RAB4B and Japanese encephalitis virus E protein interaction is essential for viral entry in early endosomes.

RAB4B (Ras-Related GTP-Binding Protein 4b) is essential for intracellular trafficking and endosomal recycling processes. Our previous study, we demonstrated that RAB4B promotes Japanese encephalitis virus (JEV) replication in PK15 cells. However, the exact mechanisms underlying the role of RAB4B in JEV internalization remain unclear. Here, a genome-wide CRISPR/Cas9 library screen was performed, which identified RAB4B, along with other significant hits like ST8SIA4 and ELAVL1, as essential mediators of JEV replication. In vitro validation using RAB4B knockout in U251 and BV2 cells showed a significant reduction in JEV genome copies and viral titers, which were restored upon reintroducing RAB4B, confirming its pivotal role in viral propagation. Further mechanistic investigation revealed that RAB4B is required for JEV internalization into early endosomes. Co-immunoprecipitation and in vitro binding assays demonstrated a direct interaction between RAB4B and the JEV E protein, highlighting the functional importance of this interaction. In vivo experiments with RAB4B knockout mice showed a reduction in viral load in the brain and improved survival rates compared to wild-type mice. Taken together, these findings provide compelling evidence that RAB4B is indispensable for JEV entry and replication.