BPS2026 – Investigating the role of RRBP1 in dengue viral RNA localization and protein translation

Dengue is a mosquito-borne viral infection that annually affects hundreds of millions of people. Each dengue virion contains one copy of a single-stranded, positive-sense genomic RNA, which upon entry into the host cell is localized to the endoplasmic reticulum and translated by the ribosome into a polyprotein. The polyprotein contains approximately twenty transmembrane domains and is processed into ten viral proteins, four of which reside at the ER. RRBP1 (ribosome binding protein 1) was identified as an intrinsically disordered human ER protein that binds to the dengue viral RNA; it was also found to be important for viral protein translation and RNA stability. However, the structure of RRBP1 and the details of its interactions with the viral RNA and the 80S ribosome are still unknown. To perform in vitro binding assays and structural characterization, we expressed the cytoplasmic domains (165 kDa) of RRBP1 in E. coli and purified the protein in vitro. We characterized the recombinant protein through various biophysical methods, including dynamic light scattering and nano differential scanning fluorimetry. We also validated formation of a complex with the 40S and 60S ribosomal subunits and the dengue RNA. We are currently investigating interactions of RRBP1 with 80S ribosome and viral RNA using single-particle cryoEM. This structural information will provide deeper understanding of how RRBP1 might recruit the viral RNA and the 80S ribosome to the ER, and how it might help the viral RNA hijack protein synthesis and processing pathways at the ER during infection.