Interaction of West Nile virus NS5 with orthoflavivirus SLA RNAs and their effects on viral replication and inhibition

West Nile virus (WNV) is a single-stranded, positive-sense RNA virus in the <i>Orthoflavivirus</i> genus, within <i>Flaviviridae</i>. The genus encompasses numerous pathogens of public health importance, including WNV, dengue virus (DENV), Zika virus (ZIKV), and Japanese encephalitis virus (JEV). Orthoflavivirus replication depends on the presence of the stem-loop A (SLA) structure in the 5' untranslated region of the genome. The viral polymerase, NS5, interacts with the SLA and initiates synthesis of the negative-strand RNA. The sequences and secondary structures of SLA and NS5 are highly conserved across orthoflaviviruses, suggesting that the viruses utilize a similar SLA-mediated replication mechanism. Here, we determined the molecular shapes of WNV and JEV SLAs and investigated WNV NS5 interaction with orthoflavivirus SLAs. Although WNV NS5 interacts with DENV, ZIKV, and JEV SLAs in binding assays, only DENV and ZIKV SLAs could replace WNV SLA for viral replication. Next, we found that the top and side loops of SLA are important regions for WNV NS5 interaction. Consequently, when these SLA mutations were introduced into a WNV replicon, genomic replication was greatly reduced. Finally, we tested whether the WNV SLA mimic could inhibit viral replication. The addition of exogenous SLA reduces replication of both WNV replicon and infectious virus, suggesting that exogenous SLA can outcompete the viral SLA for NS5 interaction. Next-generation sequencing data indicate that the presence of exogenous SLA during infection increased the genetic diversity of WNV.IMPORTANCEWest Nile virus (WNV) causes West Nile disease in humans. Approximately 1 in 150 cases develops serious neurological complications, such as meningitis or encephalitis. Currently, no vaccines or antiviral treatments are available. WNV relies on a conserved RNA element in the genome, known as stem-loop A (SLA), to recruit viral polymerase for replication. We found that WNV polymerase can bind the SLAs of other orthoflaviviruses, including dengue virus (DENV), Zika virus (ZIKV), and Japanese encephalitis virus (JEV). However, only the DENV and ZIKV SLAs supported replication when substituted into a WNV replicon. The failure of the JEV SLA to support WNV replication suggests that efficient replication requires additional virus-specific factors beyond the polymerase-SLA interaction. We then tested whether exogenous SLA could act as an RNA decoy to compete with genomic SLA and inhibit viral replication. The addition of SLA RNA in virus-infected cells significantly reduced viral replication and infection, highlighting the therapeutic potential of viral RNA mimic against WNV.