Endolysosomal two-pore channels regulate a conserved entry pathway for enteroviruses

Enteroviruses are significant human pathogens for which there are currently no effective antiviral therapies available. Two-pore channels (TPCs) are endolysosomal cation channels activated by nicotinic acid adenine dinucleotide phosphate (NAADP). These channels play a crucial role in regulating vesicular calcium (Ca²⁺) signalling and endolysosomal membrane trafficking. While endolysosomal TPCs have been implicated in the infectivity of some enveloped viruses, their role in non-enveloped virus infections remains unclear. In this study, we identify human TPC isoforms TPC1 and TPC2 as essential host factors that enhance enterovirus infection by regulating endolysosomal trafficking. Inhibiting NAADP-evoked Ca²⁺ signalling pharmacologically or using CRISPR/Cas9 to knockout either TPC isoform significantly suppressed the replication of enterovirus 71. Mechanistically, the loss of TPC function did not affect the viral binding, internalization or post-entry RNA translation and replication; instead, it delayed the viral progression through early and late endosomal compartments. Live-cell Ca²⁺ imaging confirmed that TPC-dependent NAADP signalling is necessary for endolysosomal Ca²⁺ release during infection. Our data also suggest cepharanthine, a bisbenzylisoquinoline alkaloid, inhibits enterovirus infection partly by modulating TPC-mediated Ca²⁺ signalling. Notably, the pharmacological inhibition or depletion of TPCs broadly restricted the replication of various enterovirus serotypes. Transcriptomic profiling revealed that the disruption of TPC1 or TPC2 alters gene networks associated with endocytosis and vesicular trafficking, emphasizing the vital role of TPCs in maintaining endolysosomal homeostasis. In summary, these findings establish TPC-dependent Ca²⁺ signalling as a conserved host pathway that enteroviruses exploit and highlight endolysosomal TPCs as promising targets for the development of broad-spectrum antiviral strategies.