Long non‐coding subgenomic flavivirus RNAs have extended 3D structures and are flexible in solution

Most mosquito‐borne flaviviruses, including Zika virus (ZIKV), Dengue virus (DENV), and West Nile virus (WNV), produce long non‐coding subgenomic RNAs (sfRNAs) in infected cells that link to pathogenicity and immune evasion. Until now, the structural characterization of these lncRNAs remains limited. Here, we studied the 3D structures of individual and combined subdomains of sfRNAs, and visualized the accessible 3D conformational spaces of complete sfRNAs from DENV2, ZIKV, and WNV by small angle X‐ray scattering (SAXS) and computational modeling. The individual xrRNA1s and xrRNA2s adopt similar structures in solution as the crystal structure of ZIKV xrRNA1, and all xrRNA1‐2s form compact structures with reduced flexibility. While the DB12 of DENV2 is extended, the DB12s of ZIKV and WNV are compact due to the formation of intertwined double pseudoknots. All 3′ stem‐loops (3′SLs) share similar rod‐like structures. Complete sfRNAs are extended and sample a large conformational space in solution. Our work not only provides structural insight into the function of flavivirus sfRNAs, but also highlights strategies of visualizing other lncRNAs in solution by SAXS and computational methods. Synopsis Mosquito‐borne flaviviruses produce long non‐coding subgenomic RNAs that link to pathogenicity and immune evasion. This study presents 3D structures of sfRNAs from Zika, Dengue and West Nile virus in solution and provides insight into their functions. The duplicated dumbbell structures (DB12s) in DENV2 are extended, the DB12s of ZIKV and WNV are compact. The DB12s of ZIKV and WNV form intertwined double pseudoknots. The 3′ stem‐loop structures adopt conserved rod‐like, extended conformations and do not form pseudoknots. Complete sfRNAs are extended modular molecules with high flexibility in solution. Graphical Abstract Mosquito‐borne flaviviruses produce long non‐coding subgenomic RNAs that link to pathogenicity and immune evasion. This study presents 3D structures of sfRNAs from Zika, Dengue and West Nile virus in solution and provides insight into their functions.