Therapeutic Intervention of Crimean Congo Hemorrhagic Fever: A Tick-Borne Viral Illness

Crimean Congo hemorrhagic fever (CCHF) is a tick-borne, highly contagious, viral illness with high mortality rates (40%) in humans. The illness is geographically widespread across Africa, Eastern Europe, Asia, and the Middle East. There is no treatment for this viral disease at present. CCHF virus spreads to humans either by tick bites or through contact with viraemic animal tissues. This grant application is focused to carry out a high throughput screen for the identification of chemical inhibitors for CCHFV. We recently reported that CCHF virus nucleocapsid protein (CCHFV N protein) has two distinct RNA binding sites in the stalk and head domains. The hallmark of the Nairovirus genus is the formation of a panhandle structure by the base pairing of highly conserved and complementary nucleotides at the 5' and 3' terminus of the viral genomic RNA. We demonstrated that vRNA panhandle is specifically recognized by the RNA binding site located in the stalk domain of the N protein. The N protein-panhandle interaction is required for the replication of vRNA genome during the course of infection. We also showed that N protein-panhandle interaction is Nairovirus genus specific. Interestingly, the 5' untranslated region (5'UTR) of the CCHFV mRNA also folds into a panhandle-like secondary structure, which is also recognized by the RNA binding site located in the stalk domain of N protein. The interaction between N protein and the panhandle-like structure of the viral mRNA 5' UTR selectively facilitates the translation of viral mRNA to boost the synthesis of viral proteins in the host cell. The rapid viral protein synthesis likely builds up the viral load in infected patients shortly after infection. We hypothesize that chemical interruption in N protein-panhandle interaction will inhibit virus replication by selectively interfering in the vRNA synthesis and the preferential translation of viral mRNA by the N protein. We have developed a highly sensitive, cost effective ribozyme-based fluorescence assay that monitors the N protein panhandle interaction in vitro, by generating quantitative fluorescence readout. The assay will be used to screen a chemical library for the identification of chemical inhibitors that block N protein panhandle interaction and shut down virus replication in the host cell. A secondary fluorescence polarization anisotropy based in vitro assay and in vivo GFP reporter assays are in place to exclude the false positives from the primary screen. The positive compounds will be clustered into structurally similar groups, followed by antiviral testing of each compound cluster in cell culture model. These studies will reveal chemical scaffolds that inhibit N protein-panhandle interaction and shutdown virus replication in cells. The scaffold structures will provide critical insights for further modification to synthesize inhibitors that have high target binding affinity, reduced or no cytotoxicity, and significantly improved potency. Based on our past experience in antiviral drug development and our long-term collaboration with the University of Kansas drug development team, we are confident to identify the inhibitors for CCHFV. This application, focused on treatment and prevention areas of this RFA, has direct impact on public health and the healthcare needs of military Service members, employed at geographical regions with reported cases of CCHFV infection. Less