Inhibition of potassium ion channels reduces Semliki Forest virus genome replication

Abstract

AbstractIntroductionSemliki forest virus(SFV) is a model virus used to investigate the Alphavirus genus, which includes human pathogens Chikungunya virus and Ross River virus. Viruses harness cellular machinery to facilitate various steps of their replicative cycles. Ion channels are one group of cellular proteins required for the efficient replication of some viruses, including Influenza A viruses, Ebola virus and members of theBetacoronavirusgenus. This study focussed on understanding SFV’s requirement for functional ion channels during replication.MethodsThe effect of ion channel inhibitors onin vitroSFV infections was measured to investigate the contribution of ion channels in its replication cycle.ResultsIn vitroSFV infections carried out in the presence or absence of different ion channel inhibitors showed broad-range K+channel inhibitors reproducibly attenuated virus replication and reduced its cytotoxicity in two mammalian cell lines. These broad-range K+channel inhibitors disrupted an early, post-entry step causing a delay or reduction in SFV protein and RNA synthesis. Screens using inhibitors of specific K+channel families showed that two-pore domain K+channel (2pK) inhibitors attenuated SFV replication. Confocal microscopy revealed decreased detection of dsRNA and SFV protein in the presence of inhibitor but no change in RNA and protein colocalisation, which would indicate disruption of replication complexes. Broad-range K+and 2pK inhibitors decreased viral RNA replication and transcription from the subgenomic promoter.ConclusionsK+channel inhibitors attenuatein vitroSFV replication by inhibiting an early, post-entry step of virus replication, potentially RNA synthesis.ImportanceNo antiviral therapies have been approved for clinical use against diseases caused by members of the Alphavirus genus. Work presented in this manuscript shows for the first time that SFV genome replication and virus induced cytotoxicity can be reducedin vitroby treating infected cells with K+channel inhibitors. This work provides the basis for investigating the effectiveness of K+channel inhibitors against other alphaviruses bothin vitro and in vivoand, because many ion channel inhibiting drugs are already in clinical use, rapid repurposing against alphavirus infections would be possible.