Ebola virus infection is associated with fatal
diseases in humans. For example, Ebola hemorrhagic fever is distinguished by
the pathological onset of severe inflammation and internal bleeding within
infected hosts [2]. Despite a high prevalence of human-human transmission,
Ebola is a “zoonotic” virus, meaning it enters the human population via an
animal host. For this particular virus, the animal host is a fruit bat, as
certain species have been shown to act as reservoirs— carrying an asymptomatic
infection able to manifest as pathogenic when transmitted into humans [3].
Until 2014, Ebola was endemic, or regularly found within the population, in
West Africa. However, when a mutant strain of the virus emerged, an epidemic
was established in the region. This outbreak lasted until 2016 and during this
period the virus found its way to eleven individuals within the U.S., which
resulted in two fatalities of American citizens [4]. Today, the Democratic
Republic of the Congo is currently facing an outbreak of its own, keeping
research on the virus and potential ways to control its spread at the forefront
of global public health efforts [5].
In terms of structure,
the Ebola virus genome is composed of single-stranded, minus-sense RNA that
must be converted to positive-sense by its own polymerase before translation
can occur. Additionally, its lipid membrane envelope includes just one type of
protein, known as a glycoprotein (GP), on its surface. This protein protrudes
from Ebola viral particles, playing a key role in the attachment and fusion of
the particles to host target cells. In its mature form, GP exists as two
subunits: GP1, which includes a receptor binding domain for attaching to target
cell surfaces, and GP2, which includes a fusion peptide and transmembrane
domain to facilitate this attachment during infection. In the face of such
viral infection, host immunity often works in opposition to the invading virus,
creating an arms race as each one attempts to overcome the other. For instance,
the host restriction factor, tetherin, is an antiviral gene regulated by the
innate immune system, or the host’s nonspecific defense that kicks in directly
following detection of a virus. Known to function in the suppression of many
enveloped viruses, tetherin works to interfere with a virus’s ability to
release progeny viral particles from infected cells in order to spread within
the host. Specifically, as seen below, tetherin’s unique double-anchored
structure inserts itself into both the viral envelope and infected cell
membranes in order to anchor budding virions to the cell and thereby prevent
free release into the host.
 |
| Tetherin: Traps viral-associated vesicles, as pictured, or progeny virions at the cell surface by embedding one anchor into the releasing membrane and the other into the host cell plasma membrane. [1] |
In this study, Nehls et al. were mainly focused on tetherin’s targeting of vesicle release, in addition to trapping budding virions. Previous studies these researchers built upon have reported that, like many viruses, Ebola virus infection releases microvesicles upon infection as a mechanism to emit viral proteins or nucleic acids from infected cells. Specifically, Ebola utilizes these vesicles to carry out the release of its GP protein. To better elucidate the role of these “GP-virosomes” in Ebola’s ability to infect hosts, researchers analyzed these particles and hypothesized that tetherin interacts with the GP-associated vesicles, ultimately interfering with their release.
To
specifically analyze GP-virosomes, as opposed to exosomes and smaller vesicles,
researchers first used a centrifugation technique that separated the seemingly
larger virosomes from other smaller vesicles thought to be lacking the GP viral
protein. To verify that these virosomes were both vesicles of larger size and
carriers of the Ebola virus GP, a western blot, as shown below, was performed
showcasing enriched GP in microvesicles (pelleted at 21000xg) as compared to in
exosomes and smaller vesicles (pelleted at 100000xg).
 |
| Figure 1C: Verifying Ebola virosomes as GP-containing, non-exosomal vesicles. Supernatants of transfected 293T cells were spun down at different centrifugation speeds and GP levels were detected via western blot. Microvesicles (100-1000nm in size) pelleted at 21000xg and exosomes (<100nm) pelleted at 100000xg. |
After observing via electron microscopy that the release of these GP-virosomes from host cells is similar to the budding of progeny virions, researchers aimed to investigate tetherin’s impact on this release. First, a human embryonic kidney cell line (HEK-293T) commonly used for transfections, or the introduction of DNA to eukaryotic cells, was used to analyze the coexpression of tetherin and GP in cell culture. To track and quantify the amount of GP released by virosomes, researchers performed a western blot on the cell culture media, as this is where the virosomes are expected to be located after successful release from infected cells. However, if tetherin was expressed as well, as shown below, this release of Ebola virus GP-virosomes detected within the media was significantly reduced. This result was replicated in HeLa cells, as this cell line contains endogenous levels of tetherin, and therefore verifies that the suppression of release seen is not an unintended impact of tetherin overexpression in the transfected 293T cells.
 |
| Figure 4a: Monitoring the release of GP-virosomes. 293T cells were transfected to express varying amounts of tetherin alone (mock) or in addition to cotransfection with GP. As tetherin expression increased, GP-virosome detection in media decreased in GP-transfected cells. |
Next,
researchers analyzed tetherin mutants to narrow down which components of the
protein’s unique anchor shape are vital for its ability to suppress GP-virosome
release. Tetherin mutants lacking the C-terminal anchor were discovered via
western blot to be incorporated into the cell culture media. This result
suggested that the C-terminal anchor facilitates tetherin insertion into the
host cell plasma membrane and is therefore vital for the protein’s ability to
prohibit virosome release. Without this region, tetherin is not only unable to
keep GP-virosomes from entering the cell culture media, but is also
incorporated into the virosomes themselves. After narrowing down the vitality
of tetherin’s shape to its antiviral function, transfections with tetherin
derived from various animals, from hamsters to alligators, were compared to
human tetherin functioning. Differential levels of GP detected in the media
based on tetherin variation supported a conclusion that the primary
sequence of tetherin, as it varies by species, is also important for the
efficiency of the protein’s antiviral activity. Turning their sights to regions
of the Ebola virus GP important for virosome release, researchers then analyzed
the impact of various mutations in either the receptor binding or transmembrane
domains of the protein. After transfecting HeLa cells with a GP variant
containing a change in its transmembrane domain, tetherin levels were detected
via a proximity ligation assay that uses antibodies against tetherin to
fluorescently image the protein, as shown in the figure below. Levels of
tetherin detected in the GP variant cells (ELE) were significantly reduced as
compared to cells with wild-type GP transmembrane domains. This finding implied
that this region of the Ebola virus GP is essential to tetherin’s ability to
interact and prevent the release of GP-virosomes.
 |
| Figure 5c: The GP transmembrane domain is important for tetherin binding. HeLa cells were transfected with GP or a GP variant with changes to its transmembrane domain (ELE). Cells were imaged via proximity ligation assay (PLA). Tetherin levels are shown in red, with decreased tetherin detection seen in GP-variant cells. |
Lastly, Nehls et al. aimed to determine how these virosomes contribute to Ebola virus pathogenic abilities, as well as their possible role in viral-mediated immunomodulation within hosts. Interestingly, when a GP mutant strain from the 2014-2016 Ebola outbreak in West Africa was transfected into HeLa cells, the release of GP-virosomes in both the presence and absence of tetherin was significantly amplified as compared to Ebola virus strains with wild-type GP. As demonstrated by the outbreak, this mutant, known as A82V, is highly pathogenic. Therefore, this result supports a correlation between GP-virosome release and viral pathogenicity. As interesting as this result is, the GP-virosome’s role in immunomodulation is perhaps the most fascinating finding of this paper. Using a neutralization assay, the ability of an antibody, KZ52, to neutralize GP was measured via luciferase reporter activity. In supernatants containing GP-virosomes, the neutralizing activity of the antibody was counteracted until the addition of tetherin, in which neutralization levels were restored. These results provide support for a mechanism of virosomes to trap these neutralizing antibodies, ultimately preventing their ability to restrict true viral particles that express GP on their surface. This idea of GP-virosomes as “decoys” for the immune response was further supported by an analysis of cytokine release from macrophages treated with supernatants in the presence and absence of GP. Of the eight cytokines in question, three were seen to have decreased secretion in the presence of GP-virosomes unrestricted by tetherin. This immunomodulatory function of GP-virosomes, and its antagonization by tetherin, is key to understanding the breadth of the tetherin antiviral protein’s contribution to host innate immune responses against Ebola infection.
Nehls
et al. do not highlight any future directions for their study in particular, however,
there are many remaining questions left to be investigated. For instance,
GP-virosome release was found by the researchers to be significantly inhibited
by tetherin derived from the fruit bat reservoir species. This leads to
speculation that tetherin’s role in suppressing GP-virosome release is separate
from its role in antagonizing budding viral particles. However, since fruit bat
tetherin demonstrates more efficient antiviral activity than the human form,
does the idea that these tetherin-suppression processes are separate still hold
true in human hosts facing pathogenesis? With human tetherin’s lower antiviral
activity, its actions on GP-virosomes could be impacted in the presence of
budding virions that tetherin must also work to target. To investigate this,
researchers could utilize the same methodology of transfecting with Ebola virus
GP, and analyzing its release into cell culture media, but add in the condition
of infected cells. If GP-virosome release is still inhibited by human tetherin at
the same level as in uninfected cells, this would verify that, in the infected
host, these antiviral mechanisms of tetherin are indeed independent of one
another.
Additionally,
in their initial characterization of Ebola virus GP-virosomes, Nehls et al. notice
an upregulation of the CD81 marker protein and describe the microvesicles as
“CD81-positive,” but do not expand upon this finding. As CD81 is known as an
exosomal marker, what role does it have in these non-exosomal GP-virosomes? One
possibility is that CD81 contributes to tetherin’s ability to recognize the
vesicle, as the protein has been found to also play a role in suppressing
exosomal release [6]. To determine if CD81 is important for tetherin to be able
to target exosomal and non-exosomal particles alike, the expression of the
marker protein could be attenuated using a knockout mechanism such as RNAi. If
tetherin’s ability to suppress release in the absence of CD81 is impacted,
these findings would suggest that the marker could be a common denominator
between tetherin targets.
Lastly,
with the support these findings provide toward tetherin’s strong role in the
innate immune response, it is easy to wonder if tetherin could potentially act
as an agent in antiviral therapy. Future investigations could thus consider
ways to upregulate tetherin expression on the cell surface of sites such as
dendritic and other immune cells that face Ebola in its early stages of
replication [7, 8]. Such advancements of therapy against Ebola virus infection could make a difference in facing current outbreaks, as well as future threats to international public health.
References
[1] Nehls,
J., Businger, R., Hoffmann, M., Brinkmann, C., Fehrenbacher, B., Schaller, M.,
... & Pöhlmann, S. (2019). Release of Immunomodulatory Ebola Virus
Glycoprotein-Containing Microvesicles Is Suppressed by Tetherin in a
Species-Specific Manner. Cell reports, 26(7), 1841-1853.
[2] “What is Ebola?” John Hopkins Medicine. Retrieved
from https://www.hopkinsmedicine.org/ebola/about-the-ebola-virus.html
[3] Leroy,
E. M., Kumulungui, B., Pourrut, X., Rouquet, P., Hassanin, A., Yaba, P., ...
& Swanepoel, R. (2005). Fruit bats as reservoirs of Ebola virus. Nature,
438(7068), 575.
[4] “2014-2016 Ebola Outbreak in West Africa.”
(2018, March 8). Centers for Disease Control and Prevention. Retrieved
from https://www.cdc.gov/vhf/ebola/history/2014-2016-outbreak/index.html#anchor_1515001427541
[5] Moss, K., Michaud, J., & Kates, J.
(2019, September 27). “The Current Ebola
Outbreak and the U.S. Role: An Explainer.” Kaiser Family Foundation.
Retrieved from
[6] Edgar,
J. R., Manna, P. T., Nishimura, S., Banting, G., & Robinson, M. S. (2016).
Tetherin is an exosomal tether. Elife, 5, e17180.
[7] Sauter,
D., Specht, A., & Kirchhoff, F. (2010). Tetherin: holding on and letting
go. Cell, 141(3), 392-398.
[8] Servick, K. (2014, August 13). “What
does Ebola actually do?” Science. Retrieved from https://www.sciencemag.org/news/2014/08/what-does-ebola-actually-do
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