Abstract
Many flaviviruses such as Dengue and West Nile virus undergo essential long-range interactions of their 5’ and 3’ terminal regions (TRs), mediated by a conserved complementary cyclization sequence. However, we lack insights into such long-range interactions for the Japanese Encephalitis virus (JEV). Here, we utilized an extensive, multi-faceted approach involving computational and biophysical tools. We performed multi-angle light scattering (SEC-MALS) to determine absolute molecular weights of JEV TRs, and their complex concluding they form a 1:1 complex and corroborated this interaction using analytical ultracentrifugation (AUC). The microscale thermophoresis (MST) experiments demonstrated that the 5’ and 3’ TR of JEV interact with nM affinity, which is significantly reduced without the conserved cyclization sequence. To our knowledge, this is the first study representing the application of three key biophysical methods (AUC, MST and SEC-MALS) to study RNA-RNA interactions. Furthermore, we performed computational kinetic analyses corroborating our MST studies showing the essential role of the cyclization sequence in the RNA-RNA interaction. The binding affinity of this biologically critical event is a vital pharmacological feature that can influence potential competitive inhibition by therapeutics. This evidence can also influence pharmaceutical interventions aimed at inhibiting the conserved flavivirus cyclization, thus, interrupting replication across the flavivirus family.