
==== Front
Biomed J
Biomed J
Biomedical Journal
2319-4170
2320-2890
Chang Gung University

S2319-4170(23)00096-3
10.1016/j.bj.2023.100659
100659
Review Article
The Yin and the Yang of extracellular vesicles during viral infections
Martin Charlène
Ligat Gaëtan
Malnou Cécile E. cecile.malnou@univ-tlse3.fr
∗
Institut Toulousain des Maladies Infectieuses et Inflammatoires (Infinity), Université de Toulouse, INSERM, CNRS, UPS, Toulouse, France
∗ Corresponding author. INSERM UMR1291 – CNRS UMR5051 - Université Toulouse III CHU Purpan – BP 3028, 31024, Toulouse Cedex 3, France. cecile.malnou@univ-tlse3.fr
09 9 2023
10 2024
09 9 2023
47 5 10065925 7 2023
5 9 2023
© 2023 The Authors
2023
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
The role of extracellular vesicles (EVs) as key players in the intercellular communication is a subject of growing interest in all areas of physiology and pathophysiology, and the field of viral infections is no exception to the rule. In this review, we focus on the current state of knowledge and remaining gaps regarding the entanglement of viruses and EVs during infections. These two entities share many similarities, mainly due to their intricated biogenesis pathways that are in constant interaction. EVs can promote the replication and dissemination of viruses within the organism, through the dysregulation of their cargo and the modulation of the innate and adaptive immune response that occurs upon infection, but they can also promote the mitigation of viral infections. Here, we examine how viruses hijack EV biogenesis pathways and describe the consequences of dysregulated EV secretion during viral infections, beneficial or not for viruses, revealing the duality of their possible effects.

Highlights

• Extracellular vesicles biogenesis pathways and viral replication cycles are intimately intricated.

• Viruses interfere with the secretion and content of extracellular vesicles.

• Extracellular vesicles can promote or mitigate viral infection depending on the context.

Keywords

Extracellular vesicles
Viruses
Infection
Biogenesis
Proviral
Antiviral
==== Body
pmcIntroduction

In recent years, the field of extracellular vesicles (EVs) has been in full expansion. First considered as cellular waste in the 80s, their status quickly evolved to become intercellular mediators involved in both physiological and pathological processes. These membrane vesicles include different categories, listed according to their size, content and biogenesis pathway [1]. Microvesicles (MVs, also called ectosomes) are the largest, with a diameter comprised between 50 nm and 1 μm, and are generated by direct budding from the plasma membrane. Exosomes have a smaller size, between 30 and 150 nm in diameter, and arise from intraluminal budding of the endosomal membrane, leading to the formation of multivesicular bodies (MVB) which release their content by fusion with the plasma membrane. Therefore, size alone is not sufficient to define the different subtypes of EVs, which is sometimes misleading in the literature in relation to the nature of EVs supposedly observed. According to the latest ISEV (International Society for Extracellular Vesicles) recommendations, the terms small (<200 nm) or large (>200 nm) EVs should be used in the absence of biogenesis description of a particular type of EV [2]. Once in the extracellular space, EVs can be internalized in the recipient cells by membrane fusion or endocytosis to release their content, sometimes over long distances. Their cargo includes various lipids, proteins and nucleic acids, including mitochondrial DNA, ribosomal RNAs, messenger RNAs and microRNAs and in rare cases genomic DNA (gDNA). EVs can also initiate downstream signaling pathways in cells by directly interacting with plasma membrane receptors. To date, many answers are still missing regarding the biogenesis and cargo sorting of EVs, their dynamics in the extracellular space or the functional delivery of their cargo, and are the subject of intense research [3].

The role of EVs, particularly in the context of viral infections, is currently drawing great interest. Many studies focus on the differences in secretion, composition and functional effects of EVs upon viral infections [4,5], however, similarities in structures and biochemical properties between viruses and EVs can sometimes harden the task. Indeed, many parallels can be drawn between EVs and viruses, notably their size – ranging from 30 nm for the Picornaviridae family to 1 μm for giant viruses – but also the use of the same cellular machinery for their biogenesis and their secretion into the extracellular space. Thus, one of the main challenges regarding the studies of EVs in the context of viral infections lies in the ability to successfully separate viruses from EVs, if it really can be done, and particular attention should be paid to this issue [3,6]. In this review, wepresent current knowledge and remaining questions regarding the entanglement of EVs and viruses at the level of their biogenesis pathway, and the consequences of the manipulation of EV biogenesis and content by viruses on their replication cycle and host immune response.

A duality between EVs biogenesis and virus replication cycles

The interactions between EV biogenesis pathways and viral replication cycles have been extensively shown in the literature [4,5]. The most described common pathway used by both viruses and EVs is the Endosomal Sorting Complex Required For Transport (ESCRT) complex [7]. Both enveloped and naked viruses use ESCRT to acquire their lipid (pseudo-)envelope or establish their replication complex [Fig. 1]. Additionally, many interactions between cellular and viral machineries occur during the sorting of EV cargoes, resulting in EV content dysregulation, which often contain viral components.Fig. 1 Interactions between EV biogenesis pathways and viruses. Viruses or families of viruses (represented in purple) can interact at all steps of the EVs biogenesis pathways, notably with exosome biogenesis during the formation of intraluminal vesicles via the ESCRT machinery, and with the Rab GTPase machinery complex and the syndecan-syntenin-ALIX pathway. Some viruses also interact with the formation of microvesicles and apoptotic EVs. Created with BioRender.com.

Fig. 1

The shared use of the ESCRT machinery

The ESCRT complex plays a major role in the cell, especially during the biogenesis of MVBs [1]. MVBs correspond to late endosomes that harbor intraluminal vesicles (ILVs) resulting from intraluminal budding of the endosomal membrane. Upon fusion of MVBs with the plasma membrane, these vesicles are released into the extracellular space as exosomes. The biogenesis of ILVs can be divided into two steps. The first one consists in the reorganization of the endosomal membrane which will be loaded with tetraspanins, including CD9 and CD63, often considered as canonical markers of exosomes. The second corresponds to the recruitment of the ESCRT machinery and its ESCRT-0 to III subunits [8]. ESCRT-0 initiates intraluminal budding by binding its hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) subunit to the phosphatidylinositol 3,4,5-triphosphate protein, which is exposed at the endosomal membrane. The ubiquitinated HRS-bound cargo then recruits the tumor susceptibility gene 101 (TSG101) protein from ESCRT-I. This leads to the recruitment of the ALG-2 Interacting protein X (ALIX) protein from ESCRT-III via its association with TSG101. ESCRT-II, composed of ELL associated protein (EAP) proteins, can be recruited by ESCRT-I and binds to ESCRT-III. Finally, thanks to the polymerization of its chromatin modifying protein (CHMP) subunits, ESCRT-III causes membrane fission to form the vesicle, before being disassembled by the ATPases associated with diverse cellular activities (AAA) ATPase vacuolar protein sorting 4 (VPS4) [9].

Since ESCRT complex is involved in intracellular membrane remodelling, its hijacking by viruses for their replication is often observed [7]. Most enveloped viruses divert ESCRT-III to drive the scission of their envelope, recruiting ALIX and TSG101. This mechanism has been well described in the case of the Human Immunodeficiency Virus (HIV-1) [7]. The retroviral group-specific antigen (Gag) protein harbors different short motifs called L-domains, such as YPXnL and P(T/S)AP, which allow the recruitment of ALIX and TSG101 respectively [10]. Another motif, PPXY, allows Gag to bind to an E3 ubiquitin ligase belonging to the neural precursor cell-expressed developmentally downregulated gene 4 (NEDD4) family. This leads to the ubiquitination of Gag, and therefore, the recruitment of ESCRT-I and ALIX. Similar strategies are shared within Retroviridae and among Filoviridae with VP40, Arenaviridae with Z protein or Rhabdoviridae with matrix protein. The Yellow Fever Virus (YFV) from the Flaviviridae family directly recruits ALIX via its NS3 protein, a crucial step for the release of viral particles [11]. Similar strategies have been described for Dengue Virus (DENV), ALIX being involved in the early phase of the virus replication [12]. The budding of DENV and Japanese Encephalitis Virus (JEV) also involves CHMP2 and CHMP4, directly recruited at the virus replication sites in the endoplasmic reticulum (ER) [13]. Lately, a new motif has been described among Flaviviridae, the L-domain motif LYXLA present on the NS3 protein, which may promote the recruitment of ALIX and CHMP4 [14]. Concerning the Paramyxoviridae, the Parainfluenza Virus 5 recruits the cellular protein angiomotin like 1 (AMOTL1) containing the Pro-Pro-x-Tyr (PPXY) motif, leading to a similar scenario [15]. DNA viruses are no exception with in particular the Herpesviridae family [16]. The VPS4 protein is required for the generation of the Human Herpes Simplex type 1 (HSV-1) envelope and is also found in mature virions [17]. Another study underlines the importance of a functional MVB machinery for the viral cycle, in particular for the trafficking of HSV-1 glycoprotein gB [18]. Among the Alphaherpesvirinae subfamily, the requirement of VPS4 to perform a complete replication cycle has also been described for Pseudorabies Virus (PRV) since its mutated version leads to a reduction in the ability to traffic along microtubules [19]. Human Cytomegalovirus (HCMV) also uses the ESCRT machinery during the last stages of its replication cycle, where, unlike TSG101, VPS4 and CHMP1 appear essential for the maturation of virions [20].

Recruitment of the ESCRT machinery is also shared by naked viruses, leading some of them to acquire a “pseudo-envelope”. For example, the Hepatitis A Virus (HAV) has been found inside EVs. VPS4 and ALIX are recruited to allow internal budding of HAV capsid in MVBs, leading to virus release in EVs [21]. CHMP2A of ESCRT-III also appears particularly important in this process. In the same way, Hepatitis E Virus (HEV) is found in MVBs [22]. Indeed, pORF3 interacts with TSG101, which allows the recruitment of the viral capsid and its loading into MVBs. Additionally, deletion of Rab27a and HRS leads to a reduction in the amount of HEV particles released. Enterovirus 71 (EV71) is also present in sEVs (virions found in vitro, viral RNA found in vivo) [23], as well as the full-length genome of Hepatitis C Virus (HCV) [24]. Finally, Bluetongue Virus (BTV) interferes with EV biogenesis pathways and requires MVB integrity for its replication cycle, interacting with TSG101 among others [25].

Besides its hijacking for viral budding, the ESCRT machinery can be used for viral genome replication. For instance, in the case of the Tomato Bushy Stunt Virus (TBSV), the recruitment of the ESCRT machinery is necessary for the assembly of its replication complex. The p33 replication protein binds to Vps23p from ESCRT-I, and Bro1p, an ESCRT accessory protein, resulting in the recruitment of Vps23p to the peroxisomes where viral replication takes place [26]. VPS4 and ESCRT-III also play an essential role in TBSV replication [27]. Another illustration is found with the replication cycle of the Hepatitis B Virus (HBV) [28]. Depletion of EAP20, EAP30 and EAP45 from the ESCRT-II complex leads to reduced viral egress, likely due to a reduced level of encapsidated pre-genomic RNA, highlighting the role of ESCRT-II in the formation of replication-competent nucleocapsids.

Other shared pathways

Rab GTPases, in particular Rab11, Rab35 and Rab27A/B, are other shared molecules between EVs biogenesis and viral replication cycles. In the case of HBV, its replication cycle is regulated by the Rab5B protein, allowing the inhibition of the hepatocyte transcription factor HNF4α, as well as the trafficking of the large hepatitis B surface protein (LHBs) from endoplasmic reticulum (ER) to MVB [29]. Thus, the depletion of Rab5B leads to an increase in the transcription of LHBs, its accumulation in the ER and an increasing number of viral particles released. Other viruses use these Rab GTPases to transport viral material, such as HCV with the action of Rab27a [30], Hantavirus via Rab8 and Rab11 [31], Respiratory Syncytial Virus (RSV) via Rab11-FIP2 [32], and the Influenza A Virus (IAV) thanks to Rab10 and Rab11 [33]. In this latter case, Rab11 is also particularly important both for the formation of filamentous virions and the budding of spherical ones. Finally, a close association has been found between the viral ribonucleoprotein of the Sendai Virus (SeV) and Rab11a, suggesting a role in the trafficking of viral components to the assembly site [34].

Cargo sorting of EVs is regulated by different proteins and mechanisms [1]. One of them, the syndecan-syntenin-ALIX pathway, stimulates the release of exosomes enriched in syntenin-1, syndecan and CD63 [35]. Epstein Barr Virus (EBV) hijacks this pathway, allowing the packaging of EVs with the viral oncoprotein latent membrane protein 1 (LMP1) [36]. Through its interaction with HRS and Syntenin-1, LMP1 is sorted into EVs and promotes tumor cell proliferation, migration and growth. Viral RNA and proteins have also been found in EVs in the context of Rift Valley Fever Virus (RVFV) infection [37]. At a larger scale than the EV cargo, viruses can also be found directly within the MVB as with Human Herpesvirus 6 (HHV-6), highlighting the close relationship between EV biogenesis and maturation of Herpes virions [38].

Although most studies describe interactions between small EVs and viruses, some focus on large ones. MVs arise from direct budding at the plasma membrane. Lipids in the outer layer of the membrane are repositioned and budding is allowed through a cascade reaction involving the actin-myosin machinery. Chen et al. showed the presence of a cluster of Enteroviruses found within vesicles rich in phosphatidyl-serine, close to MVs [39]. A similar observation was made in the same viral family, where Coxsackievirus B3 (CVB3) virions were also found within MVs [40]. Another example relies on HSV-1, where analysis of MVs released by infected cells showed the presence of mature virions [41]. Finally, another type of large EVs consists of the apoptotic bodies (ApoEVs), derived from apoptotic cells. Although less studied than exosomes and MVs, the biogenesis and roles of ApoEVs are increasingly described [42]. Chikungunya Virus (CHIKV) has notably been found in these apoptotic bodies and interferes with their biogenesis pathway [43].

Due to the overlapping of their modes of biogenesis, EVs and viruses both use cellular resources, thus creating a situation of competition. The pathways used for EV formation are hijacked for viral replication to increase viral production or even lead to certain enveloped forms of the virus. Altogether, this leads to the dysregulation of EV biogenesis with multiple functional consequences on the recipient cell.

The Yin of extracellular vesicles and viruses: promoting viral infection

Many studies describe a facilitating effect of EVs for viruses during infections (reviewed in Refs. [4,5,44]). This proviral effect is observed at different levels [Fig. 2]. First, as a result of the hijacking of the exosomal machinery, EVs secreted from infected cells can carry viral particles. This strategy may protect them from their environment and improve their transmission to target cells, favoring for example the transmission of virions in cells lacking receptors or an escape from the immune response. Furthermore, through their internalization in recipient cells, EVs derived from infected cells can facilitate the entry mechanisms of viral particles and provide factors that will further enhance the viral replication. Finally, EVs can interfere with the host innate or adaptive antiviral immune response.Fig. 2 Proviral effects of EVs. These effects can either be mediated by EVs carrying viral receptor, by viral particles contained within EVs, or by viral genome and/or protein and/or miRNA packaged into EVs. EVs will consequently transmit infectious genome, prime the recipient cell, trigger the apoptosis of immune cells or inhibit the IFN pathway in recipient cells. Viral proteins present at the surface of EVs can also sequester neutralizing antibodies. EVs derived from infected cells can also deplete MHC-II molecules from the surface of immune cells. Created with BioRender.com.

Fig. 2

When EVs carry entire infectious viral particles

Several viral families use EVs to carry mature viral particles which will then be delivered to host cells [44]. This is the result of a diversion of EV biogenesis pathways by viruses as developed in Part 2. It is notably illustrated with numerous naked viruses, which then exist as “quasi-enveloped” viruses. In this case, viral release from host cell will occur in a non-cytolytic manner. This strategy is found in a non-negligible number of viruses from the Picornaviridae family, like HAV [21], CVB3 [45], EV71 [23], Encephalomyocarditis Virus (EMCV) [46], but also with viruses from other families such as HEV [22] or BTV [25]. Polyomaviridae BK and JC viruses may also use a similar strategy [44], as well as HCV [24] and DENV [47], the latter two examples being enveloped viruses. Transport inside EVs provides an “invisibility cloak” to such viruses, allowing them to evade immune surveillance and antibody neutralization, as viral surface proteins are hidden by EV membranes. This may enable viral persistence and spread even in the presence of neutralizing antibodies [48].

When protected inside large EVs, viruses can also avoid degradation due to drastic environmental conditions. This is for example the case for Rotaviruses and Noroviruses, which are excreted in the stool as viral clusters within large EVs. These EVs remain intact during fecal-oral transmission, allowing the delivery of viral particles collectively to the next recipient host, enhancing both the multiplicity of infection and disease severity [49]. These vesicle-cloaked clusters of viruses have been shown to be resistant to temperature variation, detergent and even UV radiation, these characteristics being attributed to the high concentration of viral particles into the vesicles [50]. These data suggest that such viral clusters constitute unique emerging pathogenic units.

Beyond avoiding degradation and increasing resistance from the environment, the collective (“en bloc”) transmission of viral particles contained in EVs offers many advantages and is used by several viruses such as BK and JC viruses, enteroviruses, and others [51,39,52]. It enhances infectivity by increasing the local concentration of viral particles upon infection of a host cell. This potentiates the chances for the virus to productively infect its target cell. Bringing together a population of viral particles, in particular defective particles, can also favor the evolution of the virus by initiating adaptative walks. This may contribute to the evolution of viral quasi-species and viral fitness [53]. However, an in-silico study suggests that such complementation between the viral population has no positive effect on the long-term average fitness of the population and may even lead to error catastrophes [54].

Finally, another property of the transport of viral particles in EVs lies in the modulation of the viral tropism. The pseudo-envelopment of naked viruses by host membranes may extend their tropism, since EV surface proteins will now target their recipient cells rather than engaging the viral surface protein and a host cell receptor. This receptor-independent viral entry has been suggested in particular for HAV and HCV. It has also been demonstrated for the JC virus, which can target the human microglia in the central nervous system via EVs secreted from infected choroid plexus epithelial cells [55]. Finally, the presence of specific lipids, such as cholesterol or phosphatidylserine, in the vesicle membranes can also increase viral infectivity by enhancing the uptake of virus containing EVs [56]. EV uptake is also promoted for large EVs containing virions, via alternative processes like phagocytosis or micropinocytosis.

When modification of EV content primes viral transmission and replication

Even when whole infectious particles are not present in EVs, the latter, via their content, can exert proviral effects to facilitate future infection of naive recipient cells. This effect will be exerted at different levels of the replication cycle, mainly during entry, and is due to viral but also cellular components found in EVs.

EVs secreted by infected cells can transport viral compounds, in particular genomes and/or proteins, which will be internalized by neighboring cells. This is notably the case for the Foot-and-Mouth Disease Virus (FMDV), for which full-length genomic RNA and partial viral proteins were identified in purified EVs isolated from infected cells. EVs from FMDV-infected cells have been demonstrated to be infectious, capable of transmitting the virus to naive cells [57]. A similar study was carried out with Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and demonstrated the ability of EVs, containing viral genomic RNA, secreted by infected cells, to initiate productive infection in naive recipient cells [58].

Entry, one of the key steps of the viral cycle, is often facilitated by EVs secreted by infected or uninfected cells. For example, mucosal EVs produced by uninfected human nasal epithelial cells contain the SARS-CoV-2 ACE-2 receptor and the activated protease TMPRSS2, enhancing the susceptibility of naive recipient cells to the virus [59]. Similarly, the transfer of the HIV-1 CCR5 receptor from CHO cells and PBMC via microparticles towards CCR5 non-expressing cells may render them susceptible to HIV-1 infection [60]. In HTLV-1 infected cells, EVs are not infectious but promote cell-to-cell contact with uninfected cells via the presence of CD45 and ICAM-1 adhesion molecules, promoting the recruitment of naive recipient cells and viral spread [61].

Another study, conducted by our team regarding HCMV, showed that EVs secreted by infected cells contained viral proteins that play a role immediately post-entry, including the IE1 and IE2 transcription factors, in addition to proteins from the viral envelope as gB or gH. Therefore, EVs can target naive recipient cells and prime these cells with the factors necessary for the first steps after infection to facilitate early viral transcription and replication [62,63]. Similarly, EVs derived from HIV-1 infected cells have been shown to be enriched in viral and cellular factors playing a role in different steps of the viral cycle, such as Nef and ADAM17 [64], which activate bystander quiescent CD4+ T lymphocytes and make them permissive to HIV-1 replication, thereby stimulating viral spread.

Finally, EVs carrying viral elements also play a role in virus transmission to new host species. This is well illustrated in the case of arboviruses like DENV2. EVs isolated from mosquito saliva show a higher expression of a mosquito glycoprotein containing a tetraspanin-domain, Tsp29Fb, resulting in efficient EV-mediated transmission of infectious viral RNA and proteins to naive human cells [65].

When EVs counteract the immune response

One way of escaping immune detection often used by viruses has already been presented above. Many viruses, mainly naked but not only, are transported into EVs, allowing them to avoid antigen presentation to immune cells and detection by neutralizing antibodies [21,44,45,48]. This would facilitate the dissemination of the viruses into the organism and allow the persistence of some viruses even without latency properties, by permitting constant productive infection [66].

EVs delivered from infected cells may also present an altered content in proteins and/or non-coding RNAs that will manipulate both innate and adaptative host immune response. EVs secreted from infected cells can carry viral or cellular components that directly target the innate antiviral interferon (IFN) pathway. This is illustrated by EV71, which infected cells-derived EVs selectively package a high level of cellular miR-146a that can be transferred in naive target cells. Internalized miR-146a will suppress type I IFN response, facilitating viral replication [67]. Similarly, Newcastle Disease Virus (NDV) promotes the presence of miR-1273f, miR-1184, and miR-198 in EVs, suppressing IFNβ gene expression in recipient cells [68]. A recent study also demonstrated that an anti-immune subgenomic flaviviral RNA is present in mosquito salivary EVs and inhibits type I and III interferon pathways, enhancing viral transmission and replication in human dermal fibroblasts [69]. A last example is given during Hepatitis B infection, where EV-mediated transport of IFITM2 to dendritic cells inhibits IFNα pathway activation [70].

EVs derived from infected cells can also interfere with adaptative immune response. One example is given by the immunoregulatory potential of Alphaherpesvirus gB surface protein. gB is expressed in EVs secreted by infecting cells and promotes major histocompatibility complex (MHC) class II sorting to EVs, resulting in a decrease of MHC II level at the surface of immune cells [71]. Another illustration is given by HCMV-infected cells which secrete EVs containing viral gp34 protein, an Fc-γ receptor homolog which could divert neutralizing IgG antibodies from binding the viral particles [72].

Finally, EVs may divert immune response by targeting cells of the adaptative immunity to inhibit their action, either by direct infection of these cells, or by triggering apoptosis. Hence, transporting viral components into EVs allows some viruses to acquire new tropism, notably for immune cells. This is illustrated by HBV, which can target NK cells via the transport of infectious materials into EVs, resulting in the dampening of NK immune functions [73]. Another mechanism lies in the induction of apoptosis of immune cells, leading to their depletion. One example is VP40 of Ebola virus, packaged into EVs, which may be responsible for the dysfunction and eventual apoptosis of bystander lymphocytes [74]. A similar action is described upon HIV infection, with Nef containing EVs triggering apoptosis in bystander uninfected T-cells, contributing to immunosuppression observed during AIDS [64].

Apart from manipulating host innate and adaptative immune responses, EVs can also play important roles in promoting the pathogenesis of viral infections. They have been implicated in the stimulation of inflammatory response during HIV [75], Papillomavirus [76] or SARS-CoV-2 [77] infection for example. Other studies point out a role of EVs secreted by infected cells during virus-mediated oncogenesis (reviewed in Refs. [78,4]). Thus, the positive contribution of EVs secreted by infected cells during viral infections is multiple, and largely involved in the dissemination of viruses in the organism, their persistence and the physiopathology of the viral infection.

The Yang of extracellular vesicles and viruses: inhibiting viral infection

Conversely, to what has been presented in Part 3, EVs derived from uninfected or infected cells can also exert antiviral effects by interfering with the viral life cycle, thereby inhibiting viral replication and spread [Fig. 3]. They can also promote the antiviral innate and adaptive immunity in recipient cells [4,5,79,44].Fig. 3 Antiviral effects of EVs. These effects can either be mediated by the sequestration of viral particles that will inhibit virus attachment and entry, or by different proteins/RNA/miRNA packaged into EVs allowing a restriction of viral replication in the recipient cell, as well as an activation of the IFN pathway that can be induced by viral or cellular components. This leads to the induction of ISG and the synthesis of inflammatory molecules. Viruses can also be sequestered by EVs harboring viral receptor at their surface. Finally, antigen presenting cell may secrete EVs expressing MHC-II molecules loaded with a viral epitope. Created with BioRender.com.

Fig. 3

When EVs secreted by non-infected cells possess intrinsic antiviral activity

EVs secreted by non-infected cells can protect recipient cells from viral infection. Host EVs found in different body fluids can interfere with viral entry by sequestering viral particles and preventing their attachment and internalization into host cells. One example is given by human saliva EVs (saEVs), which affect Zika Virus (ZIKV) infectivity. The saEVs express canonical markers such as CD9, CD63 and CD81 and inhibit viral attachment to target cells. Hence, inhibition of ZIKV infection by saEVs has been proposed as a novel oral innate immune defense mechanism against viral pathogens. Another illustration is given by EVs isolated from semen and vaginal secretions of healthy individuals, which can prevent HIV-1 infection and block viral transfer in vitro [80].

Another interesting example of the intrinsic antiviral potential of EVs is observed during pregnancy. A microRNA cluster named C19MC is expressed exclusively in primate placenta and encodes 46 different microRNAs, which are packaged in trophoblast-derived EVs [81,82]. Several studies propose that this cluster of microRNAs may exhibit antiviral activity that can be transferred by EVs [[82], [83], [84]]. Interestingly, trophoblast EVs do not activate the type III IFN pathway [84]. Other studies have focused on individual microRNAs from the cluster C19MC. For example, miR-517a-3p inhibits HCMV replication and protein expression in vitro in both fibroblast and trophoblast cell cultures [85]. Despite several studies, some of which proposing a role for autophagy, the exact mode of action of the C19MC microRNAs contained in trophoblast EVs remains to be determined. A recent work suggests that the antiviral effect could be mainly due to the Alu Short Interspersed Nuclear Element (SINE) RNAs contained within the C19MC cluster and not to the microRNAs themselves [86].

When EVs secreted by infected cells boost innate or adaptative immune response

Additionally, EVs secreted by infected cells, containing both host and viral factors (proteins or microRNAs), can promote the antiviral innate and adaptive immune response in non-infected recipient cells.

Triggering of antiviral innate immunity is observed for example with EBV-infected B cells, which release EVs carrying viral EBER1 RNA that is transferred to non-permissive dendritic cells (DC) expressing the TIM1 phosphatidylserine receptor [87]. Upon IAV infection, the host hsa-miR-1975 is delivered by EVs into recipient cells where it induces IFN antiviral responses [88]. In the case of Rabies Virus (RABV), infected cells release miR-423-5p into EVs, which induces IFN-β expression and thus inhibits viral replication [89]. The role of host EVs in stimulating innate immune system is not limited to their ability of carrying viral compounds to trigger innate immunity, and numerous studies have described other mechanisms. EVs released by infected cells can contain intermediates of the IFN signaling pathway or IFN-stimulated genes (ISGs) which will inhibit viral replication and spread in target cells [4]. For example, cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) is produced in response to HIV-1 sensing and transported via EVs to non-infected cells, activating the Stimulator of IFN Genes (STING) pathway [90]. EVs derived from HSV-1-infected cells contain STING and other host factors that activate ISG transcription, such as ISG15 and ISG56, and inflammatory gene transcription, such as IL-6 and IL-1β [91]. EVs may also mediate the direct cell‐to‐cell transmission of IFN‐α‐induced antiviral activity [92]. Hence, EVs released by HBV-induced liver nonparenchymal cells contain IFN-α which is delivered to recipient hepatocytes by TIM-1, a HAV receptor, thereby protecting them from further HBV infection. Finally, EVs released by infected cells can also transport IFN-Induced Transmembrane protein (IFITM). IFITM are innate effector proteins restricting host cell entry of many enveloped viruses, including HCV, and activate the adaptative immune response [93]. For example, during DENV infection, infected cells released EVs, which contain IFITM2 proteins, preventing DENV entry into recipient cells [94]. IFITM carried by EVs released by IAV-infected cells stimulate the secretion of IL-6, TNF and MCP-1 by non-infected cells [95].

EVs can also stimulate the secretion of pro-inflammatory cytokines by recipient cells, leading to the induction of the adaptative immune response and recruitment of immune cells. For example, EVs derived from RSV-infected cells induce the secretion of CCL5, MCP-1 and IP-10 by recipient monocytes [96]. EVs from DENV-infected macrophages, carrying viral NS3 protein and miRNAs, promote the secretion of inflammatory cytokines by endothelial cells, eliciting a defense program against damage during the first stages of the disease [97]. DCs can also trigger the adaptive immune response upon incubation with EVs containing IAV proteins and both MHC-I and -II molecules [95]. EVs secreted by HBV-infected hepatocytes contain viral RNA, which is transported to macrophages, resulting in the expression of NKG2D ligands and induction of IFN-γ production by NK cells [98]. Last, EVs produced by antigen presenting cells can also express and present antigens on MCH-I and II to immune cells to participate in adaptative immune response [99].

Besides their action on innate and adaptative immunity, EVs can also express viral receptors that bind to viral surface proteins, thereby inhibiting the attachment of viral particles to the host cell surface. This is observed during IAV infection, where α2,3 and α2,6-linked sialic acids are present at the surface of airway EVs, which counteracts the virus from binding and entering target cells [95]. A last example is given by ZIKV-infected cells which release EVs that contain E proteins at their surface and thus attenuate antibody-dependent enhancement mediated by antibodies targeting E proteins of virions [100].

Overall, these findings suggest that EVs may represent a new hope in antiviral therapy, as they can inhibit viral infections either by inhibiting viral lifecycle and/or by promoting innate and adaptative immunity. Hence, understanding the mechanisms underlying EV-mediated antiviral effects is crucial for the development of effective antiviral strategies and therapeutics.

Conclusion

The difference between viruses and EVs is very thin and is a long-debated point in the literature [6]. The large analogies found between the two entities come from their mode of biogenesis. Viruses present a size close to EVs and enveloped viruses possess a lipid bilayer dotted with cellular proteins. Both viruses and EVs use the ESCRT machinery to leave the host cell. Both contain biological material (proteins, lipids, nucleic acids) that can be transferred to a target cell through fusion or endocytosis mechanisms. However, although the high similarities between the two entities, the original definition of a virus endows it with a replicative capacity, which EVs do not have. In a semantic way, the controversial question of the qualification between EVs and viruses can effectively be addressed when talking about naked viruses, which can be found within a pseudo-envelope similar to the lipid membrane of EVs, or EVs that carry full-length infectious viral genomes.

The continuum between EVs and viral particles currently poses a problem in the experimental realization of numerous studies, since the techniques used for virus and EV preparation are highly similar. Furthermore, there is a high heterogeneity in the different EV populations released by infected cells. Virus preparation/purification can be carried out by ultracentrifugation of the viral particles, precipitation with PEG, or by size exclusion chromatography. Thus, this type of preparation results in a suspension containing both EVs and viruses. It is possible to separate some viruses, like Herpesviridae, from certain types of EVs by performing a sucrose/iodixanol gradient (HCMV [62]; HSV-1 [91]), and more and more technology is now being developed to purify EVs with techniques based on affinity for membrane proteins [100]. However, a virus possessing an envelope harboring canonical EV membrane proteins will escape this exclusion. Conversely, upon infection, EVs can harbor viral surface proteins. It is therefore important to remain particularly attentive when reading literature and to keep in mind the continuum between EVs and viral particles and the biases caused by the various EV isolation methodologies.

Finally, as viruses can modify EV content, EVs are interesting candidates as biomarkers in liquid biopsies. The signature of microRNAs within EVs in various biological fluids is particularly currently attracting a lot of attention in the context of HCV infection, vector-borne viral diseases or during SARS-CoV-2 infection among others. A growing number of studies are now aiming to identify such signatures in EVs that could help in viral disease staging, diagnosis, or treatment adaptation. The scientific community must now address the challenges of standardizing EV preparation regarding the heterogeneity of EV subtypes, available technologies, and the constraints of time and costs of such analyses, before being able to generalize this promising approach.

Funding

Funding for CM doctoral thesis is provided by the 10.13039/501100003510 Ministry of Education and Research (MESR ) and her work is supported by the Villa M foundation. CEM received financial support from the ANR Corofet grant (ANR-21-CE14-0030 ) and Défi Clé Cell Based Biotherapies Occitanie (EXOBARRIER ). Our team received institutional grants from INSERM, CNRS and Toulouse III University.

Conflict of interest

The authors declare no conflict of interest.

Acknowledgement

We thank all the ViNeDys team members and our collaborators. We apologize in advance for not being able to cite all existing works due to space limitations. We particularly thank Vincent Broucquart and Elsa Suberbielle for their careful reading of the manuscript.

Peer review under responsibility of Chang Gung University.
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