Extracellular Vesicles and Viruses: Two Intertwined Entities

Abstract

Viruses share many attributes in common with extracellular vesicles (EVs). The cellular machinery that is used for EV production, packaging of substrates and secretion is also commonly manipulated by viruses for replication, assembly and egress. Viruses can increase EV production or manipulate EVs to spread their own genetic material or proteins, while EVs can play a key role in regulating viral infections by transporting immunomodulatory molecules and viral antigens to initiate antiviral immune responses. Ultimately, the interactions between EVs and viruses are highly interconnected, which has led to interesting discoveries in their associated roles in the progression of different diseases, as well as the new promise of combinational therapeutics. In this review, we summarize the relationships between viruses and EVs and discuss major developments from the past five years in the engineering of virus-EV therapies.

Keywords: cancer; extracellular vesicles (EVs); gene therapy; virotherapy; virus-host interactions.

Figure 1

Viruses and EVs use intertwined biogenesis pathways. (A) EVs biosynthesis pathway: from MVBs formation to the release of EVs. EV formation requires the ESCRT complex to internalize cargos in ILVs inside MVBs, the Rab-GTPases-11, -27 and -35 to transport MVBs to the plasma membrane, and the SNARE complex for MVBs fusion with the plasma membrane and release of ILVs, becoming EVs. Ubiquitinated cargos in MVB membrane recruit ESCRT-0, which in turn recruits ESCRT-I, -II and -III, mediating membrane invagination. ESCRT-III complex forms a filament-inducing ILV modeling and scission with the help of VPS4 [20,21,22]. (B) Viruses hijack the EVs biosynthesis pathway. (B1) HAV capsid enters MVBs, is secreted in EVs and forms a “quasi-enveloped” virus. HAV viral capsid domains VP2 and VP1pX recruit ALIX, ESCRT-III and VPS4 to enter MVBs. After the fusion of the MVBs with the plasma membrane, the quasi-enveloped HAV is released. The enveloped viruses SARS-CoV-2, HCMV and HBV also enter MVBs where they acquire their viral envelope [23,24,25,26,27,28]. (B2) HIV acquires its envelope by hijacking the ESCRT complex. HIV envelope proteins (Env) recruit Gag and Gag/Pol polyproteins associated with the dimerized viral RNA. Gag p6 domain then recruits the ESCRT complex, ALIX and VPS4 and buds from the plasma membrane [29]. (B3) HSV-1 escapes the nucleus by hijacking the ESCRT complex. HSV-1 viral nuclear envelopment complex (NEC) inserted in the inner nucleus membrane recruits ESCRT-III and VPS4 and buds inside the nucleus envelope. Then, the vesicles containing HSV-1 capsid fuse with the outer nuclear membrane and release HSV-1 capsid in the cytosol. HSV-1 later acquires its viral envelope by budding inside the trans-Golgi vesicles and then by exocytosis [30,31]. (B4) Ancestral retrotransposon Arc protein recruits ALIX and is secreted. Arc encodes the capsid GAG domain, which self-assembles into a virus-like-capsid containing Arc mRNA. The capsid might recruit ALIX, enhancing the budding of the plasma membrane and the secretion of EVs containing Arc capsid and mRNA [32].

Figure 2

Virus-EVs increase viral uptake by recipient cells. (A) Virus-EVs increase viral uptake and active infection by recipient cells. Both enveloped [HCMV, HHV-6, Sars-CoV-2, DV, HBV] and non-enveloped [HAV, HEV, EV71, Bluetongue virus] viruses are secreted inside virus-EVs and induce active infection of recipient cells. The transport of the viral genome inside virus-EVs can also induce active infection of recipient cells [Pegivirus] [56]. Moreover, virus-EVs can present PS at their surface, which enhances their uptake into recipient cells [HAV [59], EBV [61,62], Zika virus [57], HIV [58], Coxsackievirus B3, rhinovirus, poliovirus [60]. Virus-EVs also induce active infection of recipient cells in a receptor-independent fashion, as observed with virus-EVs containing the viral genome and proteins, the viral capsid and proteins, or viral RNAs associated with host miRNAs and proteins [EV71 [63], HCV [64], HBV [65], SFTS virus [66]. (B) EV-mediated enhancement of viral infectivity. Some virus-EVs transfer the viral receptor between recipient cells, increasing viral uptake by these cells [Sars-CoV-2 [67,68], HIV [69]. Other virus-EVs containing the viral genome, viral RNAs and proteins and host adherent proteins (ICAM-1 and its receptor LFA-1, an integrin) enhance cell-to-cell transmission of the virus [HTLV-1 [70,71]. Virus-EVs containing host adherent proteins, antigen-presenting receptors, cytokines and viral RNA does not lead to infection directly but instead enhances virus transmission in a non-viral receptor fashion instead [HIV-1 [58,72]. Virus-EVs can also enhance latent infection by inhibiting virus replication [HSV-1 [73,74]. (C) EV mediated transmission of viruses between hosts. Single virus-EVs can transport and deliver multiple viral particles to recipient cells, thus enhancing viral infection [Poliovirus, Rhinovirus, Coxsackievirus B3, Norovirus, Rotavirus [60,75]. Some of these virus-EVs mediate virus fecal-oral transmission, resisting both the stool and the gastrointestinal tract [Norovirus, Rotavirus [75]. Virus-EVs produced by mosquitos containing the viral genome, viral RNAs and proteins, and presenting PS [Zika virus [57] or an ortholog to the human CD63 tetraspanin [DV [76] can induce active infection of mammalian cells.

Figure 3

EVs and viruses modulate the immune response. (A) EVs secreted by both infected and non-infected cells promote the antiviral immune response. Virus-EVs enriched in transmembrane sialic acids bind viral particles and prevent them from infecting new cells [IAV [88]. Viral infection induces the production of molecules such as cGAMP due to viral DNA sensing [HIV [83] or miRNA due to virus-induced apoptosis [IAV [82]. These molecules can be transported via EVs to activate downstream antiviral IFN response in recipient cells. Virus-EVs also transfer IFN-induced molecules (IFIT and IFITM) that inhibit virus entry and enhance pro-inflammatory cytokine secretion [DV [87], IAV [88]. Virus-EVs containing various host and viral proteins and RNAs can also induce the secretion of pro-inflammatory cytokines by recipient immune cells [RSV [99] and endothelial cells [DV [89], activating the antiviral immune response. They also activate the expression of adherent proteins by endothelial cells, thus strengthening the endothelial barrier [DV [89]. Virus-EVs transporting viral proteins transporting virus antigens to antigen-presenting cells (APC) activate the adaptive immune response [IAV [88]. EVs secreted by APC activated by the viral infection transfer IFN to recipient cells [HBV [100]. EVs secreted by activated Vδ2-T cells transport immune proteins to infected tumor cells [EBV [101]. For instance, EVs transporting death ligands induce tumor cell death. EVs transferring NK cells activator enhance tumor cells death by NK cell cytotoxic response. EVs containing CD80/86 and MHC molecules increase the adaptive immune response against tumor cells [101]. (B) Virus-EVs enhance viral escape from the immune response. Some viruses escape degradation inside DCs phagosome and enter MVBs to be secreted in EVs [HIV-1]. Others escape degradation by bypassing the autophagolysosome of infected cells to be secreted in EVs [Poliovirus, Rhinovirus, Coxsackievirus B3, DV]. Virus-EVs containing host miRNA repress the antiviral IFN response in recipient cells, consequently enhancing viral replication [EV71 [63], NDV [92] and EV secretion [EV71 [63]. Virus-EVs containing viral proteins also inhibit the IFN pathway in recipient cells [Ebola virus [93]. Virus -EVs transporting viral proteins can inhibit MHC-II presentation and induce MHC-II secretion inside EVs by recipient cells, thus lowering potential immune recognition of infected cells [HSV-1 [94]. Virus-EVs transporting a viral Fc receptor homolog can bind neutralizing antibodies and prevent their association with viral particles [HCMV [23]. Virus-EVs also mediate the transfer of viral proteins and genome that induce active infection inside recipient cells while avoiding immune recognition by neutralizing antibodies [HCV [91], DV [90]. Virus-EVs also target immune cells by transporting viral proteins that either induce active infection [HBV [65] or apoptosis [Ebola virus [97,98] of recipient immune cells.

Figure 4

EVs mediate virus-associated disorders. (A) EVs secreted by both infected and non-infected cells lead to over-inflammation. Virus-EVs containing viral proteins can indirectly activate the inflammasome in immune cells, supporting over-inflammation [HIV [106]. Virus-EVs containing viral proteins, glycoproteins, miRNAs and or mRNAs can also activate the secretion of pro-inflammatory cytokines through PRR recognition in immune cells, leading to chronic inflammation [HIV-1 [72,102,103], EBV [61], Ebola virus [93], HTLV-1 [105]. Virus infection induces the release of pro-inflammatory cytokines in the environment, which activate the secretion of EVs by non-infected cells. These EVs contain host proteins that activates the secretion of pro-inflammatory cytokines by recipient immune cells through PRR recognition [DV [107]. Virus-EVs containing viral proteins and mRNAs induce the phenotype switch of recipient monocytes into a pro-inflammatory phenotype and promote inflammatory and coagulant pathways in recipient endothelial cells [Zika virus [57]. (B) EVs secreted by infected cells are associated with liver disease. Virus-EVs containing viral proteins and RNA or host miRNA are integrated by hepatic stellate cells and either stimulate cell proliferation or fibrosis in the liver [HBV [108], HCV [109]. (C) EVs secreted by infected tumor cells enhance tumorigenesis. Infected tumor cells expressing oncogenes can be associated with the secretion of virus-EVs transferring viral oncogene proteins [HTLV-1 [105] or host miRNAs [HPV-16 [110,111] to recipient cells, which inhibit apoptosis. These virus-EVs can also contain the viral oncogene mRNA, which induces oncogenesis in recipient cells [HPV-16 [112]. In EBV-infected cells, expression of LMP-1 viral protein induces secretion of EVs containing host proteins that induce the epithelial-mesenchymal transition in recipient cells and promote tumor migration [113]. EBV-EVs also transfer LMP-1 to B cells and induce their proliferation and differentiation into a plasma cell-like phenotype, causing IgG overproduction and a higher risk of autoimmune disorders [114].Virus-EVs transporting host miRNA also enhance migration and proliferation of recipient cancer cells by inducing expression of proinflammatory cytokines that limit apoptosis [HIV [115].

Figure 5

EV-guided improvement of virotherapies. (A) AAV-EVs-based therapies. (A1) Producer cells (HEK293 or 293T cells) are transfected with AAV plasmids expressing different therapeutic elements to be delivered to the appropriate tissue. (A2) In addition, producer cells can be co-transfected with other plasmids. For instance, PDGF-transmembrane domain fusioned to RVG allows targeting of RVG to EVs thanks to PDGT-TD. Then, RVG mediates EVs binding to the acetylcholine (Ach) receptor of neuronal cells (A5) [132]. (A3) Producer cells then naturally produce EVs, with a small proportion containing AAV particles and PDGT-TD-RVG construct. Injection of AAV-EVs into mice intratumorally or intravenously (A3), allows evasion of neutralizing antibodies (A4), increases delivery to target tissues and improves transduction efficiency (A5) [132,133,134,135,145,146]. (B) EVs improve efficacy of Ad virotherapy. PC-3 prostate and A549 lung tumor cells infected with Ad5/3-D24-GMCSF secrete EVs containing viral proteins and DNA, which actively infect recipient cells [147]. Melanoma cells (Mel526) infected with LOAd-CD40L or -4-1BB-L secrete EVs containing CD-40L or 4-1BB-L protein and mRNA respectively, which both activate DCs, thus enhancing the adaptive immune response [148]. In mice models, HCT110 primary tumor cells treated with OBP-301 Ad virotherapy secrete EVs containing the E1A viral protein and the viral DNA that can migrate to metastatic niches and induce immune cell recruitment [149]. (C) Engineered Ad virotherapy to target EVs. (C1) Producer cells are infected with the oncolytic Ad [136]. (C2) Producer cells can then naturally secrete EVs, with a certain fraction containing the oncolytic Ad. Incubation of Ad-EVs with a chemotherapeutic drug allows uptake of the drug inside the EVs (Ad-EV-Chemo) [136]. In vitro, Ad-EV-Chemo mediates tumor cell apoptosis and enhances cell transduction [136]. In vivo, intravenous injection mediates an increase in T cell activation and infiltration inside the tumor [137]. (C3) Higher quantity of Ad-EVs can be generated by passing producer cells through decreasingly smaller nanosized filters. The use of producer cells expressing VSV-G allows the formation of Ad-EVs presenting VSV-G at their surface. VSV-G then mediates binding to tumor cells through the VSV-G receptor. In vivo intraperitoneal injection of Ad-EVs in an ascitic tumor model in mice leads to both neutralizing antibody escape and successful targeting of the Ad to tumor cells in a virus receptor-independent manner [140].