Molecular Basis of Exosomal Function during Rift Valley Fever Virus Infection
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Ahsan, Noor A
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Abstract
Exosomes are small “bioactive” extracellular vesicles that play a central role in intercellular communication and have garnered tremendous interest given the recent discovery of their critical role in a variety of diseases, including infectious diseases. Among these are immunomodulation, alteration of dissemination/infectivity during infection, and modulation of pathogenesis. However, their mechanisms of action remain largely unknown and their role during infections with biodefense agents remains unexplored. We have examined the role of host exosomes during infection with the Rift Valley Fever Virus (RVFV), a Category A priority Pathogen that carries the potential for both devastating public health and agricultural impacts. To assess the role of exosomes, clones of Vero cells that show resistance to RVFV infection and are unable to release functional virions were first generated. Exosomes from these clones contained exosomal markers such as CD63 and were able to activate the TLR3 pathway in recipient reporter cells. Interestingly, these exosomes contained viral RNA (signal for L, M, and S segments). Furthermore, exosomes derived from some of the resistant clones contained viral proteins such as N. Finally, treatment of immune recipient cells (T cells and monocytic cells) with some of the exosome preparations showed a drastic rate of apoptosis through PARP cleavage and caspase-3 activation. We have already published these resistant clone findings (Ahsan et al., 2016). To further investigate these findings, we also analyzed the role of exosomes derived from cells susceptible to RVFV infection, and as part of this effort developed a highly effective purification scheme that allowed complete separation of the exosomes from the released virions. Similar to the resistant clones, we find the presence of RVFV RNA and N protein in purified exosomes from cells susceptible to RVFV. Additionally, the exosomes from infected origin are capable of inducing cell death in recipient monocytic and lymphocytic cells. Collectively, our data suggest that exosomes from RVFV-infected cells alter the dynamics of the neighboring cells and may contribute to disease pathology.
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This thesis has been embargoed for 1 year and will not be available until April 26, 2017.
Keywords
Rift Valley Fever Virus, Exosomes, Resistant clones, Immune cell viability