Japanese Encephalitis Virus (JEV) belongs to the viral family Flaviviridae, transmitted through competent mosquito vectors. JEV is responsible for ∼68,000 clinical cases each year; 25% result in death, while 20-30% of survivors have resultant neurological sequelae. This family includes many other deadly viruses such as Zika, Dengue, and West Nile. Studies have shown, and others theorized that flavivirusesundergo long-range 5' - 3' terminal region (TR) interactions mediated by a complementary cyclization sequence. This cyclization has been shown to be necessary for viral replication, and therefore a potential target for therapeutics. Since multiple flaviviruses undergo this cyclization, we wanted to gain structural insights into the duplex structure created by this RNA-RNA interaction. Therefore, we started by performing a bioinformatics approach to identify a conserved duplex structure shared by over 109 JEV isolates. We chose a specific isolate through this analysis, which resulted in the highest number of theoretic base-pair interactions. We then conducted an extensive biophysical analysis in vitro to verify the interaction using multiple complementary techniques. After validating in vitro RNA-RNA binding, we sought to gain structural information about the RNA-RNA complex by performing small-angle X-ray scattering (SAXS) to obtain a low-resolution structure. Furthermore, we performed cryogenic electron microscopy (Cryo-EM) to gain a high-resolution image of the same complex. Our research shows the first low-resolution image of JEV terminal regions interacting in vitro, along with preliminary data of Cyro-EM images which could lead to a high-resolution image. This research provides a pipeline of theoretical structure prediction, along with validation using structural biophysical techniques. We believe that using JEV as a model system and the conserved nature of flaviviral cyclization; this pipeline will be applied to other flaviviruses to gain a family-wide picture of TR cyclization.

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