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Extracell Vesicles Circ Nucleic Acids 2020;1:20-56 I http://dx.doi.org/10.20517/evcna.2020.10 Page 55

Affiliations: The Research Institute of the McGill University Health Centre, Montréal, Canada.
Abstracts:
Leishmaniasis, a complex pattern of diseases caused by sand fly-transmitted Leishmania sp. causes over 2
million new infections and 30,000 deaths each year. In mammals, Leishmania parasites establish a persistent
infection by inducing MØ dysfunction through direct manipulation of MØ signaling. We have deciphered
the mechanisms whereby Leishmania exploits MØ signaling pathways to block microbicidal functions
and innate inflammatory responses during infection. Work from my lab discovered that Leishmania major
GP63 was enriched in Leishmania exosomes and to play a pivotal role in those deactivation process of MØ
responses.

We reported that Leishmania exosomes are released in the gut of its sand fly vector and co-inoculated with
Leishmania promastigotes during blood meals. Co-egested Leishmania exosomes were found to exacerbate
cutaneous leishmaniasis skin lesions by overproducing inflammatory cytokines fueling Th17 immune
response.

Recently, Leishmania RNA virus 1 (LRV1) infecting certain Leishmania species was found to be associated
with aggressive mucocutaneous disease triggered in response to this dsRNA virus. However, it was unclear
how LRV1 is exposed to the mammalian host cells. In higher eukaryotes, some viruses are known to utilize
the host exosome pathway for their formation and cell-to-cell spread. As a result, exosomes derived from
infected cells contain viral material or particles. Recently, we found that LRV1 exploits the Leishmania
exosome pathway to reach the extracellular environment. Biochemical and electron microscopy analyses of
exosomes derived from LRV1-infected Leishmania revealed that most dsRNA LRV1 co-fractionated with
exosomes, and that a portion of viral particles was surrounded by these vesicles. Transfer assays of LRV1-
containing exosome preparations showed that a significant number of parasites were rapidly and transiently
infected by LRV1. Remarkably, these freshly infected parasites generated more severe lesions in mice
than non-infected ones. Moreover, mice co-infected with parasites and LRV1-containing exosomes also
developed a more severe disease. Overall, jpeg, with resolution of 300-this work provided evidence that Leishmania exosomes function
as viral envelopes, thereby facilitating LRV1 transmission and increasing infectivity in the mammalian host.

47. Delivery of pre-miRNA Cargo to Tumor Microvesicles

Authors: Crislyn D’Souza-Schorey, James Clancy, Ye Zhang
E-mail: Crislyn.D’Souza-Schorey.1@nd.edu
Affiliations: Department of Biological Sciences, University of Notre Dame, Notre Dame, IN, USA.
Abstracts: The regulated shedding of extracellular vesicles (EVs) is now understood to serve as an
important means of intercellular communication. Tumor-derived microvesicles (TMVs) comprise a class
of extracellular vesicles released from tumor cells that are now understood to facilitate communication
between the tumor and the surrounding microenvironment. Despite their significance, the regulation and
mechanisms governing the trafficking of bioactive cargos, including microRNAs (miRNAs), to TMVs at the
cell surface remain poorly defined. While miRNA recruitment ito exosomes has been reported, including
the sequence specific mechanisms for targeting miRNA, current understanding of miRNA loading into
TMVs has only recently begun to be elucidated. We have described a molecular pathway for the delivery of
miRNA cargo to nascent TMVs involving the dissociation of a pre-miRNA/Exportin-5 complex from Ran-
GTP following nuclear export and its subsequent transfer to a cytoplasmic shuttle comprised of ARF6-
GTP and GRP1. As such, ARF6 activation increases the pre-miRNA cargo contained within TMVs via a
process that requires casein kinase 2-mediated phosphorylation of Ran-GAP1. Further, TMVs were found
to contain pre-miRNA processing machinery including Dicer and Argonaute 2, which allow for cell-free