RNA elements within the flavivirus genome may play essential regulatory roles during virus replication. Here, recombinant West Nile virus (WNV) NS5 protein was used in combination with WNV subgenomic RNA templates to establish RNA-dependent RNA polymerase and RNA-binding assays. These assays identified mutations in the stem–loop A (SLA) region of the 5′ untranslated region (5′UTR) altering NS5 RNA synthesis and RNA-binding capability. These mutations were then introduced into the full-length WNV genome by reverse genetics. Further analysis of the mutant viruses showed that deletion of nt 46–60, which disrupted the stem and side loop of SLA, greatly compromised virus replication, whereas mutations that destroyed the top loop of SLA required for RNA synthesis did not significantly alter virus replication. These results suggest that SLA present in the 5′UTR of WNV is essential for RNA synthesis and for virus replication.


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