Genetic analysis of West Nile virus containing a complete 3′CSI RNA deletion

We report a genetic interplay among three pairs of long-distance RNA interactions that are involved in West Nile virus (WNV) genome cyclization and replication: 5′CS/3′CSI (conserved sequence), 5′UAR/3′UAR (upstream AUG region), and 5′DAR/3′DAR (downstream AUG region). Deletion of the complete 3′CSI element is lethal for WNV replication, but the replication of the 3′CSI deletion virus could be rescued by second site mutations. Functional analysis, using a genome-length RNA and replicon, mapped the compensatory mutations to the 5′UAR/3′UAR and 5′DAR/3′DAR regions. Biochemical analysis showed that the 3′CSI deletion abolished the 5′ and 3′ RNA interaction of the genome; the compensatory mutations could partially restore the 5′ and 3′ genome cyclization. These results demonstrate, for the first time, that a flavivirus without 3′CSI could restore genome cyclization and viral replication through enhancement of the 5′UAR/3′UAR and 5′DAR/3′DAR interactions.