Genome cyclization is essential for flavivirus replication. We used RNases to probe the structures formed by the 5′-terminal 190 nucleotides and the 3′-terminal 111 nucleotides of the West Nile virus (WNV) genomic RNA. When analyzed individually, the two RNAs adopt stem-loop structures as predicted by the thermodynamic-folding program. However, when mixed together, the two RNAs form a duplex that is mediated through base-pairings of two sets of RNA elements (5′CS/3′CSI and 5′UAR/3′UAR). Formation of the RNA duplex facilitates a conformational change that leaves the 3′-terminal nucleotides of the genome (position - 8 to - 16) to be single-stranded. Viral NS5 binds specifically to the 5′-terminal stem-loop (SL1) of the genomic RNA. The 5′SL1 RNA structure is essential for WNV replication. The study has provided further evidence to suggest that flavivirus genome cyclization and NS5/5′SL1 RNA interaction facilitate NS5 binding to the 3′ end of the genome for the initiation of viral minus-strand RNA synthesis.