In order to multiply and cause disease a virus must transport its genome from outside the cell into the cytosol, most commonly achieved through the endocytic network. Endosomes transport virus particles to specific cellular destinations and viruses exploit the changing environment of maturing endocytic vesicles as triggers to mediate genome release. Previously we demonstrated that several bunyaviruses, which comprise the largest family of negative sense RNA viruses, require the activity of cellular potassium (K⁺) channels to cause productive infection. Specifically, we demonstrated a surprising role for K⁺ channels during virus endosomal trafficking. In this study, we have used the prototype bunyavirus, Bunyamwera virus (BUNV), as a tool to understand why K⁺ channels are required for progression of these viruses through the endocytic network. We report three major findings: First, the production of a dual fluorescently labelled bunyavirus to visualize virus trafficking in live cells. Second, we show that BUNV traffics through endosomes containing high [K⁺] and that these K⁺ ions influence the infectivity of virions. Third, we show that K⁺ channel inhibition can alter the distribution of K⁺ across the endosomal system and arrest virus trafficking in endosomes. These data suggest high endosomal [K⁺] is a critical cue that is required for virus infection, and is controlled by cellular K⁺ channels resident within the endosome network. This highlights cellular K⁺ channels as druggable targets to impede virus entry, infection and disease. To penetrate cells and cause disease most viruses traverse the cellular endocytic network and exploit its environment to trigger the release of their genetic material into the cytosol. Endosomal pH, virus-receptor interactions, proteolytic cleavage, and lipid composition of membranes have all been previously reported to act as endosomal triggers. Here, we reveal the identity of an additional critical endosomal trigger for virus entry, namely a requirement for exposure to specific K⁺ ion concentrations, which we demonstrate is regulated by cellular K⁺ channels. We show that by blocking K⁺ channels, we can disrupt the K⁺ concentration across the endosomal system, impeding virus trafficking. Here, we reveal cellular K⁺ channels as a new druggable target to inhibit virus entry, and thus disease.

The broad range of cellular functions governed by ion channels represents an attractive target for viral manipulation. Indeed, modulation of host cell ion channel activity by viral proteins is being increasingly identified as an important virus–host interaction. Recent examples have demonstrated that virion entry, virus egress and the maintenance of a cellular environment conducive to virus persistence are, in part, dependent on virus manipulation of ion channel activity. Most excitingly, evidence has emerged that targeting ion channels pharmacologically can impede virus life cycles. Here, we discuss current examples of virus–ion channel interactions and the potential of targeting ion channel function as a new, pharmacologically safe and broad-ranging anti-viral therapeutic strategy.

Bunyaviruses are considered to be emerging pathogens facilitated by the segmented nature of their genome that allows reassortment between different species to generate novel viruses with altered pathogenicity. Bunyaviruses are transmitted via a diverse range of arthropod vectors, as well as rodents, and have established a global disease range with massive importance in healthcare, animal welfare, and economics. There are no vaccines or anti-viral therapies available to treat human bunyavirus infections and so development of new anti-viral strategies is urgently required. Bunyamwera virus (BUNV; genus Orthobunyavirus) is the model bunyavirus, sharing aspects of its molecular and cellular biology with all Bunyaviridae family members. Here, we show for the first time that BUNV activates and requires cellular potassium (K⁺) channels to infect cells. Time of addition assays using K⁺ channel modulating agents demonstrated that K⁺ channel function is critical to events shortly after virus entry but prior to viral RNA synthesis/replication. A similar K⁺ channel dependence was identified for other bunyaviruses namely Schmallenberg virus (Orthobunyavirus) as well as the more distantly related Hazara virus (Nairovirus). Using a rational pharmacological screening regimen, two-pore domain K⁺ channels (K_2P) were identified as the K⁺ channel family mediating BUNV K⁺ channel dependence. As several K_2P channel modulators are currently in clinical use, our work suggests they may represent a new and safe drug class for the treatment of potentially lethal bunyavirus disease.

Many enveloped viruses enter cells through the endocytic network, from which they must subsequently escape through fusion of viral and endosomal membranes. This membrane fusion is mediated by virus-encoded spikes that respond to the dynamic endosomal environment, which triggers conformational changes in the spikes that initiate the fusion process. Several fusion triggers have been identified and include pH, membrane composition, and endosome-resident proteins, and these cues dictate when and where viral fusion occurs. We recently reported that infection with an enveloped bunyavirus requires elevated potassium ion concentrations [K⁺], controlled by cellular K⁺ channels, that are encountered during viral transit through maturing endosomes. Here we reveal the molecular basis for the K⁺ requirement of bunyaviruses through the first direct visualization of a member of the Nairoviridae family, namely Hazara virus (HAZV), using cryo-EM. Using cryo-electron tomography, we observed HAZV spike glycoproteins within infectious HAZV particles exposed to both high and low [K⁺], which showed that exposure to K⁺ alone results in dramatic changes to the ultrastructural architecture of the virion surface. In low [K⁺], the spikes adopted a compact conformation arranged in locally ordered arrays, whereas, following exposure to high [K⁺], the spikes became extended, and spike–membrane interactions were observed. Viruses exposed to high [K⁺] also displayed enhanced infectivity, thus identifying K⁺ as a newly defined trigger that helps promote viral infection. Finally, we confirmed that K⁺ channel blockers are inhibitory to HAZV infection, highlighting the potential of K⁺ channels as anti-bunyavirus targets.

Ion channels play key roles in almost all facets of cellular physiology and have emerged as key host cell factors for a multitude of viral infections. A catalogue of ion channel-blocking drugs have been shown to possess antiviral activity, some of which are in widespread human usage for ion channel-related diseases, highlighting new potential for drug repurposing. The emergence of ion channel–virus interactions has also revealed the intriguing possibility that channelopathies may explain some commonly observed virus induced pathologies. This field is rapidly evolving and an up-to-date summary of new discoveries can inform future perspectives. We herein discuss the role of ion channels during viral lifecycles, describe the recently identified ion channel drugs that can inhibit viral infections, and highlight the potential contribution of ion channels to virus-mediated disease.

The Bunyavirales order of segmented negative-sense RNA viruses includes > 500 isolates that infect insects, animals, and plants and are often associated with severe and fatal disease in humans. To multiply and cause disease, bunyaviruses must translocate their genomes from outside the cell into the cytosol, achieved by transit through the endocytic network. We have previously shown that the model bunyaviruses Bunyamwera virus (BUNV) and Hazara virus (HAZV) exploit the changing potassium concentration ([K+]) of maturing endosomes to release their genomes at the appropriate endosomal location. K+ was identified as a biochemical cue to activate the viral fusion machinery, promoting fusion between viral and cellular membranes, consequently permitting genome release. In this study, we further define the biochemical prerequisites for BUNV and HAZV entry and their K+ dependence. Using drug–mediated cholesterol extraction along with viral-entry and K+ uptake assays, we report three major findings: (1) BUNV and HAZV require cellular cholesterol during endosomal escape; (2) cholesterol depletion from host cells impairs K+ accumulation in maturing endosomes, revealing new insights into endosomal K+ homeostasis; and (3) “priming” BUNV and HAZV virions with K+ before infection alleviates their cholesterol requirement. Taken together, our findings suggest a model in which cholesterol abundance influences endosomal K+ levels and consequently the efficiency of bunyavirus infection. The ability to inhibit bunyaviruses with existing cholesterol-lowering drugs may offer new options for future antiviral interventions for pathogenic bunyaviruses.