Critique of Primary Literature and Analysis

Samantha Wargo

Zaire ebolavirus (ZEBOV) is a filovirus that infects humans and

non-human primates. This virus causes a hemorrhagic fever and can be
fatal. Saeed et al studied three different mechanisms to prove that one of
these is most likely the source of entry: clathrin-mediated endocytosis,
caveolin-mediated endocytosis, and macropinocytosis. Clathrin-
mediated endocytosis (CME) is an endocytic pathway that involves
clathrin-coated pits. The clathrin coated vesicles bud off with the virus
cargo inside. Accessory protein-2 and Eps15 are molecules that are
associated with these pits. Dynamin is associated with the budding off of
the vesicles. These vesicles are mid-sized, compared to caveolin-
mediated endocytosis and macropinocytosis. In caveolin-mediated
endocytosis (CavME), the plasma membrane has invaginations that are
rich in caveolin protein. CavME also requires cholesterol rich lipid rafts.
The vesicles, also known as cavesomes, are indicated by caveolin. Just
like CME, dynamin is associated with the vesicle budding. Compared to
CME and macropinocytosis, these vesicles are the smallest. In
macropinocytosis, membrane ruffles can fold back and form a cavity,
called a macropinosome. Like CavME, cholesterol rich lipid rafts are
important. The budding off of the macropinosomes is controlled by
CtBP/BARS. Macropinosomes are indicated by dextran and Na+/H+
exchangers. The macropinosomes are the largest in comparison to CME
and CavME vesicles (in-class notes). It is known that ZEBOV enters
through an endocytic pathway, but it is not highly understood how and
through which pathway it enters.

Further research on ZEBOV is needed because there is no therapy

or vaccine currently available. The need for therapeutic intervention is
important because ZEBOV transmission occurs easily and outbreaks can
result in a high percentage of death. Of the people infected, death can
happen in 50% or more of the cases. The high mortality rate could pose
threats to not only the human population, but also to the animal
population. In 2014, there was a large outbreak that affected countries
in Africa, and posed a threat to the United States and many other places
around the world. The death toll was suspected to be around 11,000
people out of 28,000 total cases. The total cases included suspected,
probable, and confirmed. There were 15,000 laboratory confirmed
cases (2014 Ebola Outbreak in West Africa, 2015). The typical target for
intervention is focused on the point of entry for the virus. Information is
limited in the area of ZEBOV entry. Previous studies have used particles
that are not morphologically or biochemically the same as ZEBOV. In the
study done by Saeed et al, they used ZEBOV virus-like particles, which
are morphologically comparable. Scientists hypothesized that the entry
of ZEBOV involves mechanisms that appear to be consistent with
macropinocytosis.

Between the paper and what was learned in class, there are many
links; 3 of the links are as follows:
In class we talked about vesicular transport in chapter 15 of the
textbook. Vesicular transport involves the budding and fusing of
vesicles with the membrane. Some vesicles have a certain protein coat
on the cytosolic surface. The textbook mentions that one of the best-
studied vesicles is the clathrin-coated vesicle. Saeed et al proves in their
study that clathrin-coated vesicles are not the source of ZEBOV entry
but shows that proteins involved in these vesicles show no change in
ZEBOV entry.
In chapter 10 we learned about plasmids and what they can be used
for in experiments. A plasmid is a small circular DNA molecule that can
be cleaved open and a DNA fragment is inserted. The plasmid will then
be introduced into a bacterium where it can be copied and amplified. In
the study done by Saeed et al, the cells were transfected with plasmids
encoding the certain protein expression they were examining.
In chapter 10 we explored green fluorescence protein and how it
can be used. Green fluorescence protein is a reporter protein that can be
used to see something specific. For instance, if you would like to see
where a certain gene is expressed then you can use green fluorescence
protein and see where it glows. In the study by Saeed et al, they used
green fluorescence protein as a control in some parts of the study. This
would make it easy to see where the expression changes in reference to
the control cells.

In the article I found, Rhein and Maury, talk about targeting small
molecule inhibitors to block a certain step in the entry. Specifically, late
endosomes were targeted by stopping the transport of cholesterol.
Though the mechanism of how this happened is not really understood, it
was shown to have a decreased effect of the ZEBOV infection. Rhein and
Maury mentioned one drug, U18666A, called a cationic amphiphile drug.
This drug stopped the cholesterol transport and reduced infection.
Similar effects were shown from other cationic amphiphiles. This builds
upon Saeed et al because in their study, Saeed et al showed that ZEBOV
involves endosomal trafficking. Saeed used Rab 5 and Rab 7, which have
a role in trafficking. Saeed proved that the mechanism used early and
late endosomes by using dominant negative Rab 5 and Rab 7. When the
dominant negative Rab 5 and Rab 7 was used, the virus in cells was
lower. Rhein and Maurys information built on this by showing that
inhibiting cholesterol is one way to prevent the late endosomal
trafficking. They are also building on the fact that macropinocytosis
requires cholesterol lipid rafts. Saeed et al determined that cholesterol
lipid rafts and ZEBOV particles associate and that cholesterol
sequestering inhibits ZEBOV.

The findings discussed in the article by Saeed et al can be

influential in creating a drug to help fight ZEBOV. The main finding was
that ZEBOV enters through a pathway that is like macropinocytosis.
Since the entry is typically the target for drugs, figuring out that the
entry is like macropinocytosis gives scientists a few different targets
they can try to attack. The different targets can come from the different
proteins involved in the uptake of the virus. For example, in the article I
found, they examined drugs that inhibit cholesterol. The mechanism of
how the cationic amphphiles worked with inhibiting late endosomes
was uncertain (Rhein and Maury 2015). This is a question that another
scientist can try to answer. The contributions from both of these articles
point to cholesterol inhibition being a helpful tool in preventing
infection. Therefore, this is a new step in which scientists can work on in
finding a vaccination or drug to bring down the death rate for ZEBOV.

References
A. 2014 Ebola Outbreak in West Africa. (Updated November 12,
2015). Retrieved from
http://www.cdc.gov/vhf/ebola/outbreaks/2014-west-africa/
B. Rhein, B.A., Maury, W.J. (September 2015). Ebola Virus Entry
Into Host Cells: Identifying Therapeutic Strategies. Current Clinical
Microbiology Reports, 2(3). Retrieved from
http://link.springer.com/article/10.1007/s40588-015-0021-3#