Exosome-mediated MiR-146a Transfer Suppresses Type ? Interferon Response And Facilitates EV71 Infection

Exosomes are nanosized(30-150 nm)vesicles released by most cell types and present in different biological fluids such as blood,saliva,and urine.These vesicles carry unique cargo containing proteins,messenger RNA(mRNA),and microRNA(miRNA).Exosomes can transfer their cargo to recipient cells,which has been demonstrated to alter the biochemical composition and signaling pathways of the recipient cells.Their roles in viral infections are beginning to be appreciated.Researches have shown that exosomes released from virus-infected cells contain a variety of viral and host cellular factors that are able to modulate recipient's cellular response and result in productive infection of the recipient host.Here,we showed that EV71 infection upregulated exosome production in HT-29 and THP-1 cells.Of interest is that CA16 did not have any effect on exosome production.We provided evidence showing that exosomal viral RNA could overcome the restriction of the entry receptor and mediate productive infection of the host cells in a receptor independent manner,suggesting that by packaging viral genomic RNA into exosomes,the virus may have a broader host range because the infection is not restricted by the presence of a cellular receptor.In addition,blocking exosome secretion could lead to the intracellular accumulation of EV71 viral particles without affecting EV71 RNA replication,indicating that the exosomal pathway is required for the release of exosomal EV71 virion.Apart from viral RNAs,exosomes can deliver nucleic acid through their cargos of selectively packaged cellular miRNAs,which are the most abundant RNA in exosomes.Through deep RNA-sequencing and real-time PCR,we confirmed that EV71-induced miR-146a was enriched in exosomes and transferable to new host cells,resulting in elevation of miR-146a level in the recipient cells.Earlier study found that EV71 infection induced cellular miR-146a expression to inhibit IFN production mediated by IRAK1 and TRAF6,two key elements involved in TLR signaling and IFN production,and evade host immune defense.Our results confirmed that cells that received exosomal miR-146a could block the signaling for type I interferon production,facilitating viral pathogenesis.By co-delivering miR-146a,exosomal EV71 RNA may gain transmission advantages by suppressing IFN response immediately after viral entry,needing minimal viral inoculum and rapidly establishing productive infection.Moreover,the exosomes contained replication-competent EV71 RNA in complex with miR-146a,AG02,and GW182.The association of AG02 and GW182 with both EV71 RNA and miR-146a in the exosomes will not only stabilize the viral genomic RNA,directly facilitating its replication but also allow the miR-146a ready to act on its cognates immediately upon entry into recipient cells.Additionally,we found that the IFN-stimulated gene factors(ISGs),BST-2/tetherin,were involved in regulating EV71-induced upregulation of exosome secretion.Importantly,in vivo study showed that EV71 RNA was significantly more enriched in both liver and spleen of the animals injected with exosomal EV71 RNA than animals injected with free EV71,in which a higher level of viral RNA was identified in brain and intestine.Together,our findings provide evidence that exosomes secreted by EV71-infected cells selectively packaged high level miR-146a that can be functionally transferred to and facilitate exosomal EV71 RNA to replicate in the recipient cells by suppressing type I interferon response.