Research

Filoviruses
The filoviruses Ebola virus and Marburg virus cause severe human disease and are of concern as emerging pathogens and bioterrorism agents.  The Basler Laboratory has pioneered efforts to define at the molecular level how filoviruses evade innate antiviral defenses and to understand how this immune evasion contributes to disease.  Prior to this work, which began in 2000, little attention had been paid to how filoviruses interact with innate immune pathways.  Since we began, increased attention is paid to filoviral immune evasion mechanisms and two of the Ebola virus IFN-antagonist proteins, VP35 and VP24, have received increased interest as potential drug targets. This work has also led to Dr. Basler serving as PI on two U19 Center Grants, one that seeks to develop small molecule inhibitors of filoviral interferon-antagonist functions and one that seeks to define the role of these functions in filoviral pathogenesis.

figureOur work has also revealed novel mechanisms regulating filovirus replication, gene expression and host range.  We recently identified (1) how upstream open reading frames modulate Ebola virus gene expression in a manner regulated by host stress responses; (2) novel RNA editing events that diversify filoviral gene expression; (3) a novel interaction of Marburg virus VP24 with antioxidant response pathways; and (4) Marburg virus VP40 as an inhibitor of interferon signaling, in particular a suppressor of Jak-STAT signaling, providing evidence that this function influences virus host range.

Macrophages and dendritic cells (DCs) are targets of filovirus replication in vivo.  Macrophage inflammatory responses are thought to contribute to the vascular leakage and coagulopathy that characterize these infections.  Disruption of DC functions is thought to prevent mounting of an effective adaptive immune response.  We have identified mechanisms that explain how Ebola viruses disrupt DC maturation and function; we have demonstrated a failure of Ebola virus to enter and activate plasmacytoid DCs and we have identified the p38 MAP kinase pathway signaling as important for inflammatory cytokine production and a potential therapeutic target.  Overall, these findings influence therapeutic approaches for Ebola and Marburg virus infections.

Immune Mechanisms of Virus Control

The filoviruses, Ebola and Marburg viruses (EBOV and MARV), are emerging, zoonotic pathogens, that cause severe viral hemorrhagic fever. There are currently no approved anti-filovirus therapeutics available. Filovirus infection is characterized by uncontrolled viral replication, an aberrant innate immune response, suppression of adaptive immunity, dysregulated dendritic cell (DC) function and lymphocyte apoptosis. Filoviral interferon antagonists have been previously identified and characterized by our lab and include EBOV and MA
RV VP35, EBOV VP24 and MARV VP40. VP35 inhibits the production of interferon-alpha/beta through several mechanisms, including binding dsRNA and sequestering it from recognition by RIG-I/MDA5. EBOV VP24 and MARV VP40 block interferon signaling through two different mechanisms. EBOV VP24 interacts with the nuclear transport karyopherin alpha (KPNA), competitively inhibiting its interaction with tyrosine phosphorylated STAT1 (PY-STAT1), preventing nuclear localization of PY-STAT1 and the induction of an antiviral response. Alternatively, MARV VP40 blocks the phosphorylation and activation of Jak1 through an, as of yet, unidentified mechanism. Through their role in suppressing the innate antiviral activity of interferon, we hypothesize that these viral interferon antagonists will also have an effect on adaptive immunity, disrupting the downstream signaling required for dendritic cell (DC) maturation and effective DC:T-lymphocyte interactions.

figure Center consists of a team with extensive experience in filoviruses and the interface of virus-host immune responses. This includes the groups of Dr. Basler at GSU, Drs. Amarasinghe and Gross at WUStL, Drs. Bukreyev and Geisbert at UTMB and Dr. Messaoudi at UCR. Through the work of the Center we will define the signaling pathways affected by the viral interferon antagonists and determine their role on adaptive immunity responses.

Select Publications
Xu W, Luthra P, Wu C, Batra J, Leung DW, Basler CF, Amarasinghe GK. Ebola virus VP30 and nucleoprotein interactions modulate viral RNA synthesis. Nat Commun. 2017 Jun 8;8:15576. doi: 10.1038/ncomms15576. PubMed PMID: 28593988; PubMed Central PMCID: PMC5472179.

Menicucci AR, Versteeg K, Woolsey C, Mire CE, Geisbert JB, Cross RW, Agans KN, Jankeel A, Geisbert TW, Messaoudi I. Transcriptome Analysis of Circulating Immune Cell Subsets Highlight the Role of Monocytes in Zaire Ebola Virus Makona Pathogenesis. Front Immunol. 2017 Oct 26;8:1372. doi:10.3389/fimmu.2017.01372. eCollection 2017. PubMed PMID:29123522; PubMed Central PMCID: PMC5662559.

Versteeg K, Menicucci AR, Woolsey C, Mire CE, Geisbert JB, Cross RW, Agans KN, Jeske D, Messaoudi I, Geisbert TW. Infection with the Makona variant results in a delayed and distinct host immune response compared to previous Ebola virus variants. Sci Rep. 2017 Aug 29;7(1):9730. doi: 10.1038/s41598-017-09963-y. PubMed PMID: 28852031; PubMed Central PMCID: PMC5574898.

Schwarz T, Edwards, M.R., Diederichs, A., Alinger, J.B., Leung, D.W., Amarasinghe G.K., Basler, C.F. Binding affinities of VP24 proteins from different ebolavirus species for karyopherin alpha proteins influence interferon inhibition and VP24 stability J Virol. 2016. Accepted.

Edwards MR, Liu G, Mire CE, Sureshchandra S, Luthra P, Yen B, Shabman RS, Leung DW, Messaoudi I, Geisbert TW, Amarasinghe GK, Basler CF. Differential Regulation of Interferon Responses by Ebola and Marburg Virus VP35 Proteins. Cell Rep. 2016 Feb 23;14(7):1632-40. doi: 10.1016/j.celrep.2016.01.049. PubMed PMID: 26876165; PubMed Central PMCID: PMC4767585.

Johnson B, Li J, Adhikari J, Edwards MR, Zhang H, Schwarz T, Leung DW, Basler CF, Gross ML, Amarasinghe GK. Dimerization Controls Marburg Virus VP24-dependent Modulation of Host Antioxidative Stress Responses. J Mol Biol. 2016;428(17):3483-94. doi: 10.1016/j.jmb.2016.07.020. PubMed PMID: 27497688; PMCID: PMC5010500.

Chatterjee S, Basler CF, Amarasinghe GK, Leung DW. Molecular Mechanisms of Innate Immune Inhibition by Non-Segmented Negative-Sense RNA Viruses. J Mol Biol. 2016;428(17):3467-82. doi: 10.1016/j.jmb.2016.07.017. PubMed PMID: 27487481; PMCID: PMC5010489.

Yen BC, Basler CF. Effects of Filovirus Interferon Antagonists on Responses of Human Monocyte-Derived Dendritic Cells to RNA Virus Infection. J Virol. 2016;90(10):5108-18. doi: 10.1128/JVI.00191-16. PubMed PMID: 26962215; PMCID: PMC4859717.

Rivera A, Messaoudi I. Molecular mechanisms of Ebola pathogenesis. J Leukoc Biol. 2016;100(5):889-904. doi: 10.1189/jlb.4RI0316-099RR. PubMed PMID: 27587404.

Leung DW, Borek D, Luthra P, Binning JM, Anantpadma M, Liu G, Harvey IB, Su Z,
Endlich-Frazier A, Pan J, Shabman RS, Chiu W, Davey RA, Otwinowski Z, Basler CF,
Amarasinghe GK. An Intrinsically Disordered Peptide from Ebola Virus VP35 Controls
Viral RNA Synthesis by Modulating Nucleoprotein-RNA Interactions. Cell Rep. 2015 Apr
21;11(3):376-89. PubMed PMID: 25865894; PubMed Central PMCID: PMC4599368.

Luthra P, Jordan DS, Leung DW, Amarasinghe GK, Basler CF. Ebola virus VP35
interaction with dynein LC8 regulates viral RNA synthesis. J Virol. 2015 May;89(9):5148-
53. PubMed PMID: 25741013; PubMed Central PMCID: PMC4403485.

Ilinykh PA, Lubaki NM, Widen SG, Renn LA, Theisen TC, Rabin RL, Wood TG,
Bukreyev A. Different Temporal Effects of Ebola Virus VP35 and VP24 Proteins on

Global Gene Expression in Human Dendritic Cells. J Virol. 2015 Aug;89(15):7567-83.
PubMed PMID: 25972536; PubMed Central PMCID: PMC4505630.

Edwards MR, Basler CF. Marburg Virus VP24 Protein Relieves Suppression of the NF-
κB Pathway Through Interaction With Kelch-like ECH-Associated Protein 1. J Infect Dis.
2015 Oct 1;212 Suppl 2:S154-9. PubMed PMID: 25926686; PubMed Central PMCID:
PMC4564532.

Feagins AR, Basler CF. Amino Acid Residue at Position 79 of Marburg Virus VP40
Confers Interferon Antagonism in Mouse Cells. J Infect Dis. 2015 Oct 1;212 Suppl
2:S219-25. PubMed PMID: 25926685; PubMed Central PMCID: PMC4564529.

Messaoudi I, Amarasinghe GK, Basler CF. Filovirus pathogenesis and immune evasion:
insights from Ebola virus and Marburg virus. Nat Rev Microbiol. 2015 Nov;13(11):663-
76. PubMed PMID: 26439085.

Anti-Filoviral Therapeutics

The filoviruses, Ebola and Marburg viruses (EBOVs and MARVs), cause highly lethal hemorrhagic fever in humans with fatality rates approaching 90%. There are currently no treatments or therapeutics approved for filovirus infections, the need of which was emphasized by the recent EBOV epidemic in West Africa in 2014-2016. To address this, the Center focuses on the development of small molecule therapeutics that target viral innate immune suppression and viral transcription/replication. These approaches are expected to identify panfilovirus inhibitors targeting distinct virus functions, which when combined are expected to act synergistically.

The Center consists of a diverse team with extensive experience in filoviruses, antiviral development and medicinal chemistry. This includes the groups of Dr. Basler at GSU, Dr. Ready at UTSW, Dr. Amarasinghe at WUStL, Dr. Bukreyev at UTMB and the scientists at Microbiotix, Inc. Through this collaboration, high-throughput screening (HTS) assays are developed based on integral viral functions, including EBOV and MARV VP35 suppression of interferon-alpha/beta production in infected cells, MARV VP40 and EBOV VP24 suppression of interferon induced Jak-STAT signaling and filovirus transcription/replication machinery. These HTS assays are used to screen libraries of compounds to identify small molecule inhibitors of filovirus interferon antagonism and transcription/replication. Lead compounds will be evaluated and optimized for antiviral activity in cell culture and animal models.

Select Publications
Xu W, Luthra P, Wu C, Batra J, Leung DW, Basler CF, Amarasinghe GK. Ebola virus VP30 and nucleoprotein interactions modulate viral RNA synthesis. Nat Commun. 2017 Jun 8;8:15576. doi: 10.1038/ncomms15576. PubMed PMID: 28593988; PubMed Central PMCID: PMC5472179.

Luthra P, Aguirre S, Yen BC, Pietzsch CA, Sanchez-Aparicio MT, Tigabu B, Morlock LK, García-Sastre A, Leung DW, Williams NS, Fernandez-Sesma A, Bukreyev A, Basler CF. Topoisomerase II Inhibitors Induce DNA Damage-Dependent Interferon Responses Circumventing Ebola Virus Immune Evasion. MBio. 2017 Apr 4;8(2). pii: e00368-17. doi: 10.1128/mBio.00368-17. PubMed PMID: 28377530; PubMed Central PMCID: PMC5380843.

Younan P, Ramanathan P, Graber J, Gusovsky F, Bukreyev A. The Toll-Like Receptor 4 Antagonist Eritoran Protects Mice from Lethal Filovirus Challenge. MBio. 2017 Apr 25;8(2). pii: e00226-17. doi:
10.1128/mBio.00226-17. PubMed PMID: 28442605; PubMed Central PMCID: PMC5405229.

Liu G, Nash PJ, Johnson B, Pietzsch C, Ilagan MX, Bukreyev A, Basler CF, Bowlin TL, Moir DT, Leung DW, Amarasinghe GK. A Sensitive in Vitro High-Throughput Screen To Identify Pan-filoviral Replication Inhibitors Targeting the VP35-NP Interface. ACS Infect Dis. 2017 Mar 10;3(3):190-198. doi: 10.1021/acsinfecdis.6b00209. Epub 2017 Feb 9. PubMed PMID: 28152588.

Schwarz T, Edwards, M.R., Diederichs, A., Alinger, J.B., Leung, D.W., Amarasinghe G.K., Basler, C.F. Binding affinities of VP24 proteins from different ebolavirus species for karyopherin alpha proteins influence interferon inhibition and VP24 stability J Virol. 2016. Accepted.

Edwards MR, Liu G, Mire CE, Sureshchandra S, Luthra P, Yen B, Shabman RS, Leung DW, Messaoudi I, Geisbert TW, Amarasinghe GK, Basler CF. Differential Regulation of Interferon Responses by Ebola and Marburg Virus VP35 Proteins. Cell Rep. 2016;14(7):1632-40. doi: 10.1016/j.celrep.2016.01.049. PubMed PMID: 26876165; PMCID: PMC4767585.

Edwards MR, Pietzsch C, Vausselin T, Shaw ML, Bukreyev A, Basler CF. High-
Throughput Minigenome System for Identifying Small-Molecule Inhibitors of Ebola Virus
Replication. ACS Infect Dis. 2015 Aug 14;1(8):380-387. PubMed PMID: 26284260;
PubMed Central PMCID: PMC4537067.

Basler CF. Innate immune evasion by filoviruses. Virology. 2015 May;479-480:122-30.
PubMed PMID: 25843618; PubMed Central PMCID: PMC4424075.

Messaoudi I, Amarasinghe GK, Basler CF. Filovirus pathogenesis and immune evasion:
insights from Ebola virus and Marburg virus. Nat Rev Microbiol. 2015 Nov;13(11):663-
76. PubMed PMID: 26439085.

Leung DW, Borek D, Luthra P, Binning JM, Anantpadma M, Liu G, Harvey IB, Su Z,
Endlich-Frazier A, Pan J, Shabman RS, Chiu W, Davey RA, Otwinowski Z, Basler CF,
Amarasinghe GK. An Intrinsically Disordered Peptide from Ebola Virus VP35 Controls
Viral RNA Synthesis by Modulating Nucleoprotein-RNA Interactions. Cell Rep. 2015 Apr
21;11(3):376-89. PubMed PMID: 25865894; PubMed Central PMCID: PMC4599368.

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