Daniels, D. E.; Carr, S. M.; DeWitte-Orr, S.

Viruses make long (>40 bp) double stranded RNA (LdsRNA) during replication, which stimulates the innate antiviral immune response. In vertebrates, LdsRNA can induce the type I interferon response (IFN) or the antiviral RNA interference response (dsRNAi) to limit viral replication. Extracellular vesicles (EVs) have previously been shown to carry a variety of nucleic acids for intercellular signaling, and insects and plants have been shown to package LdsRNA in EVs as part of their antiviral immune response. We hypothesized that a similar phenomenon occurs in vertebrates in which EVs traffic LdsRNA between cells during viral infection to induce an antiviral response in naive cells. In this study we showed that both vesicular stomatitus virus (VSV)-derived and in vitro transcribed (ivt)-LdsRNA can be packaged into EVs. LdsRNA was detectable by immunoblots in EVs extracted by both differential ultracentrifugation from ivt-LdsRNA treated U937 and ExoQuick-TCTM precipitation from VSV-infected U937 cells (dsRNA-EVs) but not uninfected controls (control EVs). Isolated EVs were roughly 100 nm in diameter and were able to protect LdsRNA from degradation by RNase III. LdsRNA delivery by dsRNA-EVs was visualized in HEL-299 cells via immunocytochemistry (ICC). The LdsRNA-EVs protected against infection from HCoV-229E, while control EVs did not. Together these results indicate EVs can package and deliver long dsRNA to provide antiviral protection in naive vertebrate cells.