TY  - JOUR
TI  - Synchronized Visualization and Analysis of Intracellular Trafficking and Maturation of Orthoflavivirus Subviral Particles
AU  - Ishida, Kotaro 
AU  - Morita, Eiji 
VL  - 15
IS  - 10
PY  - 2025
DA  - 2025/05/20
SP  - e5324
C1  - Bio-protocol 2025;15:e5324
DO  - 10.21769/BioProtoc.5324
UR  - https://doi.org/10.21769/BioProtoc.5324
AB  - Orthoflavivirus 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 Orthoflavivirus 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 hook 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 Orthoflavivirus particles within cells.
KW  - Retention using selective hooks (RUSH)
KW  - Split GFP
KW  - Live imaging
KW  - Orthoflavivirus
KW  - Japanese encephalitis virus
KW  - SVP
JF  - Bio-protocol
SN  - 2331-8325
PB  - Bio-protocol LLC.
BIO101  - False