West Nile virus genome cyclization and RNA replication require two pairs of long-distance RNA interactions

West Nile virus (WNV) genome cyclization and replication require two pairs of long-distance RNA interactions. Besides the previously reported 5′CS/3′CSI (conserved sequence) interaction, a 5′UAR/3′UAR (upstream AUG region) interaction also contributes to genome cyclization and replication. WNVs containing mutant 5′UARs capable of forming the 5′/3′ viral RNA interaction were replicative. In contrast, WNV containing a 5′UAR mutation that abolished the 5′/3′ viral RNA interaction was non-replicative; however, the replication defect could be rescued by a single-nucleotide adaptation that restored the 5′/3′ RNA interaction. The 5′UAR/3′UAR interaction is critical for RNA synthesis, but not for viral translation. Antisense oligomers targeting the 5′UAR/3′UAR interaction effectively inhibited WNV replication. Phylogenic analysis showed that the 3′UAR could alternate between pairing with the 5′UAR or with the 3′ end of the flaviviral genome. Therefore, the 5′UAR/3′UAR pairing may release the 3′ end of viral genome (as a template) during the initiation of minus-strand RNA synthesis.