The influence of natural adaptation of the DCS-PK structure on the replication efficiency of Zika virus

Multiple studies have reported that different clades or strains of Zika virus (ZIKV) replicated with varied efficiency in cell culture. However, the molecular basis of these differences remains unclear. In this study, it was first shown that the downstream of the 5'-cyclization sequence pseudoknot (DCS-PK) is required for efficient viral RNA (vRNA) replication of ZIKV, similar to its function in dengue virus life-cycle by using a newly developed ZIKV replicon. Interestingly, a combination of phylogenetic analysis, RNA chemical probing, and structure prediction revealed that substitutions were accumulated in the DCS-PK sequence and its structural integrity was improved during the evolutionary history of ZIKV. By using replicons of representative strains, it was demonstrated that the more stable DCS-PK from the Asian ZIKV strains was more potent in promoting vRNA replication in comparison with the less stable version found only in the early-diverged African strains. The significance of this difference was further supported by the characterization of virus mutants generated by reverse genetics approaches. MR766 mutants harboring a stabilized DCS-PK exhibited a moderate advantage over the corresponding wild type controls in replication fitness, which was detected by direct competition and reporter virus assays. Moreover, introducing a metastable DCS-PK into the Asian FSS13025 strain attenuated viral replication in multiple in vitro models, including human neural stem cells. In summary, our work pointed to a structural adaptation of DCS-PK to enhance ZIKV replication fitness, highlighting an important role of cis-acting RNA elements in natural selection and virus evolution.