Co-selection of West Nile virus nucleotides that confer resistance to an antisense oligomer while maintaining long-distance RNA/RNA base pairings

West Nile virus (WNV) genome cyclization is mediated by two pairs of long-distance RNA/RNA interactions: the 5′CS/3′CSI (conserved sequence) and the 5′UAR/3′UAR (upstream AUG region) base pairings. Antisense peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs), designed to interfere with the 5′CS/3′CSI or 5′UAR/3′UAR base pairings, were previously shown to inhibit WNV. In this study, we selected and characterized WNVs resistant to a PPMO targeting the 3′UAR (3′UAR-PPMO). All resistant viruses accumulated one-nucleotide mutations within the 3′UAR, leading to a single-nucleotide mismatch or a weakened base-pairing interaction with the 3′UAR-PPMO. Remarkably, a one-nucleotide mutation within the 5′UAR was correspondingly co-selected; the 5′UAR mutation restored the base pairing with the 3′UAR mutation. Mutagenesis of WNV demonstrated that the single-nucleotide change within the 3′UAR-PPMO-target site conferred the resistance. RNA binding analysis indicated that the single-nucleotide change reduced the ability of 3′UAR-PPMO to block the RNA/RNA interaction required for genome cyclization. The results suggest a mechanism by which WNV develops resistance to 3′UAR-PPMO, through co-selection of the 5′UAR and 3′UAR, to create a mismatch or a weakened base-pairing interaction with the PPMO, while maintaining the 5′UAR/3′UAR base pairings.