A non-enveloped arbovirus released in lysosome-derived extracellular vesicles induces super-infection exclusion

Abstract

22Recent developments on extracellular-vesicles (EVs) containing multiple virus particles challenge 23 the rigid definition of non-enveloped viruses. However, how non-enveloped viruses hijack cell 24 machinery to promote non-lytic release in EVs, and their functional roles, remain to be clarified. Here 25 we used Bluetongue virus (BTV) as a model of a non-enveloped arthropod-borne virus and observed 26 that the majority of viruses are released in EVs, both in vitro and in the blood of infected animals. 27Based on the cellular proteins detected in these EVs, and use of inhibitors targeting the cellular 28 degradation process, we demonstrated that these extracellular vesicles are derived from secretory 29 lysosomes, in which the acidic pH is neutralized upon the infection. Moreover, we report that secreted 30EVs are more efficient than free-viruses for initiating infections, but that they trigger super-infection 31 exclusion that only free-viruses can overcome. These results suggest that secreted EVs and free-32 viruses do not share a common receptor. 33 Author summary 34Recent discoveries of non-enveloped virus secreted in EVs opened the door to new developments in 35 our understanding of the transmission and pathogenicity of these viruses. In particular, how these 36 viruses hijack the host cellular secretion machinery, and the role of these EVs compared with free-37 virus particles remained to be explored. Here, we tackled these two aspects, by studying BTV, an 38 emerging arthropod-borne virus causing epidemics worldwide. We showed that this virus is mainly 39 released in EVs, in vivo and in the blood of infected animals, and that inhibition of the cell degradation 40 machinery decreases the release of infectious EVs, but not free-virus particles. We found that BTV 41 is responsible for neutralizing the pH of lysosomes, which are important organelles of the cell 42 degradation machinery. Our results highlight unique features for a virus released in EVs, explaining 43 how BTV transits in lysosomes without being degraded. Interestingly, we observed that EVs are more 44 infectious than free-virus particles, but only free-viruses are able to overcome the super-infection 45 exclusion, which is a common cellular defense mechanism. In conclusion, our study stresses the dual 46 role played by both forms, free and vesicular, in the virus life cycle. 47 48 49 50 51 52 53

Multiple Routes of Bluetongue Virus Egress

“…During BTV infection both lytic and non-lytic virion release has been documented. Further, electron microscopy analysis revealed different forms of virion particles, naked virion particles released by cell lysis and non-lytic viruses budding through the plasma membrane [ 19 ]. In addition to individual particle budding, a recent report demonstrated virion particles are released in aggregates within extracellular vesicles (EVs), suggesting a secretory pathway [ 17 ].…”

“…Recently, we reported that in some cell types, a majority of BTV particles can be released in extracellular vesicles, both in vitro and in vivo [ 19 ]. The release of BTV containing EVs was initially confirmed by electron microscope analysis of EVs purified from infected cells culture’s supernatants and blood from BTV infected cattle.…”