Family Flaviviridae is composed of pathogenic viruses such as Dengue, West Nile, Zika, and Japanese encephalitis virus (JEV). Flaviviruses contain a single-stranded, positive-sense RNA genome that undergoes long-distance 5'-3' interaction of their terminal regions (TRs) resulting in genomic cyclization. This cyclization is hypothesized to be conserved across the various members of this family and is necessary for viral replication. However, the exact biochemical and biophysical basis underlying the 5'-3' TRs interaction has yet to be elucidated. Therefore, we have selected JEV, which is responsible for ∼18,000 deaths and ∼30,000 cases of permanent neurological damage each year as a basis to investigate 5'-3' TR cyclization. First, we performed bioinformatics analysis of a variety of JEV strains to identify the 5' and 3' TRs that could base pair with high affinity. We prepared multiple TRs and validated their homogeneity using biophysical techniques. Next, we performed interaction studies using microscale thermophoresis (MST) which established that JEV TRs interact in solution. This interaction was further investigated using various segments of each terminal region. Furthermore, we performed multi-angle laser light scattering (MALLS) on the JEV 5'-3' TR complex and demonstrated that they interact with each other. Following the identification of this complex, we aim to obtain structural information on these TRs in isolation, and in the complex through small-angle X-ray scattering (SAXS) and cryo-electron microscopy. Ultimately, our multidisciplinary work will provide a detailed understanding of JEV genomic cyclization, that could aid the designing of targeted therapeutics to treat viral infection. As climate change continues to increase global temperatures, mosquito-borne flaviviral infection will likely increase as a global health risk, and the need for effective antiviral treatments will only become more apparent.

Top