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Extracell Vesicles Circ Nucleic Acids 2020;1:20-56 I http://dx.doi.org/10.20517/evcna.2020.10 Page 49

38. Exosomes mediate Zika virus transmission through SMPD3 neutral Sphingomyelinase in
cortical neurons

Authors: Hameeda Sultana
E-mail: hsultana@odu.edu
Affiliations:
Old Dominion University, Norfolk, VA, USA.
Abstracts: The transmission dynamics of ZIKA virus (ZIKV) in or between neurons, or within the
developing brains of the fetuses are not fully understood. Using primary cultures of murine cortical
neurons, we show that ZIKV uses exosomes as mediators of viral transmission between neurons. Increased
production of exosomes from neuronal cells was noted upon ZIKV infection. Neuronal exosomes contained
both ZIKV viral RNA and protein(s) that were highly infectious to naïve cells. RNaseA and neutralizing
antibodies treatment studies suggested presence of viral RNA/proteins inside exosomes. Exosomes derived
from time- and dose-dependent incubations showed increasing viral loads suggesting higher packaging
and delivery of ZIKV RNA and proteins. Furthermore, we noted that ZIKV induced both activity and gene
expression levels of neutral Sphingomyelinase (nSMase)-2/SMPD3, an important molecule that regulates
production and release of exosomes. Silencing of SMPD3 in neurons resulted in reduced viral burden
and transmission through exosomes. Treatment with SMPD3 specific inhibitor GW4869, significantly
reduced ZIKV loads in both cortical neurons and in exosomes derived form these neuronal cells. Taken
together, our results suggests that ZIKV modulates SMPD3 activity in cortical neurons for its infection and
transmission through exosomes perhaps leading to severe neuronal death that may result in neurological
manifestations such as microcephaly in the neonatal developing embryonic brains and other complications
associated with Guillain-Barré syndrome in adults.

39. Understanding Intracellular Fate of EV-delivered Content

Authors: Killian P. O’Brien, Stefano Ughetto, Xandra O. Breakefield
E-mail: kobrien61@mgh.harvard.edu
Affiliations: Department of Neurology, Massachusetts General Hospital, Boston, MA, USA.
Abstracts: Despite much work performed on evaluating the potential effects of extracellular vesicles
(EVs), the functional uptake of their cargo is still controversial. This project aimed to demonstrate that EV
content (protein and mRNA) is protected and can be subsequently transferred with functional activity into
recipient cells, while also developing a tool to assess and quantify functional EV uptake.
Methods: Fusion proteins used were mitochondrial localized coxVIII-CFP-nanoluc(Cox) and nuclear
localized H2B-RFP-nanoluc(H2B).
Results: HEK293T cell-derived EVs protected Cox proteins from proteinase K digestion while
demonstrating significantly improved efficiency of uptake when compared to free protein, as measured by
bioluminescence that was still detectable in recipient cells 96 hrs post EV-exposure. To confirm functional
uptake, recipient cells exposed to EVs containing H2B for 72 hrs were imaged and some recipient cells
manifested fluorescent red nuclei. To demonstrate the presence of functional mRNA within EVs, producer
cells were transfected for such a duration as not to have detectable levels of protein in the EVs while still
containing detectable levels of mRNA (qPCR) even after RNaseA treatment. Transfer of these EVs to HeLa
cells showed an increase in expression of H2B which was blocked by cyclohexamide, confirming translation
of the mRNA (2.2 kb). To determine if recycling of EV delivered proteins occurs, recipient HeLa cells were
exposed to EVs containing Cox for 72 hrs. All extracellular EVs were removed and cells were trypsinized
(0.25% for 30 min) to remove any non-internalized Cox protein. 48 hrs later, EVs (CD63+ and CD9+)