Activation of store-operated calcium entry and mitochondrial respiration by enterovirus 71 is essential for efficient...

Enterovirus (EV) infections disrupt cellular calcium (Ca²⁺) homeostasis. The EV protein 2B is localized to the endoplasmic reticulum (ER) and causes depletion of ER Ca²⁺ stores. This depletion coincides with a substantial increase in cytosolic Ca²⁺ levels driven by extracellular Ca²⁺ influx. However, the precise mechanism underlying this influx remains elusive. In the present study, we demonstrated that EV71 infections induce store-operated Ca²⁺ entry (SOCE) by activating the Ca²⁺ sensor stromal interaction molecule 1 (STIM1), which subsequently interacts with Orai1, a plasma membrane (PM) Ca²⁺ channel. This finding was supported by confocal imaging, which revealed that STIM1, typically localized in the ER, becomes active and colocalizes with Orai1 at the PM in EV71-infected cells. Pharmacological inhibition of the STIM1-Orai1 interaction and knockdown of either STIM1 or Orai1 significantly reduced virus-induced cytosolic Ca²⁺ levels and viral replication. Global transcriptome analysis revealed that differentially expressed genes are primarily associated with the mitochondrial electron transport chain (ETC) upon SOCE activation, contributing to enhanced ATP generation and oxygen consumption. This increase in mitochondrial Ca²⁺ levels is correlated with the mid-stage of virus infection. Furthermore, we demonstrated that high levels of mitochondrial Ca²⁺ influx led to apoptotic cell death, favoring viral release at the late stage of virus infection. Finally, SOCE-dependent EV replication was observed in a mouse intestinal organoid culture, a more physiologically relevant cell system. Our results provide valuable insights into the mechanism through which EV infections induce SOCE-mediated spatial and temporal control of Ca²⁺ signaling, substantially affecting the virus life cycle.IMPORTANCEHost cell Ca²⁺ signals play crucial roles in various steps of virus life cycles, including entry, replication, and exit. EV requires increased cytosolic Ca²⁺ levels for efficient replication, but the precise mechanisms underlying the association between Ca²⁺ levels and EV replication remain elusive. Using EV71 as a model virus, we demonstrated that EV71 infection elevated cytosolic Ca²⁺ levels through store-operated Ca²⁺ entry activation and progressive Ca²⁺ mobilization to mitochondria. This led to the upregulation of electron transport chain activity, which is essential for efficient virus replication and apoptotic cell death, facilitating viral release during the mid and late stages of the infectious cycle, respectively. These findings substantially enhance the understanding of how EVs co-opt host cell mechanisms to promote their life cycle. STIM1 and Orai1 may be novel targets for broad-spectrum host-directed therapeutics against EVs and other viruses that employ similar replication mechanisms.