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Page 46 Extracell Vesicles Circ Nucleic Acids 2020;1:20-56 I http://dx.doi.org/10.20517/evcna.2020.10

Affiliations:
1 Laboratory of Molecular Virology, School of Systems Biology, George Mason University, Manassas, VA, USA.
2 CIRI International Center for Infectiology Research, Inserm U1111, CNRS UMR5308, Ecole Normale
Superieure de Lyon, Université Lyon, Lyon, France.
Abstracts: Recently, it has become evident that Extracellular Vesicles (EVs) play a major role in the viral
pathogenesis. Our lab has been able to elucidate the role of EVs in the pathogenesis of several different
[1-7]
viruses including HIV-1, HTLV-1, Rift Valley Fever Virus and Ebolavirus . However, timing difference
between EV and virions release from infected cells has not been previously reported. We have attempted to
address the spatiotemporal dynamics of EV and virions release from HIV-1 and HTLV-1 infected cells. The
infected cells were synchronized in G0 quiescent stage using serum starvation. Viral latency was reversed
by increasing gene expression with the addition of serum rich media and inducers (20% FBS + PMA/
PHA). Supernatants and cell pellets were collected post induction at 0, 3, 6, 12, and 24 h and assayed for
the presence of markers of EVs, autophagy and for the viral proteins and RNA transcripts. Results from
supernatants of uninfected cells showed a peak of tetraspanin proteins (CD63, CD81, and CD9) at 6 h
and a gradual decrease of all EV associated proteins by 24 h. However, the EV from HIV-1 infected cells
showed all three tetraspanins present at 3 h and expression gradually increased up to 24 h. When compared
to HTLV-1 infected cells, the three tetraspanin proteins peaked at 6 h and expression continued to decrease
up to 24 h. HTLV-1 infected cells also showed a unique pattern of CD81 expression. Autophagy associated
proteins (LC3A, LC3B and p62) from uninfected cells and HTLV-1 infected cells plateaued at 6 h, whereas
in HIV-1 infected cells their expression continued to increase and peaked at 24 h. HIV-1 viral proteins
(p24, gp120, Nef) expression was present at 6 h and continued to increase and peaked at 24 h. HTLV-
1 proteins (p19 and gp46/61) peaked at 6 h and decreased overtime. HIV-1 and HTLV-1 gene expression
was quantified, and data correlated with viral protein expression. EV release was analyzed by nanoparticle
tracking analysis and significant increase of EV concentration overtime in both uninfected and infected
samples was observed. Finally, virus rescue assay with the use of naïve cells was performed on 6- and
24-h supernatants. HIV-1 supernatant from 6-h sample was found not to be infectious, however the virus
from 24-h sample was rescued. Our data indicates that EV release may occur prior to virion release from
infected cells. The released EVs can be implicated in the facilitation of virus spread and deleterious effect
on the naive recipient cells.

REFERENCES
1. Narayanan A, Iordanskiy S, Das R, et al. Exosomes derived from HIV-1-infected cells contain trans-activation response element RNA. J
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2. Sampey GC, Saifuddin M, Schwab A, et al. Exosomes from HIV-1-infected cells stimulate production of pro-inflammatory cytokines
through trans-activating response (TAR) RNA. J Biol Chem 2016;291:1251-66.
3. Ahsan NA, Sampey GC, Lepene B, et al. Presence of viral RNA and proteins in exosomes from cellular clones resistant to rift valley
fever virus infection. Front Microbiol 2016;7:139.
4. Barclay RA, Schwab A, DeMarino C, et al. Exosomes from uninfected cells activate transcription of latent HIV-1. J Biol Chem
2017;292:11682-701.
5. DeMarino C, Pleet ML, Cowen M, et al. Antiretroviral drugs alter the content of extracellular vesicles from HIV-1-infected cells. Sci Rep
2018;8:7653.
6. Pleet ML, Erickson J, DeMarino C, et al. Ebola virus VP40 modulates cell cycle and biogenesis of extracellular vesicles. J Infect Dis
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7. Pinto DO, DeMarino C, Pleet ML, et al. HTLV-1 extracellular vesicles promote cell-to-cell contact. Front Microbiol 2019;10:2147.

35. Separation of EVs from virions in coronavirus infections

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Authors: James Erickson , Maria Cowen , Yuriy Kim , Anoop K. Pal , Heather Branscome , Archana
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Gupta , Fatah Kashanchi 1