRAL FUSION WITH THE PLASMAMEMBRANE

Direct Fusion of Viruses: a Model for EV Uptake?

Common mechanism of fusion. Enveloped virus particles canfuse directly with the plasma membrane of target cells at neutralpH and after interaction with cell surface receptors. Endocytosisand subsequent endosomal fusion will be discussed later. HIV andEBV are examples of viruses that are capable of direct fusion at thecell surface to deliver their RNA genomes. The envelopes of theseviruses consist of host membrane lipids, sterols, and proteins. Fu-sion with the plasma membrane is mediated by virus-encodedglycoproteins (74, 87) and fusogenic host factors that include tet-raspanins. The first step to membrane fusion is tethering or at-tachment, as described above. This sets up the initial contact be-tween virus and target cell or between other fusing entities.Docking follows, as the fusion machinery activates and connectsthe two membranes. To bring the membranes close together, thetertiary structure of the viral glycoproteins changes, culminatingin fusion pore formation and dilatation (Fig. 2) (88). Direct evi-dence for direct fusion of EVs with the PM is limited, and themolecules involved await discovery. Nevertheless, EV fusion withthe PM has been suggested by live fluorescence microscopy usinga general lipophilic dye (R18), which increases in intensity in re-cipient cells (89). Viruses might provide clues as to how EVs rec-ognize and attach to the PM and undergo fusion.

Human immunodeficiency virus. The HIV-1 envelope (Env)gene encodes a protein of approximately 160 kDa that becomesdecorated with sugar residues and is hence referred to as glycopro-tein 160 (gp160). This protein is cleaved by host proteases into

gp120 and gp41 subunits. Heterodimers of these subunits clusterin threes on the surface of the virion, resulting in EM-visible“spikes” that are essential for HIV cell entry. The glycoproteinsfunction together with several host cofactors (i.e., CD4, CXCR4,and CCR5) that act as the main determinants of viral tropism.Buried within the gp41 subunit of HIV Env, itself embedded in theviral membrane, is a hydrophobic fusion peptide. When exposedto fusion-activating conditions, the fusion peptide is exposed, un-dergoing a conformational change and insertion into the targetmembrane. Fusion activation starts when gp120 stabilizes the vi-rus to cell attachment by binding to the primary receptor CD4,initiating conformational changes that facilitate interactions ofgp120 with CXCR4 or CCR5 (Fig. 3). In turn, this event triggersconformational changes in gp41. Two well-separated �-helicesfold to form a hairpin-like �-helical bundle. These bundles bringthe viral and cell membrane close together, causing actual fusionof the virus and target cell membranes (90, 91). While most EVswill lack such glycoproteins, other molecules, such as tetraspanins,may have fusogenic properties (79). In agreement with the in-volvement of CD9 and CD81 in the membrane fusion processes(92), it has been suggested that expression of tetraspanins can alterHIV-1 progression. Tetraspanins incorporated into the mem-branes of virus particles may collaborate in fusion (93). In addi-tion to its presence in transmission electron micrographs at theplasma membrane, CD63 also plays an early postentry role priorto or at the reverse transcription step of HIV (94). HIV fusion canalso occur after endocytosis, as we describe below (2).

Epstein-Barr virus. In addition to HIV, human herpesvirusesshow plasticity in their mechanism of entry. The enveloped hu-man large DNA herpesvirus EBV exploits CD21 (complement re-ceptor 2) to target resting naive B cells via EBV gp350/220 glyco-proteins present on its envelope. It does so in combination withother receptor-ligand reactions, including those with HLA-DR1.CD35, another complement receptor, also participates in the tar-get specificity of EBV (95), whereas the virus can enter nasopha-ryngeal epithelial cells through neuropilin 1 (NRP1)-facilitatedinternalization and fusion (96).

A Molecular Viral Toolbox for EV Fusion

For many animal viruses, including the examples we have justvisited, target cell range is limited and defined by the interaction ofviral proteins with a small number of host factors; the target rangeof EVs remains substantially less clear. How could the above in-

FIG 2 Common mechanism of fusion. Here, fusion of a virus with the plasmamembrane is shown, giving an example of the common (not virus-specific)mechanism of fusion. Initial contact is made through tethering. Involved mol-ecules can contribute to target specificity and are regularly linked to fusionmachinery. Tethering is followed by docking, as the fusion machinery con-nects the two membranes. Changes in tertiary structure of this machinery,noted as protein folding, culminate in fusion pore formation and subsequentdilatation. Insertion of fusion loops into the host cell membrane is necessaryfor entry.

FIG 3 HIV cell entry. HIV enters target cells via fusion with the concertedaction of viral glycoproteins gp120 and gp41 and host cofactors such as CD4,CXCR4, and CCR5. The glycoproteins are visible as spikes on electron micros-copy images.

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formation be relevant for EV uptake by direct fusion? Althoughcanonical host exosomal proteins, including CD81, appear to beinvolved in some instances of viral fusion, it is not yet establishedthat they can achieve fusion alone. At the same time, mammalshave a deep and diverse relationship with viruses, endogenous andexogenous. Is it possible that viral factors incorporated into EVscould support fusion?

Fusion is utilized as an entry mechanism for subtypes of EVs.We found that inhibition of exosomal uptake using dynasore, apotent endocytosis inhibitor, cannot completely block the uptakeof exosomes produced by EBV-infected cells (7). As proposedpreviously (97), then, it appears that exosomes have multiple en-try options, including through the PM. EVs, in particular thosereleased by virus-infected cells, share molecular characteristicswith fusogenic viruses (i.e., incorporation of viral glycoproteins)that suggest fusion is utilized as an entry mechanism for at leastsome subtypes of EVs produced by virus-infected cells (Fig. 4). Forexample, exosomes from EBV-infected cells have a plethora of theTSPANs CD9 and CD81, but they also seem to incorporate certainamounts of viral gp350. Thus, viral envelope factors that aresorted into EVs support EV attachment and possibly fusion withtarget cells (98). This process was found to be so efficient that EVsoutcompeted actual viral particles for entry (98). Furthermore,HCV-infected cell-derived exosomes seem to harbor CD81 andE2 glycoproteins, possibly exploiting these molecules’ fusogeniccapabilities (37). However, other studies claim that it is unknownwhether HCV entry receptors are involved in exosome uptake (36,43). Finally, a glycoprotein of herpes simplex virus (HSV) is alsoincorporated into exosomes of HSV-infected cells. Although thismay serve more as an immune escape function, the fate of thesevirus-modified exosomes has not been studied (99).

The propensity of EBV gp350 to mediate targeting of exosomeshas recently been applied in clinical settings (100). The conceptcould certainly be expanded to other virus-derived molecules(29). Nor are we limited to glycoproteins. The EBV latent mem-brane protein 1 (LMP1) is also selectively incorporated intoCD63-enriched exosomes and may have several purposes (101).Besides signaling domains, LMP1 contains a hydrophobic peptide

motif that resembles that of fusogenic retrovirus proteins (Fig. 4)(102).

Endogenous retroviruses. Interestingly, human endogenousretroviruses (HERVs), partial sequences at least of which areabundant but usually inactive in mammalian genomes, can be-come activated in tumor cells and under certain physiologic con-ditions. Products of these ancient viruses have been detected inEVs (103). Is it possible that these endogenous viral products en-dow EVs with the ability to choose and enter target cells? If so,tumor cells may exploit the viral machinery not only to producevesicles but also to direct their ability to transmit signals via an-cient viral fusion factors. Standard proteomic analyses may misssuch molecules, matching acquired data with characterized, com-monly expressed human or mouse peptides.

Provocatively, a physiological role for “virus-driven” modifi-cation of exosomes has been described. In recent years, severalreports have demonstrated that endogenous retrovirus group W,member 1, commonly known as syncytin-1, and endogenous ret-rovirus group FRD, member 1, referred to as syncytin-2, are en-coded by HERV genes and are important players in syncytiotro-phoblast formation (16–19). Both syncytin-1 and syncytin-2 arepresumably former retroviral envelope (Env) genes that have re-tained a fusogenic ability. The fusion events facilitated by syncytinare crucial for maintenance and formation of the syncytiotropho-blast. Crucially, use of a human/hamster radiation hybrid panelidentified as major facilitator superfamily domain containing 2(MFSD2) as the gene coding for a receptor that contains multiplemembrane-spanning domains and mediates cell-cell fusion andinfection by retroviral pseudotypes bearing syncytin-2. Remark-ably, screening human tissues by reverse transcription-PCR dem-onstrated that MFSD2 expression is placenta-specific and, moreprecisely, localized in the syncytiotrophoblast as revealed by in situhybridization (104). Interestingly, villous trophoblasts in the pla-centa produce exosomes that seem to specifically incorporate bothsyncytin 1 and 2, which enter their target cells not through directfusion with the PM but first through endocytosis followed by fu-sion with the endosomal membrane (Fig. 5). This suggests that aninternal receptor may mediate fusion. Moreover, circulating syn-cytin 2 is reduced in circulating exosomes isolated from womenwith preeclampsia, a placental pathology specific to the humanspecies and that can culminate in maternal and perinatal morbid-ity (105). Finally, both syncytin 1 and 2 are downregulated inplacentas from pregnant women, suggesting placental exosomesmay have a physiological role in human placental morphogenesis(78). Thus, apart from direct fusion of exosomes with the PM,endocytosis is a second internalization pathway used by EVs andmany viruses.

THE ENDOCYTIC PATHWAY AS ENTRY MECHANISM

Many viruses exploit endocytic pathways to enter host cells, asdescribed and compared in excellent reviews elsewhere (2, 106).The entry pathways of EVs have many similarities, but because ofthe lack of a common method to isolate and purify EV (subtypes)(107), it is difficult to find a common pathway or strategy of EVuptake and function (97). Endosomal trafficking is thought tooffer several advantages for viruses which, upon cell entry, side-step degradation in the unfriendly environment of the endolyso-somal pathway. For example, viruses entering the endosomalpathway can escape immune surveillance and bypass restrictionfactors or physical obstacles, such as the actin cortex. This route

FIG 4 Extracellular vesicle entry via fusion. Different viral and nonviral mol-ecules can facilitate fusion of vesicles with the target cell. HCV-infected cell-derived exosomes are enriched in CD81 and HCV glycoproteins and couldpossibly be taken up via fusion (37). EBV-infected cell-derived exosomes areenriched for LMP1 and gp350, possibly facilitating fusion (98, 101). Further-more, HSV gB, CD9, and other tetraspanins could have a role in EV entry viafusion (84, 93, 99).



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also provides viral particles with rapid transport to the cell nu-cleus. As we have seen, HIV enters endosomal compartments ofDCs and, upon cellular entry into lymphoid tissue, releases this“stored” and protected HIV to infect T cells. Intriguingly, an an-alogue of this “trans-infection” has also been described for exo-somes (26), endowing the rereleased vesicles with an intercellularWnt signaling capacity.

The internalization of viruses into the cytosol by endocytosishas been divided into several mechanistic categories: (i) clathrin-mediated endocytosis (CME), (ii) macropinocytosis, (iii) caveo-lar/lipid raft-mediated endocytosis and, albeit less characterized,(iv) clathrin- and caveolin-independent mechanisms. Generally,after internalization within the primary endocytic vesicles, the en-gulfed material, including viruses, ligands, hormones, growth fac-tors, lipids, etc., follows the same intracellular pathways, and thecomplex endosomal trafficking system will determine the finaldestination and function of the internalized molecule (108). Thefate of internalized material could be subject to sorting, process-ing, recycling, or degradation in lysosomes that frequently reveal alaminar ultrastructure. The realization that endosomal mem-branes also act as platforms for signaling complexes and possiblyhave a direct role in gene regulation (109, 110) suggests a divisionof tasks that could have important implications for EV and virus-mediated delivery of functional biomolecules.

The endosomal system is classically categorized into compart-ments known as early endosomes (EEs), late endosomes (LEs),recycling endosomes (REs), and lysosomes (Lys), each with dis-tinct membrane domains and internal structures. Due to diversedomains, association with distinct Rab GTPases traditionally de-fines the endosomal compartments. Rabs are the main regulatoryproteins in membrane trafficking, vesicle formation, vesicle

movement along actin and tubulin networks, and membrane fu-sion (111, 112). EEs are generally defined as RAB5-containingcompartments, and they are the first to receive endocytosed ma-terial from the PM. EEs reveal a complex tubular-like ultrastruc-ture and are responsible for sorting and trafficking of internalizedmolecules to different intracellular targets. Some endosomes un-dergo maturation and are RAB5 and RAB7 positive. These belongto a class of intermediate organelles, serve as precursors for LEs,and are suspected to play a key role in entry of many viruses (113,114). A specialized form of maturing LEs are the so-called multi-vesicular bodies, which are highly dynamic sorting platforms thatsupport the budding process of viruses and exosomes (115).

Clathrin-Mediated Endocytosis

A common infection pathway exploited by many animal viruses isCME, which involves the internalization of molecules throughclathrin-coated vesicles (116). Clathrin is a molecular scaffoldprotein that forms transport vesicles of material at the plasmamembrane. These vesicles are eventually pinched off by a mecha-nism called membrane scission and undergo clathrin uncoatingthrough GTP hydrolysis-mediated changes of dynamin 2. The un-coated vesicles become fusogenic, allowing release of their contentwithin EEs (117).

CME of viruses. In CME, viruses and their transmembrane re-ceptors are packaged into clathrin-coated vesicles. CME as amechanism for viral entry was first observed in Semliki forest virus(SFV) infection (118–121). Investigating entry led to the discoverythat CME is used as the entry mechanism by many, mainly single-stranded RNA enveloped animal viruses, including dengue, hep-atitis C, and influenza A viruses (122, 123). However, double-stranded DNA viruses, including adenovirus 2 and 5 and thedsRNA reovirus (120, 124, 125), exploit this pathway, albeit usingdifferent receptors. Importantly, Kaposi’s sarcoma-associatedherpesvirus (KSHV) enters human fibroblast cells by dynamin-dependent CME, but in dermal endothelial cells, entry is oddlydynamin independent (126). Thus, CME-exploiting virusessometimes have alternative entry routes, depending on the targetcell.

CME of EVs. For EVs, type-specific dissimilar entry routes havenot yet been described, partially because it remains challenging toisolate a pure single EV type. The main integral component of aclathrin-coated pit is a complex of epidermal growth factor recep-tor pathway substrate clone 15 (EPS15) and adaptor protein 2(AP2). CME inhibition by a dominant-negative EPS15 results in asignificant reduction of EV uptake (34), but not complete inhibi-tion. Those authors explained this result as an indication thatEPS15 has a partial role in uptake of EVs through CME. An alter-native explanation may simply be that not all EVs require EPS15for uptake into their target cell, stressing the need for better char-acterization and identification of EV subtypes. Indeed, recentstudies showed that not all EV uptake can be inhibited by targetingmultiple endocytic pathways. Besides CME, uptake by macropi-nocytosis and caveolin-mediated endocytosis are main routes forcellular internalization of viruses (2). For EVs, this seems to besimilar. Tian and colleagues showed that PC12 cell-derived exo-somes enter target cells through CME and macropinocytosis(127). They identified CME to be involved in the internalizationby using different methods, such as pretreatment with K� deple-tion buffer, which blocks the clathrin-coated pits, knocking downthe clathrin heavy chain, the basic subunit of clathrin, and �2, the

FIG 5 Clathrin-mediated endocytosis used by EVs. EPS15, one of the twomain integral components of a clathrin-coated pit, has a partial role in uptakeof possible subtypes of EVs via CME. Blocking endocytic pathways partiallyinhibits EV uptake (34, 127). Villous trophoblast-derived exosomes seem toincorporate syncytins 1 and 2, which could facilitate endocytosis of these EVs(78, 103, 105).

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subunit of clathrin adaptor complex AP2, all resulting in signifi-cant decrease in exosome uptake by PC12 cells (127). Moreover,another study indicated that exosomes can be taken up via lipidraft-mediated endocytosis (128). Disrupting lipid rafts and inhib-iting tyrosine protein kinase are two of the most widely used tech-niques for blocking this endocytic pathway. Even so, in PC12 tu-mor cells, disruption of this pathway through general means didnot affect PC12 exosome uptake.

As previously mentioned, studying EV uptake is challenging,because of the heterogeneity in size, shape, and composition ofEVs. However, one of the most reliable methods of studying EVentry is fluorescent labeling. In a study by Nanbo et al., thismethod was used to clarify the exact mechanism of EV releasefrom EBV-positive cells and their function in recipient cells.Those authors analyzed the internalization of fluorescently la-beled exosomes derived from EBV-uninfected and type I and typeIII latency EBV-infected cells into EBV-negative epithelial cells.CME and caveolin-mediated endocytosis were identified as possi-ble EV entry pathways, targeting dynamin 2, which plays a crucialrole in both pathways. Treating cells with dynasore (dynamin-specific inhibitor) resulted in a decrease but not total blockage ofEV uptake (129), supporting the outcome of other studies thatthese endocytic pathways are partially involved in EV entry inspecific cell types (Fig. 5).

Dynamin 2 also has a crucial role in membrane curving duringCME, especially in uptake into specialized professional phagocyticcells, such as macrophages, microglia, neutrophils, and mono-cytes. The studies by Feng et al. suggested that exosomes are, per-haps not surprisingly, internalized more efficiently by phagocyticcells (34). Labeled exosomes moved to phagosomes together withphagocytic polystyrene carboxylate-modified latex beads and ap-peared to be sorted into lysosomes. In these studies, exosome in-ternalization was dependent on actin remodelling and phosphati-dylinositol 3-kinase (Pi3K), and the entry process was sensitive toboth knockdown of dynamin 2 and overexpression of a domi-nant-negative form of dynamin 2. However, in other studies,short hairpin RNA against dynamin 2 also reduced to 50% theuptake efficiency of tumor EVs, indicating that dynamin-depen-dent endocytosis was involved. Control experiments with latexbeads excluded phagocytosis as the main mechanism for tumorexosome uptake by PC12 cells (127). Despite these observationsthat suggest some selectivity for EV cell entry, strong evidence forselective CME-dependent EV transfer is still lacking. Becausemany viruses use CME as the main entry pathway upon surfacereceptor attachment, largely explaining cellular tropism (2), itseems logical to focus mechanistic EV entry studies in this direc-tion. Notably, for RNA-carrying EVs, the CME pathway may beessential for function as the host intercellular messenger or as analternative stealth transmission mode for viral genomes, as shownfor HCV (3).

HCV Cell Entry as a Model To Study EV Uptake Mechanisms

Hepatitis C virus. Like the heterogeneous population of EVs, the40- to 80-nm pleomorphic HCV particles are difficult to charac-terize (38, 39). As we expect for EVs, HCV is known for its varietyof uptake-associated molecules. HCV cell entry (Fig. 6) is a com-plex multistep process involving the presence of many entry fac-tors, which are tightly coordinated in time and space (2, 130);therefore, this process provides a possible model to study EV up-take.

The complex process of HCV cell entry. HCV is believed toinitially infect the liver through the basolateral side of hepatocytes,where it engages its main attachment factors, heparan sulfate pro-teoglycan (HSPG), low-density lipoprotein receptor (LDLR), andscavenger receptor class B type I (SR-BI) (131). Subsequently,HCV interacts with coreceptor CD81, which is composed of fourtransmembrane passages, a small extracellular loop (SEL) and alarge extracellular loop (LEL), and the tight junction protein clau-din-1 (CLDN-1) (132). Many have studied the mechanisms ofHCV cell entrance in detail, all pointing to receptor-mediatedendocytosis of HCV by CME (133). CD81, reported as a bindingreceptor for HCV in 1998 (134), has proven to be a central playerin HCV entry in part by binding directly to the HCV E2 surfaceglycoprotein that mediates endosomal fusion (134–136). Despitenumerous studies of the CD81-HCV E2 (envelope) interaction, itwas argued initially that the role of CD81 as a putative virus recep-tor was doubtful, because of its ubiquitous expression in vivo.Indeed, CD81 is far from the only determinant of HCV tropism, asrecently illustrated by the discovery of six CD81 binding partnersthat promote HCV infection, including serum response factorbinding protein 1 (SRFBP1) (137). With a comprehensive quan-titative proteomics protocol, those authors identified 26 dynamicbinding partners of HCV-triggered interactions with CD81. Asmany as six of these proteins promoted HCV infection, as indi-cated by RNA interference (RNAi) studies (137). SRFBP1 is re-cruited to CD81 during HCV uptake, supporting HCV infectionof hepatoma cells and primary human hepatocytes. While all vi-ruses have evolved elaborate entry strategies, HCV is the currentchampion in complexity, requiring a plethora of molecules, in-cluding HSPG, liver/lymph node-specific intercellular adhesionmolecule-3-grabbing integrin (L-SIGN), dendritic cell-specificintercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN), LDL-R, transferrin receptor 1 (TfR1), Niemann-Pick C1-

FIG 6 HCV cell entry. HCV has a plethora of molecules involved in entry viareceptor-mediated endocytosis. Initial attachment to the target cell is facili-tated by HSPG, LDLR, or L-SIGN/DC-SIGN. Followed by interaction with themain receptors SR-B1 and CD81, TfR1 (not shown) could have a post-CD81role in HCV entry, and SRFBP1 (not shown) is possibly recruited to CD81during HCV uptake, coordinating host cell penetration. Late actors in theentry process are CLDN-1 and OCLN. NPC1L1 (not shown) might act in theentry process concerning cholesterol transport.



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like 1 (NPC1L1), SR-B1, CD81, CLDN-1. and occludin (OCLN),highlighting the difficulties in understanding possible entry mech-anisms of EVs (130). Resembling HCV, EV target selectivity isunlikely to be determined by a single target cell factor. Interest-ingly, however, it has been very recently demonstrated that exo-somes enriched in CD81 from HCV-infected cells are capable oftransmitting infection to naive human hepatoma cells even in thepresence of neutralizing antibodies (36, 43), while anti-CD81 an-tibodies seem to prevent infection in vivo (138, 139).

Because both HCV virions and host exosomes have a role inHCV infection, it may be highly informative to investigatewhether exosomes produced by HCV-infected cells have a simi-larly complex receptor usage. To date, most studies have used lipiddyes to study EV uptake by target cells; since this tracing methodwill not discriminate between EV subtypes, it will probably beessential to develop better purification and reporter methods toidentify any EV-specific entry receptors.

MACROPINOCYTOSIS

Macropinocytosis mediates nonselective uptake of small solublemolecules such as nutrients, antigens, and other components ofthe extracellular medium, leading to macropinosome formation.Macropinosomes are relatively large and therefore provide an ef-ficient route for nonselective endocytosis (140). Macropinocyto-sis is accompanied by actin reorganization, ruffling of the plasmamembrane, and engulfment of relatively large volumes of extra-cellular fluid. Like CME, macropinocytosis facilitates cell entry forviruses, bacteria, and some protozoa (141). In particular, virusesbelonging to the vaccinia virus, adenovirus, picornavirus, andother families have been reported to take advantage of macropi-nocytosis, either as a direct endocytic route for productive infec-tion or indirectly, in order to assist in penetration of particles thathave already entered by other endocytic mechanisms. Viruses thatbenefit from this route are mainly adenoviruses 2 and 5 and ru-bella virus (142, 143). Dendritic cells have been shown to usemacropinocytosis in antigen presentation, since this mechanismis useful in uptake of large fluid volumes and opsonized particles,

more so than can be accommodated within clathrin-coated pits.This realization may explain the usefulness of this entry route formicroorganisms and EVs. Exosomes, as defined by immunodetec-tion of marker proteins, have been shown to be specifically andefficiently taken up and transported from oligodendrocytes to mi-croglias by macropinocytosis (10). In addition, it was recentlydemonstrated that the active induction of macropinocytosis uponstimulation by surface epidermal growth factor receptor signifi-cantly enhances the cellular uptake of exosomes into target cells(144).

While it is thus clear that exosomes and other EVs exploit thesame endocytic entry routes as animal viruses, it could be conjec-tured that EVs produced by particular cell types may select theirtarget cells by specific receptors. Despite the added complexitythat EVs are innately heterogeneous, viruses and EVs appear toshare receptors for cell entry.

SHARED RECEPTORS FOR VIRUS AND EV ENTRY

Lectins

Accumulating evidence suggests that HIV and EVs both rely onlectins as target cell receptors (Fig. 7). Apart from T cells andmacrophages, HIV can target myeloid dendritic cells via the re-ceptor CD169/Siglec-1 (a lectin), which binds to the ganglioside 3(GM3) present on the surface of virus particles (145). HIV captureby CD169 is enhanced upon DC maturation with lipopolysaccha-ride, and viral particles, rather than inserting their capsids into thecytoplasm, are protected from degradation in CD169� virus-con-taining compartments. From there, they may later be released anddisseminated to CD4� T cells, a mechanism of DC-mediatedHIV-1 trans-infection (146). Sialic acid binding immunoglobulinlectins are sialic acid binding molecules expressed on a variety ofleukocytes and stromal cells, in particular by subcapsular sinusand medullary macrophages in lymph nodes and on marginalmacrophages in the marginal zone of the spleen. In an elegant invivo study using CD169 knockout mice as controls, CD169 wasidentified as a key mediator of B cell-secreted exosome capture by

FIG 7 Apoptotic mimicry and receptor-mediated EV entry. (A) Tetraspanins and integrins. The Tspan8-CD49d (ITG�4�1) complex on exosomes possiblybinds to VCAM-1 on target cells (84). In general, EVs could attach to the target cell via ITG�1�1, ICAM-1, or VCAM-1 (71). ICAM-1 on DC-derived exosomescould bind to LFA-1 (ITG�L�2) on T cells/DCs and ITG�v�3 on DCs (61, 72, 73). (B) Lectins. GM3 on B-cell-derived exosomes may bind to Siglec-1 for uptakeby macrophages (67, 147). (C) Phosphatidylserine receptors. PtdSer is exposed on the outer leaflet of the exosomal membrane, and the Tam family of PtdSerreceptors, which include but are not restricted to KIM-1, TIM-1, and TIM-4, mediates EV uptake. MFG-E8, enriched on exosomes, might mediate EV uptake byacting as a bridge between PtdSer and integrins or via Gas6 interactions (10, 176). (D) HSPG. Heparan sulfate proteoglycans act as internalizing receptors ofcancer cell-derived EVs (152).

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these macrophages (147). Although a possible role for GM3 wasnot studied, early proteomic studies identified GM3 on the surfaceof B cell exosomes (67). The intracellular fate of these exosomesand whether they follow a similar route as proposed for engulfedHIV particles (28) remain to be investigated. Apart from pro-teomic studies, classical biochemical techniques also revealed thepresence of several glycan binding proteins. Apart from CD169,CD62, a C-type P-selectin, is present on the surface of platelet-derived EVs that bind to target cells via classical P-selectin glyco-protein ligand-1 (PSGL-1) (148). Of note, the latter study wasactually one of the first to clearly describe the existence of differentpathways for vesicle release.

To escape the mucosal clearance system and reach its targetcells, HIV-1 has evolved strategies to circumvent deleterious hostfactors. Galectin-1, a soluble lectin found in the underlayers of theepithelium, increases HIV-1 infectivity by accelerating its bindingto susceptible cells. In its normal function, galectin-1 binds toglycans on the CD4 coreceptor of T cells to prevent autorecogni-tion. However, when HIV is present, the galectin bridges the CD4coreceptor and gp120 ligands, thus facilitating HIV infection ofthe T cell (149). There might also be a link between galectins andEV attachment. In a proteomic analysis, galectin-3 was associatedwith DC-derived exosomes (69); galectin-3 may also have adhe-sion properties (150). However, in a recent study, it was observedthat galectin-5 functionality involved in the exosomal sortingpathway during rat reticulocyte maturation actually delayed up-take by macrophages (151).

Heparan Sulfate Proteoglycans

Heparan sulfate (HS) is a linear polysaccharide that occurs as aproteoglycan (HSPG) when HS chains are attached in closeproximity to cell surface or extracellular matrix proteins.HSPGs were recently discovered as internalizing receptors ofcancer cell-derived EVs with exosome-like characteristics (Fig.7) (152). Prior to these studies, it was known that many envel-oped viruses, notably herpesviruses, can enter cells by inducingfusion between the viral envelope and a cell membrane (153).Studies of the interaction of the fusion peptide domain of HIVgp41 with the surface of T lymphocytes showed that heparinasecould block these interactions and cell entry (154). Likewise, ifheparan sulfate is present on proteoglycans exposed at the cellsurface, these molecules capture herpesviruses, facilitating in-teractions with coreceptors. For these viruses, it was observedthat, depending on the nature of the interaction and the size ofthe heparan sulfate chain, a single chain might bind multipleviral ligands on a virion. For example, the mass of heparinsulfate chains on human HEp-2 cells averaged 105 kDa (155).This molecular mass corresponds roughly to 420 sugar residuesper chain, or a length of almost 200 nm, which is larger than thediameter of many virions (like HIV) and EV subtypes. By usingseveral cell mutants and enzymatic depletion, Christianson andcolleagues provided the first genetic and biochemical evidenceof a receptor function for HSPG in exosome uptake, which wasdependent on intact HS (152). This was highly specific for the2-O and N-sulfation groups. Enzymatic depletion of HS at thecell surface or pharmacological inhibition of endogenous pro-teoglycan biosynthesis significantly attenuated exosome up-take. Not yet defined are the structures on the surface of EVsthat could bind to HSPGs, but speculations can be made. Dueto their high negative charge, HS chains can bind to a great

amount of proteins (156). For example, HSPGs at the cellmembrane can activate adhesion mechanisms, via receptor ty-rosine kinases (RTKs) or via presentation of chemokines,which can activate ITG complexes present on EVs. There isevidence that EVs incorporate RTKs, suggesting that EV adhe-sion and possibly uptake can be facilitated by interactions be-tween RTKs and HSPG (157). Finally, while the specificity forparticular subtypes of EVs was not studied, it is known thatsome viruses lack binding affinity for HSPG altogether. Forexample, EBV does not require HSPG for cell entry (158).Thus, the role of HS in EV targeting may depend highly on theEV (sub)type. Are there any common uptake pathways andmolecules identified that mediate EV uptake and biology?

Apoptotic Mimicry by EVs

While EVs have the same membrane topology as the plasma mem-brane, many studies have revealed that cell-secreted exosomescontain phosphatidylserine (PtdSer) facing the extracellular envi-ronment (10, 35, 61, 159, 160). PtdSer is a ubiquitous membranephospholipid that has a key role in initiating the removal of apop-totic bodies by phagocytosis (161) and participates in the clear-ance of viruses by macropinocytosis (162). In healthy cells, PtdSeris actively kept within the inner membrane leaflet such that itscharged head faces the cytosol. Due to membrane dynamics, PtdSermay become exposed on the outer leaflet, but in healthy cells spe-cial enzymes maintain lipid asymmetry. In diseased cells, how-ever, membrane integrity is compromised. In early apoptoticanimal cells, PtdSer becomes exposed to the extracellular environ-ment, serving as an “eat me” signal for professional phagocyticcells. Fitzner and colleagues tested the involvement of PtdSer inexosome internalization. Using liposomes that contained a mix-ture of phosphatidylcholine and PtdSer, those authors showedthat uptake of exosomes by microglia was significantly reduced inthe presence of PtdSer-containing liposomes (10). Importantly, in2008 Zhang and colleagues showed that T cell immunoglobulinand mucin domain proteins 1 and 4 (TIM-1 and TIM-4) cancapture exosomes, though internalization and delivery of cargowere not studied (35). B-cell-derived exosomes express PtdSerand are taken up by TIM receptor-expressing monocyte-derivedDCs (MoDCs) and pDCs (60). The observation that TIM recep-tors bind exosomes sparked interest in these molecules as entryreceptors for enveloped viruses (Fig. 7).

Enveloped viruses cover their capsids in a lipid bilayer that isobtained during virus budding from either plasma or organellemembranes. Although enveloped viruses are more sensitive toharsh environmental conditions, acquisition of an envelopecan be an advantageous strategy to (i) protect viral structuralproteins from immune recognition and neutralizing antibod-ies, (ii) increase surface area, allowing optimal display of viralproteins for early steps of infection, and (iii) provide a mech-anism for virus egress that does not lyse and kill infected cells.In addition, a more recently appreciated benefit is the incorpo-ration of phospholipids into viral envelopes. Presentation ofPtdSer on the outer leaflet of these membranes disguises vi-ruses as apoptotic bodies, thereby confusing cells into engulf-ing virions through cell clearance mechanisms. This mecha-nism of enhanced virus entry is sometimes termed apoptoticmimicry (163). The first viral receptor identified that turnedout to be a PtdSer receptor was hepatitis A virus cellular recep-tor 1 (HAVCR1), also known as kidney injury molecule-1



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(KIM-1) and later as TIM-1, used by HAV for cellular entry(164). The cellular function of KIM-1 was believed to be as anepithelial cell adhesion molecule specifically upregulated inproximal renal tubular cells after injury (165). It was later dis-covered that KIM-1 serves as a specific PtdSer receptor, con-ferring a phagocytic phenotype to epithelial cells (166). Using alibrary of monoclonal antibodies against mouse peritonealmacrophages, it was observed that an antibody against TIM-4strongly inhibited the PtdSer-dependent engulfment of apop-totic cells.

Since the discovery of TIM-1 and TIM-4 as PtdSer receptors,additional family members, such as TAM, were identified thatsupport cell entry of many enveloped viruses (33, 167–171).For dengue virus (DV) infection, TAM-mediated infection re-lies on indirect DV recognition, in which growth arrest-specific6 (Gas6), a known TAM ligand, acts as a bridging molecule.This dual mode of virus recognition by TIM and TAM recep-tors reveals a clear example of how viruses usurp the apoptoticcell clearance pathway for infectious entry. Again, a similaritybetween viruses, EVs, and cellular fragments becomes appar-ent. The interaction between phosphatidylserine, Gas6, andAxl was originally shown to be a molecular mechanism throughwhich phagocytes recognize phosphatidylserine exposed ondead cells (172). However, it is now known that Axl/Gas6, aswell as other phosphatidylserine receptors, facilitates entry ofdengue, West Nile, and Ebola viruses (173). Viral envelopePtdSer exposure is a viral entry mechanism generalized tomany families of viruses, and apart from Axl/Gas6, variousother molecules are known to recognize PtdSer. The effects ofAxl/GAS6 on virus binding and entry have not been compre-hensively evaluated and compared (174), including the PtdSer-bridging molecule milk fat globule EGF factor 8 protein (MFG-E8), also known as lactadherin. MFG-E8 was discovered to actas a bridge by binding to PtdSer on apoptotic cells and to theintegrins �V�3 and �V�5 on phagocytes (175). While the ev-idence for the GAS6 interaction with exosomes and EVs is notoverwhelming, in most, if not all proteomic studies MFG-E8 isidentified (ExoCarta), suggesting that this molecule is an at-tractive candidate for mediating EV uptake (176). However, inrecent studies of viral infection efficiency, it was found thatMFG-E8 can increase the infectivity of PtdSer-pseudotypedlentiviral vectors by 5- to 10-fold, much less than mediation byGas6 (200-fold), although this may depend on the specific set-ting (174). In sum, recent advances in the molecular mecha-nisms of viral entry yield another physiological link betweenviruses and EVs (Fig. 7).

PERSPECTIVES

While it is clear that designated pathways exist for EV entryinto target cells, we are only at the beginning of understandingthe complexities involved. Major questions remain, includinghow many entry-capable EV subtypes there are and the extentto which they use common or diverse, EV-specific entry routes,as seen with viruses. Are all cells “partially” susceptible to EV-mediated communication? Is signaling mainly local, or couldEVs function systemically, as suggested by studies of tumor-bearing mice (14, 177, 178)? Research into the biodistributionof EVs has revealed that some selectivity may exist (10, 147),but many details remain unknown. There are reasons to beoptimistic that specific pathways will be discovered. In recent

years, accumulating evidence suggests that the biogenesis andrelease of exosomes and other EVs is tightly regulated, althoughthe molecular details need further clarification. Moreover, asmany reports describe, EV composition is not uniform, in par-ticular that of EVs found in biological fluids such as plasma, asrecently described after high-resolution electron microscopy(179). New high-throughput techniques, currently under de-velopment, will provide more insight into their heterogeneityand details on their molecular composition, which will be im-portant to discern the physiological role of EVs in vivo.

We have not discussed in detail the fate of EVs once inter-nalized by recipient cells. More precisely, the intercellular des-tination of EV cargo upon cell entry is heavily understudied.Presumably, EVs that fuse with the PM deliver their contentdirectly into the cytosol. While EVs carry diverse cargo, RNAmolecules have been among the most studied. In terms of func-tional RNA delivery, one might wonder how efficient such amechanism could be. Would these RNAs not be attacked bynucleases, or are they protected, e.g., by surrounding proteins,in the EV and upon delivery? And could these RNAs and theirassociated proteins easily incorporate into recipient cell ma-chinery for translation (mRNA) or inhibition (miRNA)? Sim-ilar questions could be asked of other cargoes.

While there is some evidence that EVs and exosomes are endo-cytosed and end up in internal compartments, whether these canescape endosomes by fusion with the delimiting membrane, as isrequired for viruses, is unknown. For certain viruses, the pH en-vironment provides a cue for conformational changes in glyco-proteins initiating fusion. The low pH of endosomes triggers con-formational changes in the envelope proteins of HCV, whichcauses the HCV viral genome to get released into the cytosol. Thatthe acidic environment of the endosome is required for these con-formational changes was confirmed by studies using endosomeacidification inhibitors. It may be interesting to know whethersuch inhibitors would also affect EV-mediated functional RNAtransfer.

Perhaps the most important message to emphasize is thesynergism of virus and EV research. The hard-won details ofviral fusogenic processes will be an important Rosetta stone tohelp us decipher mechanisms of EV docking and fusion. Sim-ilarly, EV-related discoveries may give fresh insights into virus-host interactions. Witness, for example, the case of the TIMreceptors, which were first identified as putative exosome re-ceptors but have now been shown to act as key viral receptors.As new results come in, we may very well find that viruses andEVs share more compelling similarities than we are now able tocomprehend.

ACKNOWLEDGMENTS

This work was supported by the Dutch Arthritis Association (13-02-401) and the Dutch Cancer Society KWF (VU2012-5510) awarded toD.M.P.

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Helena M. van Dongen is a medical student atthe VU Medical Center (VUmc) Amsterdamand a research intern at Exosomes ResearchGroup at the Pathology Department of VUmcand Cancer Center Amsterdam, under the su-pervision of D. M. Pegtel. Her research focuseson extracellular vesicles and on biomarker dis-covery concerning Epstein-Barr virus antibodyprofiling in systemic lupus erythematosus. Sheis interested in oncogenesis and autoimmunityrelated to viruses and virus-infected cell-de-rived exosomes.

Niala Masoumi is a Ph.D. candidate at the VUMedical Center Amsterdam, Exosomes Re-search Group, in the Pathology Department.The focus of her Ph.D. project is the role ofEBV-modified exosomes and their content, es-pecially small RNAs, in autoimmune diseasessuch as systemic lupus erythematosus and lupusnephritis. She obtained her M.Sc. and Ing. titlesat Utrecht University, The Netherlands, on thesubjects of cancer genomics and developmentalbiology and on molecular biology, respectively.She has worked on multiple molecular research projects, as an intern andresearch technician, at the University of California, Santa Barbara, RIVM,The Netherlands, University Medical Center Utrecht, and Hubrecht Re-search Institute.

Kenneth W. Witwer is an Assistant Professor ofMolecular and Comparative Pathobiology andNeurology at the Johns Hopkins UniversitySchool of Medicine. His research focuses on ex-tracellular vesicles and RNA, biomarker discov-ery, and therapeutic modulation of innate andintrinsic defenses. Dr. Witwer trained at PennState University and at Johns Hopkins Univer-sity. He is an active member of the InternationalSociety for Extracellular Vesicles and is pursu-ing several funded research projects related toexosomes and HIV infection.

Dirk M. Pegtel is a P.I. at the Department ofPathology at the VU University Medical Center(VUmc) in Amsterdam, The Netherlands. Heobtained his Ph.D. degree from Tufts Univer-sity (Boston, MA, USA), studying Epstein-Barrvirus infection, and performed postdoctoraltraining in cell biology at the Netherlands Can-cer Institute. For his original work on viral mi-croRNA transport via exosomes, he wasawarded the Bijerinck Premium from the RoyalDutch Academy of Sciences. His research in-cludes basic principles on cell-cell communication via extracellular vesiclesand exosomes and the development of liquid biopsy approaches for cancerdiagnosis.

van Dongen et al.


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