Activation of IRE1α-mediated ER stress pathway enhances the intestinal barrier to restrain viral replication in response to enterovirus infection

Background: Human enteroviruses, including enterovirus 71 (EV71), are major pathogens of hand-foot-mouth disease (HFMD), which primarily spread through the fecal-oral route and target intestinal epithelial cells (IECs), disrupting the intestinal barrier and transmitting to multiple organs. In response to endoplasmic reticulum (ER) stress caused by viral infection, the unfolded protein response (UPR) via inositol-requiring enzyme type 1 (IRE1), protein kinase R-like endoplasmic reticulum kinase (PERK), and activating transcription factor 6 (ATF6) pathways are activated in host cells. However, the role of ER stress in regulating the intestinal barrier during enteroviuses infections remains elusive. Methods: We assessed EV71-induced ER stress and intestinal barrier by Western blot, FITC-Dextran permeability measurement,and immunofluorescence assay in SW48 cells, and immumohistochemical and Periodic Acid-Schiff (PAS) staining in mice and human intestinal tissues, respectively. Using lentiviral packaging technology, PERK, ATF6, and IRE1α were stably knocked-down to investigate which ER stress pathway mediated the enhancement of intestinal barrier and inhibition of EV71 replication by Western blot. Next, an ER inducer, Thapsigargin (TG) was employed to examine the antiviral activityagainst EV71 in SW48 cells by Western blot, Qualitative real-time PCR (qRT-PCR), TCID50 assay, and transmission electron microscope (TEM), and further confirm the anti-EV71 activity in mice. Results: We demonstrate that EV71 infects intestinal epithelial cells (IECs) to activate ER stress, upregulate the expression of cell junction proteins, and enhance the intestinal barrier in cultured cells, mouse intestine, and human specimens. Specifically, we clarified that the IRE1α-mediated ER stress pathway drives this process, promoting cell junctions and intestinal barrier, inhibiting EV71 entry and further viral replication. In the treatment with the Thapsigargin (TG), the intestinal epithelial cell barrier was increased, which restrained EV71 infection and replication both in vitro and in vivo. Additionally, TG comprehensively inhibited the replication of other enteroviruses, including Coxsackievirus B3 (CVB3) and Echovirus 6 (ECHO6). Conclusions: Our findings provide new insights into the cellular mechanisms underlying the antiviral effects of the activation of ER stress, offering a novel strategy for combating early enteroviruses infections. Moreover, TG effectively suppresses the replication of various enteroviruses, suggesting its potential as a broad-spectrum antiviral agent.