The long-term objectives of our research are to understand the mechanisms of filovirus gene expression. Much of our current understanding of gene expression is extrapolated from findings in related Rhabdovirus and Paramyxovirus systems. Work in these systems has revealed that the mechanism of formation of the 5' mRNA cap structure and 3'poly A tail are unique and suggests that they may represent attractive targets for antiviral intervention. In this proposal, we will build upon a novel in vitro assay that we have recently developed to study mRNA cap methylation. We will extend this system to permit an examination of each step of mRNA synthesis in filoviruses by reconstituting Ebola and Marburg virus transcription in vitro from purified recombinant components.
In aim 1, we will reconstitute mRNA cap addition and mRNA cap methylation from purified recombinant L protein and purified RNA. We will use this system to define the requirements in L and the RNA for cap addition and mRNA cap methylation.
In aim 2, we will reconstitute mRNA synthesis from purified templates and recombinant polymerase. We will use this system to determine the mechanism by which the transcription factor VP30 functions, determine how the polymerase complex assembles, and define the c/s and frans-acting requirements for mRNA synthesis.
In aim 3, we will screen small molecule libraries to identify candidate inhibitors of the polymerase and define their mechanism of action. These experiments will lead to a new mechanistic understanding of filovirus mRNA synthesis and they will reveal the viral requirements for mRNA cap addition and poly A tail formation. These studies will therefore provide detailed information regarding new targets for antiviral drug development as well as identifying candidate small molecule inhibitors of filovirus polymerases. This work will also provide new tools and reagents that will prove useful in our longer-term objective of understanding the structure and function of filovirus polymerases.

Public Health Relevance

Ebola and Marburg viruses are causative agents of devastating hemorrhagic fever. There are no vaccines or antiviral drugs to combat these deadly infectious diseases. The large RNA dependent RNA polymerases of these viruses are thought to possess a set of unique enzymatic activities that are essential for mRNA cap formation. Here we will determine how mRNA capping is accomplished and identify small molecule inhibitors that interfere with this process.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Specialized Center--Cooperative Agreements (U54)
Project #
5U54AI057159-08
Application #
8233441
Study Section
Special Emphasis Panel (ZAI1)
Project Start
2011-03-01
Project End
2014-02-28
Budget Start
2011-03-01
Budget End
2012-02-29
Support Year
8
Fiscal Year
2011
Total Cost
$324,553
Indirect Cost
Name
Harvard University
Department
Type
DUNS #
047006379
City
Boston
State
MA
Country
United States
Zip Code
02115
Nair, Dhanalakshmi R; Chen, Ji; Monteiro, João M et al. (2017) A quinolinol-based small molecule with anti-MRSA activity that targets bacterial membrane and promotes fermentative metabolism. J Antibiot (Tokyo) 70:1009-1019
Huang, Nai-Jia; Pishesha, Novalia; Mukherjee, Jean et al. (2017) Genetically engineered red cells expressing single domain camelid antibodies confer long-term protection against botulinum neurotoxin. Nat Commun 8:423
Mertins, Philipp; Przybylski, Dariusz; Yosef, Nir et al. (2017) An Integrative Framework Reveals Signaling-to-Transcription Events in Toll-like Receptor Signaling. Cell Rep 19:2853-2866
de Wispelaere, Mélissanne; Carocci, Margot; Liang, Yanke et al. (2017) Discovery of host-targeted covalent inhibitors of dengue virus. Antiviral Res 139:171-179
Choo, Min-Kyung; Sano, Yasuyo; Kim, Changhoon et al. (2017) TLR sensing of bacterial spore-associated RNA triggers host immune responses with detrimental effects. J Exp Med 214:1297-1311
Zheng, Huiqing; Colvin, Christopher J; Johnson, Benjamin K et al. (2017) Inhibitors of Mycobacterium tuberculosis DosRST signaling and persistence. Nat Chem Biol 13:218-225
Coulson, Garry B; Johnson, Benjamin K; Zheng, Huiqing et al. (2017) Targeting Mycobacterium tuberculosis Sensitivity to Thiol Stress at Acidic pH Kills the Bacterium and Potentiates Antibiotics. Cell Chem Biol 24:993-1004.e4
Vrentas, Catherine E; Moayeri, Mahtab; Keefer, Andrea B et al. (2016) A Diverse Set of Single-domain Antibodies (VHHs) against the Anthrax Toxin Lethal and Edema Factors Provides a Basis for Construction of a Bispecific Agent That Protects against Anthrax Infection. J Biol Chem 291:21596-21606
Helenius, Iiro Taneli; Nair, Aisha; Bittar, Humberto E Trejo et al. (2016) Focused Screening Identifies Evoxine as a Small Molecule That Counteracts CO2-Induced Immune Suppression. J Biomol Screen 21:363-71
Fink, Avner; Hassan, Musa A; Okan, Nihal A et al. (2016) Early Interactions of Murine Macrophages with Francisella tularensis Map to Mouse Chromosome 19. MBio 7:e02243

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