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 #
5U54AI057159-10
Application #
8441643
Study Section
Special Emphasis Panel (ZAI1-DDS-M)
Project Start
Project End
Budget Start
2013-03-01
Budget End
2014-02-28
Support Year
10
Fiscal Year
2013
Total Cost
$314,279
Indirect Cost
$97,574
Name
Harvard University
Department
Type
DUNS #
047006379
City
Boston
State
MA
Country
United States
Zip Code
02115
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