The majority of the nucleotides in the top loop of the genomic 3′ terminal stem loop structure are cis-acting in a West Nile virus infectious clone

The flavivirus genome RNA terminates with a conserved 3′ stem loop (SL) structure that was shown to be essential for virus replication. A stretch of conserved nts is located in the top loop (TL) of this structure. Mutation of the TL nts (5′ ACAGUGC 3′) in a WNV infectious clone indicated that 3 of the 7 TL nts (5′ A CA GUG C 3′) are critical for virus replication. Mutation of 3 of the other nts reduced the efficiency of virus replication. The four 5′ TL nts are conserved in both mosquito- and tick-borne flavivirus genomes, while the TL 3′ C is conserved in mosquito-borne viruses. The conservation of two or three G–C base pairs in the TL flanking sequences suggests that a stable stem is necessary for precise presentation of the TL sequence. The TL may participate in RNA as well as protein interactions.