Digoxin is a potent inhibitor of Bunyamwera virus infection in cell culture

Drug repurposing is a valuable source of new antivirals because many compounds used to treat a variety of pathologies can also inhibit viral infections. In this work, we have tested the antiviral capacity of four repurposed drugs to treat Bunyamw-era virus (BUNV) infection in cell cultures. BUNV is the prototype of the Bunyavirales order, a large group of RNA viruses that includes important pathogens for humans, animals and plants. Mock-and BUNV-infected Vero and HEK293T cells were treated with non- toxic concentrations of digoxin, cyclosporin A, sunitinib and chloroquine. The four drugs inhibited BUNV infection with varying potency in Vero cells, and all except sunitinib also in HEK293T cells, with digoxin rendering the lowest half maximal inhibitory concentration (IC50). Since digoxin rendered the best results, we selected this drug for a more detailed study. Digoxin is an inhibitor of the Na+/K+ ATPase, a plasma membrane enzyme responsible for the energy-dependent exchange of cytoplasmic Na+ for extracellular K+ in mammalian cells and involved in many signalling pathways. Digoxin was shown to act at an early time point after viral entry reducing the expression of the viral proteins Gc and N. Effects on the cell cycle caused by BUNV and digoxin were also analysed. In Vero cells, digoxin favoured the transition from G1 phase of the cell cycle to S phase, an effect that might contribute to the anti- BUNV effect of digoxin in this cell type. Transmission electron microscopy showed that digoxin impedes the assembly of the characteristic spherules that harbour the BUNV replication complexes and the morphogenesis of new viral particles. Both BUNV and digoxin induce similar changes in the morphology of mitochondria that become more electron- dense and have swollen cristae. The alterations of this essential organelle might be one of the factors responsible for digoxin-induced inhibition of viral infection. Digoxin did not inhibit BUNV infection in BHK- 21 cells that have a digoxin-resistant Na+/K+ ATPase, which suggests that the effects of the blockade of this enzyme is a key factor of the antiviral activity of digoxin in BUNV-infected Vero cells.