Investigating the Role of Vaccinia Virus-derived Small Extracellular Vesicle Cargo in Infection

Abstract

Poxviruses, such as vaccinia virus, encode a variety of proteins that inactivate their host’s antiviral response. Small extracellular vesicles (sEV), including exosomes and microvesicles, contain a variety of proteins, lipids, and nucleic acids that enable intercellular crosstalk. Recent evidence suggests that viruses can hijack these sEV pathways upon infection and use them to their advantage. Here, we found that upon vaccinia virus infection of a variety of cancer cell lines, sEV production and release increases drastically. We hypothesized that these VacV-infected cell sEVs contain RNA molecules that may play an important role in VacV’s virulence. We identified two VacV mRNAs with enriched reads in the small RNA-Seq analysis of MiaPACA-2 cells and sEVs following VacV infection. By qPCR analysis, we confirmed that these two immunomodulatory VacV mRNAs, B19R and C12L, were present in sEVs. With the use of a Vaccinia Virus lacking functional B19R and an exosome-deficient 786-O cell line, we have shown that sEV-associated B19R is functionally present in exosomes and plays a role in dampening the antiviral response prior to infection. This is the first time a poxvirus has been shown to enhance sEV production, packaging viral mRNAs into sEVs, and potentially exploit sEVs for their own advantage. i