The RNA promoter for pathogenic orthoflaviviruses replication is universal and serves as target for viral inhibition.

Orthoflaviviruses comprise a diverse genus of positive-strand RNA viruses that includes major human pathogens such as dengue, yellow fever, Zika, Japanese encephalitis, tick-borne encephalitis, and West Nile viruses. Despite their global impact, the molecular constraints that preserve viral replication across distinct vectors and host environments remain incompletely understood. Viral RNA replication depends on Stem-Loop A (SLA), a structured RNA element located at the 5' end of the genome that recruits the viral polymerase NS5. Here, we examine the structural diversity of this RNA promoter across orthoflaviviruses. Using infectious clones, reporter viruses, and computational structural analyses, we show that SLAs from diverse mosquito- and tick-borne viruses are functionally interchangeable in the context of dengue and Zika virus infectious clones. Structure-function analyses reveal that conserved nucleotide contacts between the SLA top loop and the NS5 polymerase domain form a conserved interaction interface maintained across orthoflaviviruses, including insect-specific viruses. In contrast, other SLA sub-elements, such as the three-way junction and side stem, have diverged in a group-specific manner, yet co-evolution in mosquito and tick-borne viruses preserves the three-dimensional architecture and NS5 binding competence for viral replication. Guided by this conservation, we identify small molecules that bind SLA and inhibit replication across multiple pathogenic orthoflaviviruses. These findings uncover fundamental principles governing viral RNA promoter evolution and establish conserved RNA structures as promising targets for viral control.