| Many flaviviruses are globally important human pathogens that cause significant morbidity and mortality; therefore, it is necessary to develop efficacious antiviral therapeutics to combat flavivirus infections. Here I report the inhibition of flavivirus infections by antisense phosphorodiamidate morpholino oligomers (PMOs) whose sequences are complementary to RNA elements located in the 5' and 3'-termini of the West Nile (WN) virus genome. A novel arginine-rich P007 peptide was conjugated to the 5'end of each PMO (resulting in PPMO) to ensure efficient cellular delivery. The PPMOs exhibited various degrees of antiviral activity upon incubation with a WN virus luciferase reporting replicon. Two PPMOs, targeting the 5' terminal 20 nucleotides (5'End PPMO) or targeting the 3' terminal element involved in genome cyclization (3'CSI) exhibited the greatest potency. The 5'End and 3'CSI PPMOs were then tested for their ability to inhibit the replication of an epidemic strain of West Nile virus using a virus-reduction assay. When cells infected with WN virus were treated with a 5 muM concentration of either the 5'End or the 3'CSI PPMO, virus titers were reduced by approximately 5 to 6 logs without cytotoxicity. Viral-specific 5'End PPMOs targeting the first 20 nucleotides of the genomes of St. Louis encephalitis or Yellow Fever viruses also greatly reduced virus titers. Additionally, a MFLAV (multi-flavivirus) PPMO was designed to target the 3'CSI element that is conserved among mosquito-borne flaviviruses also showed antiflavivirus activity; the antiviral activity correlated with the conservation of the targeted 3'CSI sequences of the specific viruses.;WN viruses resistant to the 5'End or 3'CSI PPMOs were selected in vitro and characterized. Sequence analysis of the 5'End- and 3'CSI PPMO-resistant viruses identified escape mutations located within and outside of the targeted region, respectively. Site-directed mutagenesis of a WILT virus infectious clone demonstrated that the mismatches within the PPMO-targeted region were responsible for conferring resistance to the 5'End PPMO. In contrast, a U insertion or G deletion located within the 3'-terminal stem-loop of the viral genome was the determinant of resistance to the 3'CSI PPMO. The results demonstrate a feasibility to develop a PPMO-mediated therapy for treatment of flavivirus infections. |
