Structural dynamics of the dengue virus non-structural 5 (NS5) interactions with promoter stem-loop A (SLA).

The dengue virus (DENV) NS5 protein, essential for viral RNA synthesis, is an attractive antiviral drug target. DENV NS5 interacts with the stem-loop A (SLA) promoter at the 5'-untranslated region of the viral genome to initiate negative-strand synthesis. However, the conformational dynamics of this interaction remains unclear. Our study explores the structural dynamics of DENV serotype 2 NS5 (DENV2 NS5) in complex with SLA, employing surface plasmon resonance (SPR), hydrogen-deuterium exchange mass spectrometry (HDX-MS), computational modeling, and cryoEM. Our findings reveal that DENV2 NS5 binds SLA in a closed conformation, with interdomain cooperation between its methyltransferase (MTase) and RNA-dependent RNA polymerase (RdRp) domains, critical for the interaction. SLA binding induces conformational changes in both domains, highlighting NS5's multifunctional role in viral replication. Our cryoEM results visualizes the DENV2 NS5-SLA complex, confirming a conserved SLA binding across DENV serotypes and provides key insights for antiviral strategies targeting NS5's conformational states.