Michael C. Pribula, Andras K. Ponti, and Erin E. Johnson 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 Background Results 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. - + IFN-β: KH02: 84 kDa 34 kDa Phospho-STAT1 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 Background - - + + - - - - + + + + IFN-β: KH01: KH02: Fraction: 84 kDa 62 kDa 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. - - - - + + - - + + - - - - - - - - + + - - + + C N C N C N C N C N C N Specific Aims STAT-1 HDAC-1 (Loading Control) To purify plasmids encoding wild type eVP24 and a cluster 3 mutant (negative control) and express these proteins in mammalian cells To demonstrate that the expression of eVP24 and not the cluster 3 mutant impairs nuclear transport of pSTAT1 To determine if small molecules that target the JAK-STAT pathway can overcome the eVP24-induced block in pSTAT1 shuttling 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. 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 Conclusions Results 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. Mutant NT Wild-Type eVP24 eVP24 Future Research 27 kDa 34 kDa HA-TAG GAPDH (Loading Control) 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. References 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 KPN-α5 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. 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. 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>>.