Role of Extracellular Vesicles in Viral Infections

Extracellular vesicles (EVs) are membrane-encapsulated structures released by cells which carry signaling factors, proteins and microRNAs that mediate intercellular communication. Accumulating evidence supports an important role of EVs in the spread and pathogenesis of infectious diseases. Viruses have been reported to usurp host EV biogenesis pathways to package viral products, which are capable of modulating tumor microenvironments. In addition, EVs have been shown to cross blood-brain and placental barriers, thus viruses may utilize normal EV biology to gain access to previous impassable barriers. Chapter 2 of the thesis focuses on how Zika virus infection exploits host EV biogenesis pathways to increase infectivity, evade the host immune system and possibly alter tropism of the virus. We demonstrate that Zika virus infected cells secrete distinct EV sub-populations with specific viral protein profiles and infectious genomes. Zika virus infection resulted in the enhanced production of EVs with varying sizes and density compared to those released from non-infected cells. We also show that the EV enriched tetraspanin CD63 regulates the release of EVs, and Zika viral genomes and capsids following infection. Overall, these findings provide evidence for an alternative means of Zika virus transmission and demonstrate the role of EV biogenesis and trafficking proteins in the modulation of Zika infection and virion morphogenesis. In chapter 3 of the thesis, the role of ceramide in Latent membrane protein 1 (LMP1) EV secretion and signaling is explored. Recently, the tetraspanin protein CD63 has been found to form a complex with LMP1 and knock-out of CD63 in epithelial cell lines results in reduced exosomal LMP1. In certain cell lines, CD63 is trafficked to EVs through a ceramide-dependent manner. Therefore, we hypothesized that ceramide is required for efficient packaging of LMP1 into EVs. Following treatment with GW4869, a compound that inhibits the enzyme important for ceramide synthesis, EVs from Epstein-Barr (EBV-infected lymphoblastoid (LCL) and HEK293 cells carrying doxycycline-inducible LMP1 were analyzed by nanoparticle tracking analysis (NTA) for particle numbers and immunoblotting for EV content. EVs were further purified on density gradients to examine vesicle subpopulations. LMP1 was determined to be secreted in small EV populations by gradient purification. NTA of EVs from the cells treated with GW4869 demonstrated a significant decrease in particle secretion. LMP1 localization was disrupted in the treated cells and immunoblotting of EV lysates revealed a significant reduction in extracellular LMP1. Electron microscopy of density fractions illustrated morphological disruptions to CD63- and LMP1-enriched vesicle subpopulations. Additionally, ceramide inhibition resulted in enhanced LMP1-mediated Nuclear Factor kappa-light-chain-enhancer of activated B cells (NFkB) activation in exosome producing cells. Taken together, these data reveal a critical role for the lipid ceramide in LMP1 exosomal trafficking and the oncogenic signaling properties of the viral protein.