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bioRxiv · 10.1101/2025.05.08.652984

Endophilin mediated endocytosis and Epidermal growth factor receptor govern Japanese encephalitis virus entry and infection in neuronal cells

Sharma, P.·Tanwar, M.·Choudhary, E.·Sundaram, N. M.·Ojha, D.·Mishra, L.·Nain, M.·Rode, A.·Prasad, V. M.·Vrati, S.·Kalia, M.
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Abstract

Several trafficking pathways are operational at the plasma membrane, and both clathrin-dependent, and clathrin-independent endocytosis (CIE), can serve as virus entry portals. Our research has shown that the neurotropic flavivirus: Japanese encephalitis virus (JEV), infects neuronal cells via CIE. Here we have identified and characterized two essential host-factors for JEV trafficking in neuronal cells: Endophilin & Epidermal Growth Factor Receptor (EGFR). Through quantitative estimation of viral RNA copy number, we demonstrate that JEV entry in neuronal cells, was blocked by knock-down of Endophilin A isoforms, while activation of Endophilin-mediated endocytosis using a specific inhibitor of GSK-3{beta} enhanced virus entry. Deletion mutants of Endophilin showed an essential role of SH3, BAR, H0 domains for virus entry. High resolution fluorescence imaging of virions showed overlap with Endophilin A2 puncta. Virus entry led to rearrangements of the actin cytoskeleton, and was highly sensitive to any pharmacological actin perturbation. Virus endocytosis activated EGFR, and the specific kinase inhibitors Erlotinib, and Gefitinib, reduced virus entry and replication in cultured cells and mouse primary cortical neurons. Silencing of EGFR, competitive inhibition with receptor ligand EGF, and EGFR specific antibodies significantly impaired JEV binding and entry, indicating the crucial role of its ligand binding domain for virus attachment/receptor interaction. EGFR colocalized with virions at early time-points of infection, and the ED3 domain of the JEV-envelope protein showed specific interaction with EGFR through BLI. Our study provides evidence for JEV entry in neuronal cells through an endocytic pathway involving Endophilin and EGFR.

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Sharma, P., Tanwar, M., Choudhary, E., Sundaram, N. M., Ojha, D., Mishra, L., Nain, M., Rode, A., Prasad, V. M., Vrati, S., Kalia, M.. 2025-05-09. Endophilin mediated endocytosis and Epidermal growth factor receptor govern Japanese encephalitis virus entry and infection in neuronal cells. https://doi.org/10.1101/2025.05.08.652984

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