Immunomodulatory effects of exosomes produced by virus-infected cells

ARTICLE IN PRESS Transfusion and Apheresis Science ■■ (2016) ■■–■■

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Transfusion and Apheresis Science j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / t r a n s c i

Review

Immunomodulatory effects of exosomes produced by virus-infected cells Juraj Petrik * Scottish National Blood Transfusion Service and University of Edinburgh, Edinburgh, UK

A R T I C L E

I N F O

Keywords: Virus Extracellular vesicle Exosome Infection Immune response Cellular communication

A B S T R A C T

Viruses have developed a spectrum of ways to modify cellular pathways to hijack the cell machinery for the synthesis of their nucleic acid and proteins. Similarly, they use intracellular vesicular mechanisms of trafficking for their assembly and eventual release, with a number of viruses acquiring their envelope from internal or plasma cell membranes. There is an increasing number of reports on viral exploitation of cell secretome pathways to avoid recognition and stimulation of the immune response. Extracellular vesicles (EV) containing viral particles have been shown to shield viruses after exiting the host cell, in some cases challenging the boundaries between viral groups traditionally characterised as enveloped and non-enveloped. Apart from viral particles, EV can spread the virus also carrying viral genome and can modify the target cells through their cargo of virus-coded miRNAs and proteins as well as selectively packaged cellular mRNAs, miRNAs, proteins and lipids, differing in composition and quantities from the cell of origin. © 2016 Published by Elsevier Ltd.

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Extracellular vesicles .............................................................................................................................................................................................................. Exosome biogenesis, content, sorting, release and uptake ....................................................................................................................................... Exosomes and viral infection .............................................................................................................................................................................................. Interference of the virus-infected cell-produced exosomes with immune responses ................................................................................... 4.1. Exosome-mediated viral evasion ......................................................................................................................................................................... 4.1.1. Exosomes containing virus particles of enveloped viruses ........................................................................................................ 4.1.2. Exosomes containing virus particles of non-enveloped viruses .............................................................................................. 4.1.3. Exosomes containing viral genomes and fragments .................................................................................................................... 4.2. Effects of exosomes containing viral products on immune cells .............................................................................................................. 4.2.1. Exosomes containing virus-coded miRNAs ..................................................................................................................................... 4.2.2. Exosomes containing viral proteins ................................................................................................................................................... 4.2.3. Viral infection-affected changes in exosome content of cellular molecules ....................................................................... Conclusions ............................................................................................................................................................................................................................... References ..................................................................................................................................................................................................................................

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Abbreviations: CMV, cytomegalovirus; DC, dendritic cells; EBV, Epstein–Barr virus; ESCRT, endosomal sorting complexes responsible for transport; EV, extracellular vesicles; HAV, hepatitis A virus; HBV, hepatitis B virus; HCV, hepatitis C virus; HEV, hepatitis E virus; HHV, human herpesvirus; HIV, human immunodeficiency virus; HPgV, human pegivirus (formerly known as GBV-C or hepatitis G virus); HPV, human papillomavirus; IFN, interferon; IL, interleukin; ILV, intraluminal vesicles; miRNA, micro RNA; MV, microvesicles; MVB, multivesicular bodies; SNARE, SNAP [soluble NSF (N-ethylmaleimid-sensitive factor) association protein] receptor; TCR, T-cell receptor; TLR, toll-like receptor. * Scottish National Blood Transfusion Service and University of Edinburgh, Edinburgh, UK. Fax: +441313145583. E-mail address: [email protected]. http://dx.doi.org/10.1016/j.transci.2016.07.014 1473-0502/© 2016 Published by Elsevier Ltd.

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1. Extracellular vesicles Practically all cells have been shown to release EVs under physiological conditions and to increase this release under various stress stimuli. This supports one of the main functions proposed for EVs – intercellular communication with neighbouring cells as well as more distant target cells which they can reach via their presence in body fluids. Among early descriptions of EVs were Wolf’s paper on “platelet dust” [1], investigation of erythrocyte transferring receptor shedding [2] and vesicle formation during erythrocyte maturation [3]. Three main types of EVs are currently recognised: microvesicles, exosomes and apoptotic bodies. Apoptotic bodies are the largest EVs (1–5 μM). As their name suggests they are produced during programmed cell death. Apart from their size they can be distinguished from other vesicles by their higher density (1.24–1.28 g/ml) and DNA and histone content [4]. They are not a subject of this review. Microvesicles (MV; sometimes termed shedding microvesicles) are formed by blebbing from the plasma membrane. They are intermediate in density (1.16 g/ml) and size (100–1000 nm). Due to the process of their formation almost no general markers have been described for MV. Rather, the cargo corresponds to the cell of origin, albeit the content and quantities of molecules may not be identical to average content and quantities of the cell of origin. It may reflect local plasma membrane changes and environment, with particular molecular species accumulating at the sites of shedding. The exceptions seem to be some integrins and matrix metalloproteins, in particular MMP2 which can be considered a microvesicle (ectosome) marker [5]. Exosomes are the smallest among EVs (40–100 nm), many less dense than MV (1.10–1.19 g/ ml). They also differ by their biogenesis, originating from internal membranes. Consequently, there are a number of specific markers in exosomes produced in different types of cells. Differential centrifugation of pre-cleared conditioned media, plasma, and other body fluids is the most frequently used way of preparation of MVs and exosomes. However, MV sedimentation at 15–20,000g and subsequent exosome sedimentation at 100–110,000g do not provide a perfect separation of the two types of EVs and same is true for the commercially available kits. Additional methods such as filtration, density gradient centrifugation, chromatography or affinity capture are therefore frequently used to obtain better defined EV subpopulations [6–8]. This review will focus on exosomes, with specific attention given to exosomes produced during viral infection. 2. Exosome biogenesis, content, sorting, release and uptake As mentioned earlier, exosomes are the only EVs formed from internal membranes, by inward budding of endosomal membrane of multivesicular bodies (MVB; or late endosomes). They correspond to intraluminal vesicles (ILV) which can be destined either for degradation in lysosomes, for recycling or secretion after being trafficked to the plasma membrane and released into extracellular space upon fusion [9]. Then they are termed exosomes. Distinct subpopulations destined for different processing appear to differ in

cholesterol content – high in MVB for exosome secretion, low in those for degradation [10], and lysobiphosphatidic acid which is missing in exosomes [11]. Exosome biogenesis is intertwined with the processes of the selective cargo transport between organelles. Four ESCRT (endosomal sorting complexes responsible for transport) were described for MVB destined for degradation: ESCRT 0, I, II and III. The first three recognise and sequester ubiquitinated membrane proteins, while ESCRT III takes part in ILV scission and budding [12]. For MVB destined for secretion, several parallel mechanisms seem to operate. In addition to the ESCRT pathway described above, an ESCRTindependent mechanism, dependent on ceramide producing enzyme sphingomyelinase, was described [13]. Another mechanism, independent of the first two, involves tetraspanins [14]. Functions of the ESCRT system may be different for lysosomal sorting and EV production. Based on the authors’ unpublished data from a study of RNAi targeting 23 components of ESCRT 0/I/II/III and associated proteins, only a few involved in the EV production were identified: STAM (signal transducing adaptor molecule), TSG101, ALIX, HRS (hepatocyte growth factor-associated tyrosine kinase) and VPS4 [12]. Sorting signals in the cytoplasmic domains of transmembrane cargo proteins interact with vesicle “coats” – protein complexes coating the surface of vesicle (COP I, COP II, clathrin). Signals are quite diverse – from short hydrophobic sequences of two amino acids to 5–9 amino acid signals which may imply multiple binding sites on recognition proteins [9]. The composition of these coating proteins involving a number of adaptor molecules is rather complex and changes during the transport stages. The uncoating of clathrin vesicles is mediated by cytosolic chaperones (Hsc70, auxilin) and the association of membrane proteins with chaperones (Hsc70, hsp90, 14-3-3 epsilon, PKM2) in the process of recruitment of cytosolic proteins has been suggested for protein sorting during the exosome secretory pathway of vesicle production [15]. Less clear is the situation with sorting the RNA species into exosomes. Certain nucleotide patterns and hnRNPA2B1 interaction with target miRNA sequences were implicated in RNA sorting mechanisms [16,17]. They are unlikely to explain the presence of all RNA species in exosomes and further studies are necessary to decipher RNA targeting into exosomes. SNARES (SNAP receptors) are mostly C-terminally anchored molecules with cytosolic N-terminal domain, containing the “SNARE motif” of 60–70 amino acids involved in coiled-coil formation [18]. They appear to have two main functions: to promote fusion and to ensure the specificity of membrane fusions. Or in general terms, they act to overcome the energy barrier to fusion. These are complex processes involving a number of additional proteins and factors. But the specificity comes from pairing of different v (vesicular) and t (target) SNARES. They form a four-helix bundle, with one alpha helix contributed by the monomeric v-SNARE and three by the oligomeric t-SNARE. This very stable complex brings the membranes together, facilitating fusion. The specificity of membrane fusion is partly mediated by different v- and t-SNARE pairing. However, it has been shown that they can pair promiscuously in vitro,

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indicating a role for additional determinants of membrane fusion such as tethering proteins (COG, EEA1, golgins) which assemble with the help of Rab GTPases which participate at different stages of transport [9]. The fusion of correct membranes is a result of cooperation between Rabs, tethering proteins and SNAREs. There are around 60 human Rabs and an RNAi screen identified a knockdown of Rab 27 a and b as significantly reducing the amount of secreted exosomes [19]. Rab 11 and Rab 35 were similarly implicated [20]. As a result of sorting the exosomes contain various groups of proteins involved in antigen presentation (MHC class I, MHC class II); adhesion (tetraspanins CD63, CD81, CD9, CD37, CD53, CD82 and integrins a3, a4, aM, aL, b1, b2, MFG-E8); membrane trafficking (annexins I, II, IV, V, VI, VII, XI, syndecan-1, Rab 2, Rab 5c, Rab 10, Rab 7, Arf 3, Arf 6, Arf 5, clathrin); ESCRT proteins (Alix, Tsg101); heat-shock proteins (Hsc70, Hsp90); cytoskeletal proteins (actin, cofilin 1, moesin, tubulin a1, a2, a6, b5, b3); enzymes (pyruvate kinase, alpha enolase, GAPDH); signal transduction proteins (143-3 j, g, e Gb1, Gi2a, Syntenin-1); lipid raft proteins (flotillin1, flotillin-2) and others [21]. RNA content seems similarly complex. Apart from larger RNA–mRNA, mitochondrial RNA and long noncoding RNA or rather their fragments, a number of small RNA species were detected: microRNA (miRNA) which plays a key role in intercellular communication; URNA, involved in splicing; small nucleolar RNA (snoRNA); YRNA involved in DNA replication, vault RNA [22]. Exosomes can interact with target cells through several mechanisms. For cell-to-cell communication the exosomes have the advantage of being able to deliver a number of different molecules in their cargo simultaneously, including molecules which would not be able to be secreted by other mechanisms, due to for example a lack of signal sequence. The effect of exosome internalisation and cargo release into a target cell can therefore be quite profound. Endocytosis appears to be the most frequent way to internalise the extracellular material, including exosomes. It is important to realise that these processes are commonly taking part during normal cellular maintenance. An area equivalent to entire cell surface can be internalised and replenished every few hours [23]. There are several endocytic pathways: clathrin-dependent endocytosis (also called receptor-mediated endocytosis); caveolinmediated endocytosis, macropinocytosis (extracellular fluid uptake); phagocytosis (opsonised particulate matter uptake); lipid raft-mediated internalisation (mediate the membrane protein trafficking). The experiments using specific inhibitors revealed that EVs including exosomes can be taken up by more than one mechanism [24]. In the clathrin pathway, the specific receptors are concentrated at plasma membrane regions called clathrin-coated pits and the vesicles are formed by the invagination of the membrane and scission. Clathrincoated vesicles are then un-coated, fuse with early endosomes and proceed to degradation (lysosomal pathway), recycling for some protein content or exosomal pathway, as mentioned earlier in relation to exosome biogenesis. Other endocytic pathways are less specific or non-specific (macropinocytosis). An alternative method of exosome content internalisation is the fusion of exosome and plasma membrane, facilitated by SNAREs, Rab proteins and SM-proteins. Fusion, however, does not appear to be the main entry route [24]. Internalisation is

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not always a pre-condition for the exosome-mediated effect on the target cell. In some cases the receptor–ligand interaction at the cell surface can lead to signal transduction and downstream signalling effects. Exosomes can modulate both innate and acquired immunity. Constitutively produced exosomes by dendritic cells (DC) have been shown to participate in the activation of innate immune response. TNF superfamily members in exosomes can bind NK cell receptors to activate them [25]. As mentioned earlier, exosomes produced by DC or macrophages contain MHC class I and II molecules, as well as T-cell co-stimulatory molecules which makes them an important factor in antigen presentation. Out of three models for this function, the first two (cross-dressing pattern and cross-presentation pattern) require initial capture of exosomes by recipient cells, while the third model postulates direct CD4+ and CD8+ T-cell activation [26]. However, the T-cell stimulatory activity by vesicles alone was 10 to 20fold less efficient when compared to antigen presenting cells [27]. Exosome effects also depend on the state of the cell of origin, as shown for exosomes released by mature and immature DCs [28]. Consequently, they can have either immunoactivation or immuno-suppression effects and the balance between two exosome populations may play an important role in disease pathogenesis [26]. Presence of miRNA and mRNA in exosomes and demonstration of mRNA functionality by translation were described by Valadi and co-authors [29]. They also demonstrated a presence of new protein products in recipient cells, originating from the exosomal mRNA. Since then a number of studies confirmed transfer of functional RNAs via exosomes, as documented by the identifiable effects in recipient cells (see Refs. [22, 30]). The effects of mesenchymal stromal cell-derived EVs on immune regulation mechanisms and their therapeutic potential are described in more detail in two papers in this issue [31,32]. 3. Exosomes and viral infection Most enveloped viruses normally code for the enzymes involved in virus entry into the host cell, but generally not for those involved in viral egress. They usually assemble and bud at the plasma membrane, but a significant number of enveloped viruses bud into internal compartments. There are a number of common features between assembly and release of viruses and exosomes [26]. The components of the ESCRT pathway mentioned earlier play a crucial role in these processes and three groups which act sequentially include adaptor proteins, early-acting factors and lateacting factors. The adaptors define sites of action at specific membranes. Examples are the HRS/STAM in MVBs, viral proteins (HIV-1 gag, arenaviral Z proteins), syntenin/syndecan in exosomes. Early-acting factors initiate ESCRT assembly and stabilise membrane curvature (Bro1 domain proteins such as ALIX; ESCRTI complexes always containing TSG101; ESCRTII complexes). Late-acting factors are responsible for membrane constriction and fission (eight subfamilies of ESCRT-III subunits: CHMP1–7 and IST1). ESCRT III subunits engage in a number of pairwise protein–protein interactions. VPS4 ATPases, which bind to ESCRT-III subunit C-terminal tails, provide energy for the ESCRT pathway [33].

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What are the mechanisms by which viruses utilise the ESCRT system? Viral (usually structural) proteins recruit the ESCRT members via the interaction of short motifs, the late assembly domains. Analogous domains have been described in cellular ESCRT-interacting proteins, indicating that viruses mimicked these motifs during their evolution. At least five classes of late assembly domains were described and they can function interchangeably (redundancy), which provides an advantage in case of the motif mutations. P(T/S)AP tetrapeptide late assembly domain was identified in structural proteins of HIV-1, filoviruses, arenaviruses, rhabdoviruses, reoviruses and binds to the UEV domain of ESCRT I subunit TSG101. It has also been identified in cellular adaptor proteins HRS/STAM, GGA3 and TOM1L1. However, in case of Bluetongue virus, the PSAP motif is located in the non-structural protein NS3. Like most TSG101-recruiting viral proteins, it contains also PPXY motif, mediating the binding to NEDD4 ligases (see below) [34]. YPXL has been identified in some retroviruses (EIAV, MLV) and binds to ALIX which is known to facilitate release of paramyxoviruses, arenaviruses, flaviviruses and some other viral groups. Analogous ALIX-recruiting motifs have been described in cellular proteins (MVB/exosome adaptor, Syndecan/Syntenin, PAR1). PPXY tetrapeptide motif was first identified in Rous sarcoma virus, later in other retro-, rhabdo- arena- hepadnaand other viruses. These motifs bind to WW domains of NEDD4 family of ubiquitin E3 ligases involved together with ESCRT factors in the release of shedding vesicles from plasma membrane. Viral assembly, when the membrane is wrapped around the assembling virion, and membrane fission allowing budding are linked processes [33]. Some ESCRT factors (ALIX, ESCRT I, II) contain ubiquitin binding domains (UBDs) and HIV-1 Gag protein can fuse directly to ubiquitin, but domains in ubiquitin are not well defined. There are some additional late assembly domains but these are not well characterised, yet. Many, but not all enveloped viruses use the ESCRT pathway for egress. HIV-1 appears to recruit some of the ESCRT factors and then bud from plasma membrane. Best studied example is the EIAV. It does not use ESCRT-I, but recruits subsequent ESCRT factors via YPDL late assembly domain, in the following order: gag:ALIX:CHMP4:CHMP2:VPS4. A recent paper identified the trans-Golgi network, but not early or late endosomes as the site of EIAV Gag accumulation and although egress was mediated by the cellular vesicle transport system, it did not involve MVBs. Actin depolymerisation appeared to be associated with EIAV production [35]. Semliki forest virus budding was independent of both ubiquitin and the activity of VPS4, pointing at possible role for the highly organised envelope protein lattice during alphavirus budding [36]. Influenza A virus M2 transmembrane protein appears to manage its own processes related to membrane fission [37]. Respiratory syncytial virus, influenza A virus and andes virus all seem to require a functional Rab11 pathway for budding [33]. 4. Interference of the virus-infected cell-produced exosomes with immune responses Exosomes containing viral particles, genomes, cellular and viral mRNAs, miRNAs, or proteins can have effects on target cells in a number of ways. Those harbouring virions or genomes can provide another route for transmission of viral infection. At the same time they can protect them from

immune recognition which appears especially important from non-enveloped viruses. Exosomes containing viral components/products can also exhibit various pro- and antiinflammatory effects on target cells [30,38]. 4.1. Exosome-mediated viral evasion 4.1.1. Exosomes containing virus particles of enveloped viruses An association of hepatitis C virus (HCV, Flaviviridae) with lipids was described a couple of decades ago [39]. HCV RNA from patient sera was identified in sucrose gradient fractions with different densities. In some sera the RNA was present in fractions with densities between 1.03 and 1.08 g/ cm3, in other sera additional densities of HCV RNA-containing fractions were found with peaks at 1.12 and 1.17 and at 1.19– 1.20 g/cm3. HCV RNA banding at low densities appeared completely co-precipitated with anti-beta lipoprotein, whereas HCV RNA fractions of higher densities were only partially precipitated or not at all. More recently, purified exosomes from HCV- infected cells contained full-length viral RNA, viral protein and viral particles and were able to establish infection in uninfected human hepatoma cells. A degree of immune evasion was demonstrated by exosomemediated infection neutralisation after exosome treatment with patient IgGs. Interestingly, in parallel transfection experiments using subgenomic viral replicons, the released exosomes did not contain viral structural proteins and consequently did not produce particles but still were able to establish viral replication in naive cells [40]. This was an important experiment due to difficulties in separating exosomes and true viral particles since their densities partly overlap in density gradients [41]. Live cell imaging studies of cells infected with another enveloped virus, SFTS (severe fever with thrombocytopenia syndrome) virus (Bunyaviridae) revealed the release of the extracellular vesicles containing virions. EVs were taken up by neighbouring uninfected cells, initiating SFTS virus replication. Considering the high fatality rate for this virus (12–30%) this could open new avenues for antiviral therapeutic strategies [42]. HHV-6 virus appears to induce formation of MVBs and mature virions may be released together with ILVs. The released virions were shown to contain AP-1, TGN46 and CD63 proteins [43]. While HIV seems to exploit certain proteins involved in the formation of MVB, it is now generally accepted that virus budding occurs primarily from the plasma membrane, despite some reports of occasional budding into endosomes, possibly varying with the cell type [44]. 4.1.2. Exosomes containing virus particles of non-enveloped viruses The cell membrane-derived envelope of enveloped viruses contains also viral surface glycoproteins responsible for interactions with cell receptors. This structure is crucial for the entry and exit of enveloped viruses, allowing for a non-lytic replication cycle. On the other hand, sensitivity of the envelope to environmental factors (drying, solvents, detergents) makes the enveloped viruses less resistant. It appears that some non-enveloped viruses

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developed a mechanism how to benefit from the best of these two worlds [45]. The classic distinction between enveloped and nonenveloped viruses was challenged with the publication of a paper describing an acquisition of cellular membranederived envelope by non-enveloped hepatitis A virus (HAV; Picornaviridae) [46]. Conditioned medium of HAV-infected hepatoma cell cultures contained two populations of viruscontaining particles in a density gradient, a lower density population indicating a membrane association (1.06–1.10 g/ cm3). Electron microscopy revealed virus-like particles enclosed in membranes. The infectivity of these particles (labelled eHAV) was identical to HAV virions, but chloroform treatment reduced it by two logs, while the infectivity of virions was unchanged. Seventy-nine percent of produced virus in medium was eHAV. While virus in faeces of infected humans and chimpanzees was non-enveloped, the virus circulating in blood was eHAV. The knockdown experiments indicated ESCRT-dependent process, when a knockdown of VPS4B and ALIX inhibited the release of both, enveloped and non-enveloped particles. The ESCRT dependence seems partial, since the knockdown of HRS and TSG101 acting in the earlier ESCRT complexes did not inhibit eHAV release. Consistent with recruitment of ESCRT factors via late assembly domains, two motifs (YPX3L) responsible for ALIX interactions were identified in HAV VP2 capsid protein. An anti-HAV neutralising monoclonal antibody failed to neutralise eHAV indicating this is a novel mechanism to evade neutralising antibodies [46]. As to the existence of non-enveloped virus only in the bile and faeces and enveloped in the bloodstream, the authors favour the view that virus is produced enveloped in hepatocytes and loses the envelope in canaliculus due to the high bile salt concentration [45]. Paradoxically, on one hand eHAV seems to coexist with neutralising antibodies in blood, on the other hand passive antibody transfer even as late as two weeks after exposure seems to provide protection against symptomatic hepatitis [47]. A possible explanation of the antibody effect is a transient exposure of the capsid during the eHAV membrane degradation in lysosome, if antibody is present in the same compartment. The mechanism is not known, but it is possible that antibody enters the endocytic pathway via receptor-independent macropinocytosis [45]. Hepatitis E virus (HEV) is another non-enveloped hepatotropic virus (Hepeviridae). As in case of HAV, HEV exhibits two populations upon density gradient centrifugation. Virus circulating in blood is present in fractions with density ~1.15 g/cm3, indicating membrane association, while virus from faeces bands at density ~1.27 g/cm3. Again, the monoclonal antibodies neutralise the infectivity of faeces-derived virus but not membrane-associated virus from serum (eHEV). Small ORF3 protein, important for the release of virus in cell culture, is present in serum-derived virus, but not in non-enveloped faeces-derived virus [48]. ORF3 interacts with ORF2 capsid protein upon phosphorylation. HEV recruits ESCRT system for virus egress and ORF3 contains a PSAP late assembly domain motif for interaction with TSG101. Its disruption significantly impairs eHEV release [49]. eHEV appears to bud in endosomal or MVB space as indicated by co-localisation of ORF3. Interference with VPS4 was shown to inhibit eHEV release [50]. Additional supporting evidence for eHEV intra-

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cellular vesicle budding comes from the presence of TGOLN2 protein which is expressed in trans Golgi network, on the surface of eHEV particles [51]. There are many parallels between HAV and HEV, and it is possible that eHEV enters the cell and is neutralised by antibodies in a similar way proposed above for HAV. It seems also likely that HEV is produced membrane-associated and the membrane is lost during transport from liver to biliary canaliculus [45]. Robinson and co-authors [52] used an ingenious technique of labelling the Coxsackie virus (Picornaviridae) CVB3 clone virion stock with an engineered “fluorescent timer” protein which can be used to track viral infection and dissemination. Infection of partially differentiated myoblast cells caused the release of extracellular microvesicles (EMVs) containing alongside the “fluorescent timer” protein infectious viral particles. This appears to be a novel route of virus dissemination independent of ESCRT system. CVB3 virions were identified within low-density iodixanol gradient fractions indicating membrane association. The autophagy pathway appears to play a crucial role in microvesicle shedding and virus release [52]. 4.1.3. Exosomes containing viral genomes and fragments As discussed above, infection of naive cells by exosomes containing complete HCV viral particles has been demonstrated [40]. Such transfer, however, can also be mediated by exosomes containing not complete particles, but viral genomic RNA, or even subgenomic constructs. Virionindependent transfer of functional HCV RNA and replicationcompetent subgenomic RNA has been described [40,53]. However, the relevance to the in vivo infection processes and HCV pathogenesis is still to be evaluated. Liver resident plasmatocytoid DCs can be activated and produce type I IFN when in contact with infected hepatocytes. This transfer appears mediated via the exosomal transfer of HCV RNA to plasmacytoid DCs and is dependent on ESCRT machinery and annexin-A2 [54,55]. During HIV-1 infection the effect of the specific subgenomic RNA of HIV–TAR contained in the T-cell produced exosomes from patient sera and cultured cells was observed. TAR RNA was able to down-regulate apoptotic signals, leading to enhanced HIV replication in recipient cells [56]. Human pegivirus (HPgV; formerly known as GBV-C or hepatitis G virus) causes persistent infection, although no proven disease association has been documented to date. HPgV RNA is produced by T- and B-lymphocytes, but primary permissive cells are not known. Exosomes containing HPgV RNA were shown to deliver RNA to uninfected monocytes, NK cells and T- and B-lymphocytes, with evidence of subsequent replication. EV-mediated delivery of HPgV to PBMCs in vivo could explain HPgV’s broad tropism [57]. 4.2. Effects of exosomes containing viral products on immune cells 4.2.1. Exosomes containing virus-coded miRNAs Exosomes are just one known miRNA carrier. miRNAs were shown to circulate in association with high density lipoproteins (HDL), low density lipoproteins (LDL), Argonaute 2 (AGO2) and other nucleoproteins. It can be argued, though, that exosomes (and microvesicles) provide better protection,

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and possibly a more efficient delivery system than other mentioned complexes. Viral miRNAs are coded predominantly by DNA viruses, especially herpesviruses. There are two types of viral miRNAs – host cell analogues and virusspecific miRNAs. The effects of most of them are directed at limiting the lytic cycle, evading the immune response and prolonging the life of virus-infected cells. All these effects are pre-conditions for establishing the persistent infection. This is facilitated by the fact that miRNAs are not readily recognised by the immune system. The obvious target is cell death. A number of viruses (Kaposi-sarcoma-associated herpesvirus, EBV, Marek’s disease virus) code for miRNAs directed at pro-apoptotic host genes [58]. Gamma herpesviruses usually code for 30–40 miRNAs. Exosome-contained EBV miRNA BHRF1-3 was able to suppress the expression of immunostimulatory gene CXCL11 in exosome dose-dependent manner. Another EBV miRNA, BART15, appears to target same site as miR223. This miRNA targets NLR proteins or the inflammasomes and the target site has been identified in the NLRP3 3′-untranslated region. miR 223 restricts the inflammasome activation. Targeting host miRNA binding sites seems to be favoured by viral miRNAs as any sequence variation is less likely because it would affect the host miRNA binding affinity too. miR BART15 target site proved to be identical to miR223 and this was confirmed at functional level as this miRNA was transferred by exosomes to uninfected cells and shown to decrease NLRP3 protein expression [59]. Despite being an RNA virus, HIV is reported to code for several miRNAs. VmiR88, vmiR99 and vmiR-TAR were detected not only in primary alveolar macrophage-produced exosomes but also in the exosome fraction of sera from HIVinfected persons. vmiR88 and vmiR99 stimulated TLR8dependent macrophage TNFa release likely contributing to chronic immune activation in HIV-infected persons and accelerating HIV-associated co-morbidities. Antagomirs complementary to HIV-derived vmiRNAs dramatically reduce macrophage TNFa release, confirming the mechanism [60]. 4.2.2. Exosomes containing viral proteins Antigen-presenting cells (APC) routinely sort MHC class II molecule to intracellular compartments for loading with peptides present to T cells. HSV-1 has developed a way to hijack the antigen presenting machinery by complexing its glycoprotein B with HLA-DR leading to sorting via exosome pathway rather than a presentation on cell surface [61]. Glycoprotein B plays a role in a different mechanism developed by CMV. In this case it complexes with CD-SIGN from dendritic cells and the complex is distributed by microvesicles to other cells, increasing their susceptibility to CMV infection [62]. An increased sensitivity to infection and increased virus-fusing ability were described in previously naive cells after uptake of EVs containing a complex of HCV E2 glycoprotein with CD81 [63]. EBV can be considered a very successful example of virus spread and survival. Over 90% of population is persistently infected with EBV. Persistent herpesviruses evolved a strategy of encouraging immune recognition during the proliferative stages of their life cycle and avoiding it at later stages [44]. Consequently, the exosomes produced by virus (e.g. herpesvirus)infected cells can have stimulatory or inhibitory effects on

immune response, depending on the exosome cargo. EBVpositive tumours secrete T-cell inhibitory exosomes containing the viral protein LMP1 facilitating immune evasion. LMP1 activates several signalling pathways (EGFR, STAT3, ERK) and this activation can be mediated by LMP1 containing exosomes in recipient cells, leading to cytokines and chemokines expression, and inflammation. These exosomes also inhibited the PBMC proliferation [64,65]. Another EBV-coded protein, dUTPase, seems to modulate innate immunity in primary monocyte-derived macrophages via toll-like receptor (TLR)2 interaction, leading to activation of NF-κB and production of pro-inflammatory cytokines. Such activation was mediated by dUTPase in exosomes produced by chemically induced cells when exosomes were taken up by primary DCs and PBMCs. EBV dUTPase can modulate cellular microenvironment as a result of exosome-mediated intercellular communication, facilitating EBV infection establishment [66]. One of the main features of HIV pathogenesis is T-cell depletion. It has been shown that the exosomes containing HIV Nef protein, a key protein in the HIV life cycle, can induce CD4+ T-cell apoptosis in vitro, suggesting one possible mechanism for depletion [67]. It has been known for some time that a co-infection of HIV-1 infected patients with human Pegi virus (HPgV, formerly known as GBV-C or hepatitis G virus) appears to be associated with prolonged survival. HPgV reduces T cell activation which is a critical component of HIV pathogenesis. T cell activation inhibition is mediated by a conserved peptide motif in HPgV envelope glycoprotein E2 capable to inhibit TCR-mediated signalling. This phenomenon was demonstrated in recipient bystander T cells by E2proteincontaining EVs from HPgV-infected cells [68]. 4.2.3. Viral infection-affected changes in exosome content of cellular molecules Viral infection may change the exosome content of cellular lipids, miRNAs and proteins, modifying intercellular communication [44]. Apart from secreting exosomes containing virus-coded immunoregulatory proteins mentioned above, the EBVinfected nasopharyngeal carcinoma cells were shown to secrete exosomes with high content of host cell protein galectin-9, which is an immunoregulatory protein able to induce apoptosis in EBV-specific T-cells [69]. Another clever EBV strategy limiting the innate immunity activation appears to be a removal of innate immunity effectors (IFI16, caspase-1, IL-1b, IL-18, IL33) from infected cells via released exosomes [70]. Nef protein is a key protein in the HIV life cycle, with many functions. During the HIV replication in infected cells Nef increased the number of intracellular vesicles and MVBs. Exosomes and other EVs from HIV-infected cells were shown to contain HIV co-receptors CCR5 and CXCR4 and the susceptibility of the target cells to HIV infection increased upon the co-receptor transfer. Exosomes from HIV-infected cells were shown to contain APOBEC3G, a cytidine deaminase, which is involved in cellular anti-retroviral defence mechanism. The recipient cells of such exosomes can inhibit HIV replication [71]. Modification of the exosome composition of hostderived miRNAs by viral product can significantly affect the outcome mediated by taken-up exosomes. Nef expression

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in HIV-infected cells appears to affect miRNA content in exosomes, reducing the RNA interference in recipient cells [72]. HPV is known to have a large number of types, some of them tumourigenic. Exosomes isolated from cancer patients infected with these viruses revealed specific exosomal miRNA compositions depending on the viral oncogene E6/ E7 expression [73]. Yet different mechanism appears to be exploited by HBV, when the HBV surface antigen particles can apparently carry hepatocellular miRNAs complexed with AGO2 [74]. 5. Conclusions Growing evidence of interplay between extracellular vesicles, especially exosomes, and viral infections contributes to better understanding and appreciation of this phenomenon. Numerous examples of viruses taking advantage of cellular transport and degradation mechanisms to progress their infection cycle provide a fascinating insight into this complex interaction network. This review has discussed ways of exosome-mediated viral evasion, sometimes blurring the accepted grouping of viruses into enveloped and nonenveloped, as well as modulating immune responses in recipient target cells via exosome cargo of viral and selectively packed cellular RNAs and proteins. These intercellular communication mechanisms provide new valuable targets for potential interventions with viral infections. A key precondition for such interventions is to re-evaluate viral infections in respect of establishing which of the mentioned evasion and immunomodulatory mechanisms play significant or decisive roles for in vivo viral pathogenesis. Future applications may utilise the therapeutic potential of exosomes. Compared to lysosomes they have some potential advantages in being natural cellular products providing better protection of their cargo from immune system as well as having longer half-life. The exosome content can be selectively modified to deliver desired therapeutic molecules such as engineered therapeutics [75] either by in vitro loading of purified exosomes, or by tagging drugs for in vivo-targeted exosome incorporation [76]. New technological approaches to exosome purification and characterisation, including more detailed studies of subpopulations, will facilitate more targeted and efficient applications of these exciting messengers/ effectors of numerous biological processes.

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