Insights into the coordinated interplay of the sHP hairpin and its co-existing and mutually-exclusive dengue virus terminal RNA elements for viral replication.

Terminal RNA elements of the dengue virus (DENV) genome are necessary for balanced stability of linear and circular conformations during replication. We examined the small hairpin (sHP) and co-existing and mutually-exclusive terminal RNA elements by mutagenesis analysis, compensatory mutation screening, and by probing with RNA fragments to explore localized RNA folding and long-range RNA interactions. We found that the first base pair of the sHP and the stability of SLB and the 3'SL bottom stem affected circularization; sHPgc /C10631G+G10644C prohibited circularization, sHPuG accelerated and stabilized 5'-to-3' RNA hybridization, while C94A and A97G and C10649 mutations loosened SLB and 3'SL, respectively, for circularization. sHPuG +C10649G induced circularization and impeded replication, whereas point mutations that loosened the UAR or DAR ds region, strengthened the sHP, or reinforced the 3'SL bottom stem, rescued the replication deficiency. Overall, we reveal structural and sequence features and interplay of DENV genome terminal RNA elements essential to viral replication.