Download Ebola Virus Protein 24 Interactions with Phosphorylated STAT1

Use of Small Molecules to Abrogate the Intracellular Effects of
Ebolavirus Protein 24
Michael C. Pribula, Andras K. Ponti, and Erin E. Johnson
Department of Biology, John Carroll University, University Heights, Ohio 44118
Abstract
There have been 28,639 cases and 11,316 deaths as a result of the 2014
Ebolavirus outbreak1. Ebolavirus inhibits the immune system, which
contributes to its severity. Specifically, the Ebolavirus Protein 24 (eVP24)
interferes with the JAK-STAT signaling pathway, which governs
production of antiviral proteins. There are currently no FDA approved
treatments for Ebolavirus. Our lab identified several molecules that
function to enhance the JAK-STAT pathway. It is my hypothesis that these
compounds could counter the effects of eVP24. To date, I have expressed
eVP24 in mammalian cells. In the future, I will use the subcellular
localization of phosphorylated-STAT1 to demonstrate the ability of eVP24
to disrupt nuclear shuttling. Further studies will then address the ability of
our compounds to overcome this eVP24-mediated disturbance in
trafficking. It is my goal to determine if modulation of the JAK-STAT
pathway serves as a viable therapeutic intervention for this deadly disease.
Background
Background
IFN-β:
KH02:
-
+
-
+
+
84 kDa
Phospho-STAT1
34 kDa
GAPDH
(Loading Control)
Figure 3. There is more pSTAT-1 in the presence of KH compounds. These Western
blots show an increase in expression of phosphorylated STAT-1 (pSTAT-1) in the
presence of KH02 and Interferon-β (IFN-β). IFN-β alone increases pSTAT-1 expression
to augment antiviral responses, but the addition of KH02 further increases the response.
Figure 5. eVP24 suppresses ISRE-Mediated Luciferase Expression. A luciferase
assay was performed to analyze the expression of ISREs, gene elements associated with
the antiviral response activated by Type I Interferons. Increased expression of wildtype
eVP24 decreased ISRE expression, but mutant had little effect.
Hypothesis
This study explores whether or not KH molecules may be used to overcome the
eVP24-induced block on pSTAT1 nuclear localization. If successful, KH
compounds may be used as a viable therapeutic intervention for Ebolavirus
diseases.
Specific Aims
Figure 1. The Ebola Outbreak in West Africa. The outbreak that began in 2014 has
reached record numbers. The countries most affected are Liberia, Sierra Leone, and
Guinea.2
Results
1.To purify plasmids encoding wild type eVP24 and a cluster 3 mutant
(negative control) and express these proteins in mammalian cells
2.To demonstrate that the expression of eVP24 and not the cluster 3 mutant
impairs nuclear transport of pSTAT1
3.To determine if small molecules that target the JAK-STAT pathway can
overcome the eVP24-induced block in pSTAT1 shuttling
27 kDa
-
-
+
-
+ - - - + + - - - +
- + + + +
- + + - + - - + +
C
N
C
N
N
C N
C
C
N C N
84 kDa
STAT-1
62 kDa
HDAC-1
(Loading
Control)
Figure 6. Addition of KH compounds shows significant increases in nuclear levels of
STAT-1. Cytoplasmic-Nuclear fractionation was performed on 293T cells. The results of
the fractionation were analyzed via a Western Blot. The results showed a clear increase
in the nuclear STAT-1 level in cells treated with KH compounds.
Conclusions
Results
Mutant
eVP24
IFN-β:
KH01:
KH02:
Fraction:
Preliminary results suggest that the presence of KH01 and KH02 for the treatment of
cells causes a greater increase in STAT-1 in the nucleus than interferon by itself.
NT Wild-Type
eVP24
HA-TAG
Future Research
To determine if the STAT-1 increases observed in cells uninfected with eVP24 will also
be seen in mutant and wild-type cells infected with this protein.
34 kDa
Figure 2. The JAK-STAT signaling pathway. IFN-α binds to IFNaR1 and IFNaR2 that
stimulate JAK1 and TYK2 to phosphorylate STAT1 and STAT2, which then associate
with IRF9 to make the complex ISGF3. ISGF3 is transported into the nucleus via KPNα5 where it can activate the transcription of antiviral genes. In the presence of eVP24,
nuclear import is disrupted, thus preventing the activation of antiviral genes.
GAPDH
(Loading Control)
Figure 4. Expression check for eVP24 in mammalian cells. 293T cells were either
treated with mutant eVP24 DNA, treated with wild-type eVP24 DNA, or left untreated.
The results of these treatments were analyzed using a western blot. Cells treated with
both the wild-type and mutant DNA produced protein, while the cells left untreated
failed to produce protein.
References
1. “2014 Ebola Outbreak in West Africa - Case Counts.” CDC. March 15, 2016.
<<www.cdc.gov>>.
2. “Ebola in Graphics: The Toll of a Tragedy.” The Economist. April 2, 2015.
<<www.economist.com>>.