A conserved arginine in NS5 binds genomic 3′ stem–loop RNA for primer-independent initiation of flavivirus RNA replication

The commitment to replicate the RNA genome of flaviviruses without a primer involves RNA–protein interactions that have been shown to include the recognition of the stem–loop A (SLA) in the 5′ untranslated region (UTR) by the nonstructural protein NS5. We show that DENV2 NS5 arginine 888, located within the carboxy-terminal 18 residues, is completely conserved in all flaviviruses and interacts specifically with the top-loop of 3′SL in the 3′UTR which contains the pentanucleotide 5′-CACAG-3′ previously shown to be critical for flavivirus RNA replication. We present virological and biochemical data showing the importance of this Arg 888 in virus viability and de novo initiation of RNA polymerase activity in vitro. Based on our binding studies, we hypothesize that ternary complex formation of NS5 with 3′SL, followed by dimerization, leads to the formation of the de novo initiation complex that could be regulated by the reversible zipping and unzipping of cis-acting RNA elements.