| Abstract: |
Flavivirus are vector-borne single-strand RNA viruses associated with several diseases worldwide. The ~11 kb genome is flanked by 5’ and 3’ terminal regions (TR), which are intimately tied to human RNA-binding proteins and proteins several processes in the viral life cycle. Flavivirus 3’ TR harbours duplicated stem-loops and dumbbells along with a short-hairpin/3’stem-loop (sh/3’SL). In a pull-down assay, we pinpointed an interaction between Nucleolin and Zika virus (ZIKV) 3’TR. Nucleolin is a human multifunctional protein involved in Epstein-Barr virus immune system evasion, transcription silencing of HIV-1 and translational support of Foot-and-Mouth disease virus. To investigate if nucleolin can interact with interact with ZIKV 3’ TR directly, we expressed a domain containing four RNA-recognition motifs and in vitro transcribed ZIKV 3’ TR segments, followed by their purification using size-exclusion chromatography (SEC). Subsequently, we verified a tight nanomolar interaction with the sh/3’SL than a micromolar with a ZIKV dumbbell using microscale thermophoresis. Since sh/3’SL is the most conserved sequence in Flavivirus 3’TR, we asked if the 3’ TRs of Japanese encephalitis (JEV) and Powassan virus (POW) could also be recognized by nucleolin. Microscale thermophoresis studies demonstrated that both viral 3’ TRs bind with nanomolar affinity with nucleolin. Our following goals are to biophysically characterize RNA-protein complex using AUC and SEC-MALLS, as well as study the functional relevance of nucleolin – 3’ TR interactions using viral replicon systems and determine the high-resolution structures of the protein-RNA complexes. |
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