Synchronized Visualization and Analysis of Intracellular Trafficking and Maturation of <em>Orthoflavivirus</em> Subviral Particles

<i>Orthoflavivirus</i> is an enveloped, positive-stranded RNA virus that buds into the endoplasmic reticulum (ER) lumen. The budded virus particles are subsequently transported to the Golgi apparatus and secreted into the extracellular environment via the conventional secretion pathway. In this protocol, we describe a method for monitoring the secretion of <i>Orthoflavivirus</i> particles from the ER. To visualize intracellular membrane trafficking, we combine two distinct imaging techniques: the retention using selective hooks (RUSH) system and the split green fluorescent protein (GFP) system. In this approach, GFP11, a peptide tag fused to prME, the outer coat structural protein of Japanese encephalitis virus particles, was co-expressed in HeLa cells along with two additional components: GFP1-10 fused to a streptavidin-binding peptide and a <i>hook</i> construct consisting of streptavidin fused to the ER retention sequence KDEL. Time-lapse imaging was performed after the addition of biotin, which releases the captured GFP-labeled subviral particles from the ER. This method enables synchronized visualization of intracellular subviral particle trafficking and serves as a valuable tool for analyzing the maturation process of <i>Orthoflavivirus</i> particles within cells. Key features • Synchronized intracellular movement of <i>Orthoflavivirus</i> particles is visualized by the retention using selective hooks (RUSH) system. • Split GFP system is used to label viral particles. • This protocol has broader applications in investigating the transport of secretory proteins, especially those that are challenging to tag with full-length fluorescent proteins.