Characterization and targeting rift valley fever virus N protein-RNA interactions

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Rift Valley Fever is a clinically and agriculturally important illness caused by Rift Valley Fever Virus (RVFV), a member of the Bunyaviridae endemic primarily in sub-Saharan Africa. Because of the severe illness it causes in humans and because of recent instances of natural spread outside of Africa, it has been identified as emerging disease agent and/or a potential agent of bioterrorism. The symptoms of RVF in humans can include severe fever, malaise, neurological damage, liver damage, hemorrhagic events, and death. Currently there are no clinically proven treatments for RVFV infection. Development of antiviral strategies requires understanding of the viral replication cycle so that specific steps can be targeted. In the research proposed here, we will characterize an essential molecular interaction in the RVFV replication cycle wherein nucleocapsid protein must recognize and bind to viral RNA sequences. To achieve this goal, we will perform truncation analysis on viral RNA transcripts to determine the minimum requirements for efficient RNA-protein binding. We will also perform a combinatorial in vitro selection scheme (SELEX) to obtain a high affinity RNA aptamer(s) to nucleocapsid protein. Sequence analysis of the aptamers will shed light on nucleocapsid target recognition and will subsequently be used to construct a tool to screen drugs that inhibit the nucleocapsid-RNA interaction. Specifically, the aptamer will be ligated to an RNA module that emits a fluorescent signal when the aptamer is displaced from its nucleocapsid binding site. The drug screening tool will then be used to screen compounds at NSRB. Molecules giving promising results in the aptamer displacement assay will be tested against RVFV replication in cell culture. Owing to its central role in replication, nucleocapsid protein has been recognized as a good potential antiviral target. A novel class of drug targeted against nucleocapsid protein has recently been shown to be effective in inhibiting replication of respiratory syncytial virus, also a negative-sense RNA virus, strongly supporting the potential for developing nucleocapsid as a viable drug target in RVFV (Chapman et al, 2007). This research project fits within the RMRCE Integrated Research Focus on Viral Therapeutics, and will interact directly with PI Brian Gowen (RP 3.2) and the NSRB drug screening core.