Targeting the US21 viroporin of Human Cytomegalovirus by calcium channel blockers as a new antiviral strategy

• CCBs bind the inner pore of the HCMV US21 viroporin and abolish pUS21-mediated ER Ca 2+ leakage • CCBs inhibit HCMV replication • CCBs prevent the cytobiological consequences of pUS21 expression • CCBs treatment restores ER Ca 2+ levels to those of control cells • CCBs do not affect basal intracellular Ca 2+ homeostasis The Human cytomegalovirus (HCMV) US21 protein is a calcium-conducting viroporin that modulates intracellular Ca 2+ homeostasis, safeguards cells from apoptosis, stimulates cell migration, and supports efficient HCMV replication. To validate pUS21 as a novel target for the identification of antiviral agents, in silico structure-based virtual screening was performed using its predicted structure to identify small molecules capable of engaging the inner part of the pore. Four dihydropyridine compounds (azelnidipine, efonidipine, lercanidipine, and niguldipine) were selected from 249 Calcium Channel Blockers (CCBs) in the DrugBank database. Molecular dynamics simulations of pUS21-ligand complexes predicted that the four selected CCBs formed dynamically stable and low-mobility interactions within the US21 pore, whereas the weak CCB binder felodipine remained highly mobile, supporting the predicted docking-based binding mode. The selected CCBs showed dose-dependent inhibition of HCMV replication in both fibroblasts and endothelial cells, with low micromolar EC 50 values. Their antiviral effect was neither cell type- nor strain-dependent, as confirmed against two different clinical isolates, TRwt and VR1814, and was observed to be reduced against a US21-deficient virus, suggesting the specificity of pUS21 as a molecular target. Consistent with the predicted engagement within the pUS21 pore, CCBs prevented pUS21-mediated Ca 2+ leakage from the endoplasmic reticulum and impaired both pUS21-induced cell migration and anti-apoptotic activity. Finally, drug combination studies revealed synergistic interactions between CCBs and maribavir treatment. Together, these findings support the hypothesis that clinically used CCBs may target pUS21 viroporin activity and hamper HCMV replication, thus offering a novel and promising antiviral strategy against HCMV, including drug-resistant strains.