Identification of specific host cell potassium ion channels that mediate escape of Bunyamwera virus from late endosomal compartments

The Orthobunyavirus genus within the Peribunyaviridae family of enveloped arthropod-borne negative-sense RNA viruses includes species associated with serious or fatal disease in both animals and humans such as Schmallenberg, La Crosse and Oropouche viruses. Orthobunyaviruses (OBVs) are internalised into cells by endocytosis and release their genomes following fusion with late endosomal (LE) membranes, triggered by low pH of the luminal milieu. There is mounting evidence to suggest OBV endosomal escape is also influenced by potassium ions (K⁺), which increase in concentration as endosomes mature. Endosomal K⁺ flux is controlled by cellular K⁺ channels, and we previously showed that K⁺ channel blockade using broad spectrum pharmacological inhibition abrogates OBV infection, with virions trapped within the endosomes. However, the K⁺ channels regulating this process are unknown. Herein, to identify the K⁺ channels involved, we studied Bunyamwera virus (BUNV), the prototypical OBV, and screened a siRNA library targeting 342 human ion channels. We identified 19 K⁺ channels whose knockdown inhibited BUNV gene expression by over 50%, with at least seven channels affecting BUNV at the entry stage, suggesting they exert a combinatorial influence. Of these seven channels, we used both pharmacological inhibition and genetic channel manipulation to show that OBV escape from CD63- and Rab7-positive LEs is controlled by KCNJ2 (K_IR2.1), KCNJ13 (K_IR7.1) and KCNMA1 (BK_Ca). These studies add to the understanding of host factors influencing OBV entry, regulation of endosomal ion flux, and could aid in the design or repurposing of therapeutics for the treatment of OBV-associated disease.