Na,K-ATPase inhibition inhibits influenza A viral replication

Rationale: Influenza is a highly contagious disease with significant morbidity and mortality. Currently, there are limited therapeutic strategies to reduce the risk of severe illness and death. The development of new drugs is a necessity, preferably targeted to modulate the host response which should avoid the development of virus resistance. Na,K-ATPase inhibitors, such as cardiac glycosides, have been shown to inhibit viral replication. Here we set out to explore mechanisms by which the inhibition of Na,K-ATPase prevents influenza A virus replication. Methods: Cell culture experiments: A549 cells were infected for 1 h with influenza A virus (IAV: A/WSN/33, H1N1) at 1 MOI and incubated with ouabain at different time-points. Western blots were used to assess host and viral protein expression, and plaque assays were performed to evaluate viral titers. Real time PCR was used to determine viral mRNA in infected cells. Inductively coupled plasma/mass spectrometry was used to measure intracellular sodium and potassium. Ex vivo experiments: Lung slices from transgenic mice expressing an ouabain-sensitive Na,K-ATPase were infected ex vivo and treated with ouabain and images were taken using a confocal microscopy. In vivo experiments: Transgenic mice mentioned above were infected with IAV by intra-tracheal instillation and treated with ouabain or saline solution by intraperitoneal injection and bronchoalveolar lavage (BAL) was collected and lung homogenates were used to measure viral titer. Results: In vitro experiments revealed that influenza viral protein expression was blunted when Na,K-ATPase was inhibited. The effect of Na,K-ATPase inhibition on influenza replication was determined to be 4 to 6 h post infection and independently of mRNA transcription, protein degradation, the Src signaling pathway and intracellular calcium changes. We found that Na,K-ATPase inhibition led to decrease in the intracellular potassium affecting the cell translational machinery and consequently viral protein synthesis. Mice infected with IAV and treated with ouabain for 2 days post infection had decreased inflammation markers in BAL and lower viral titer in lung tissue compared to saline injected mice. Conclusions: Na,K-ATPase inhibition by decreasing intracellular potassium concentrations inhibits influenza viral protein synthesis post-transcriptionally. Accordingly, Na,K-ATPase inhibition impairs influenza virus replication and appears to decrease e lung inflammation and viral titers of influenza infected mice. Supported in part by HL-48129 and HL-071643