ICAM-1-mediated Src signaling pathway plays a pivotal role in encephalomyocarditis virus entry

Encephalomyocarditis virus (EMCV), a potential zoonotic pathogen with a broad host range, has been reported to enter cells via caveolin-mediated endocytosis (CavME), a process initiated by caveolae detachment from the plasma membrane. However, the molecular mechanism underlying EMCV's cellular entry remains incompletely characterized. Here, we identified for the first time that the overexpression of Src kinase and Fyn kinase individually enhanced the replication and proliferation of EMCV. The treatment of specific small interfering RNA or inhibitors resulted in the downregulation of EMCV internalization, indicating the involvement of Src, Fyn, and Ezrin in the viral entry process. By treating different cell lines (BHK-21, hCMEC/D3, and PK15 cells) from various species with inhibitors of Src or Ezrin, as well as using siCav-1, and subsequently performing EMCV invasion experiments, we demonstrated the sequential activation of Src, Ezrin, and Cav-1 during EMCV internalization, indicating that this signaling pathway is widely present during EMCV infection of human-, hamster-, and pig-derived cells. Interestingly, the candidate receptor protein ICAM-1 interacts with Src and regulates its phosphorylation. Compared to the wild-type cells, lower levels of the phosphorylation of Src, Ezrin, and Cav-1 induced by EMCV were observed in ICAM-1 knockout BHK-21 cells. These findings indicate that EMCV entry into BHK-21 cells relies on the activation of the ICAM-1-Src-Ezrin-Cav-1 pathway. Our study first elucidates the molecular mechanism of EMCV invading into BHK-21 cells, providing potential targets for limiting EMCV infection and further refining the regulatory mechanism of the Src pathway on CavME.IMPORTANCEEMCV has a wide host range and is a potential zoonotic pathogen, yet the mechanism of its entry into cells remains unclear. This study has found that the host proteins Src, Fyn, and Ezrin are involved in viral replication and proliferation, and their activity inhibitors hinder EMCV invasion into cells. With the participation of the cell surface adhesion factor ICAM-1, EMCV infects host cells by sequentially activating the phosphorylation of Src, Ezrin, and Cav-1, mediating the entry of EMCV into host cells through CavME. We have demonstrated for the first time the existence of an ICAM-1-Src-Ezrin-Cav-1 signaling pathway during EMCV entry into susceptible cells, which mediates the internalization process of the virus. Our research not only unravels the molecular mechanism of EMCV invasion, filling a gap in related research, but also provides a theoretical basis for the development of specific antiviral therapeutics.