Evolutionary refinement of the ZIKV 5′ UTR dictates a trade-off between RNA stability and promoter accessibility

Abstract Zika virus (ZIKV) has evolved from a sporadically circulating pathogen into a global health threat associated with severe neurological complications. While research has been focused on amino acid substitutions, the impact of non-coding RNA evolution on ZIKV fitness remains largely unexplored. Here, we characterize the structural and functional evolution of stem-loop A (SLA), the essential 5′ terminal promoter for the viral polymerase. We identified four distinct evolutionary variants – ancestral (Anc), intermediate (Int), contemporary (Con) and alternative (Alt) that exhibit a progressive shift in thermodynamic stability. Notably, SLACon – dominant in recent outbreaks- displays increased binding affinity for the NS5 polymerase, whereas the divergent SLAAlt variant markedly increases 5′ exoribonuclease resistance at the cost of replicative efficiency. Although the SLAAlt variant proved lethal in a contemporary ZIKV backbone, 5′ rapid amplification of cDNA ends (RACE) and phylogenetic analysis confirmed its viability in nature. These findings reveal a previously unrecognized plasticity in the ZIKV promoter and demonstrate that non-coding architecture undergoes adaptive refinement to balance genome stability with kinetic accessibility. Ultimately, this work illustrates how the structural evolution of RNA serves as a key determinant of viral fitness and the emergence of isolate-specific replication strategies.