The 5′ and 3′ downstream AUG region elements are required for mosquito-borne flavivirus RNA replication

Flaviviruses require complementarity between the 5′ and 3′ ends of the genome for RNA replication. For mosquito-borne flaviviruses, the cyclization sequences (CS) and upstream of AUG region (UAR) elements at the genomic termini are necessary for viral RNA replication, and a third motif, the downstream of AUG region (DAR), was recently designated for dengue virus. The 3′ DAR sequence is also part of a hairpin (HP-3′SL), and both complementarity between 5′ and 3′ DAR motifs and formation of the HP-3′SL in the absence of the 5′ end are conserved among mosquito-borne flaviviruses. Using West Nile virus as a model, we demonstrate that 5′-3′ DAR complementarity and HP-3′SL formation are essential for viral RNA replication.