WNV is a flavivirus of considerable medical importance. Intercellular transduction of antiviral signal in response to flaviviruses in vertebrates primarily relies upon production of IFN-a/b. However, recent findings indicated that extracellular vesicles (EVs) can also be involved in cell-to-cell transfer of antiviral signals. Here we applied systems biology approach to elucidate the role of EVs in WNV infection.
EVs were isolated from culture fluids of A549 cells infected with WNV or mock-infected and were shown to have characteristics of exosomes. They were free of infectious viral particles and were shown to be capable of transferring their RNA content into bystander cells. RNA-Seq analysis of A549 cells treated with EVs produced by WNV-infected cells revealed differential expression of 664 genes. GO analysis demonstrated that this set of genes is enriched in GO terms associated with antiviral response and miRNA-dependent regulations, indicating that EVs produced upon WNV-infection induce antiviral response in bystander cells and suggested the link between antiviral response and miRNAs.
To determine the RNA content of EVs, we performed RNA-Seq analysis of small RNAs isolated from EVs produced by WNV-infected and mock-infected cells. We found that infection significantly alters the level of 47 mature miRNAs, 38 pre-miRNAs and 35 sncRNAs in EVs. To analyse the role of differentially expressed miRNAs in regulation of antiviral genes in EV-treated cells we first reconstructed the network of direct interactions between identified differentially expressed genes using Metacore software. Then the list of validated targets for miRNAs enriched in EVs cells was obtained from Mirwalk database and aligned to the network. We found a significant overlap between the network activated in cells treated with EVs and network of miRNA targets, which indicates that miRNAs incorporated into EVs upon WNV infection can trigger antiviral response in recipient cells and thus contribute to transduction of antiviral signal.