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 has 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.

Ionic Balance in the Endosomal System

Most enveloped viruses enter cells by endocytosis. The endolysosomal system is a dynamic series of intracellular membranous compartments that facilitate the uptake, degradation, and recycling of cellular cargoes and membrane proteins. Na/K-ATPases are present on early endosomes where they transport Na into the lumen to limit proton influx, and in turn, acidification. Ca^++++++2+ plays a number of regulatory roles throughout the endosomal network, including the regulation of fusion and fission events, lipid trafficking, and lysosomal activity. The role of K influx into endosomes is to-date uncharacterised, but an increase in luminal K occurs as endosomes mature. Whilst the role of low pH in viral entry is well documented, the role of other endolysosomal ions is only beginning to be appreciated.

Fig. 1 Overview of ion channels implicated in viral entry.

Ion Channels Involved in Viral Entry

• Ca²⁺ Channels and Viral Entry
  The involvement of Ca²⁺ channels during viral entry is now well-documented. For example, ebola virus (EBOV) requires Ca²⁺ channels for its entry into host cells. EBOV enters cells through endolysosomes positive for both Niemann-Pick C1 (NPC1) and two-pore Ca²⁺ channel 2 (TPC2). The pharmacology of TPC was intensively studied by a virtual screen of ~1500 FDA-approved drugs. All identified TPC modulators were cross-referenced with two recent anti-EBOV screens, with four dopamine receptor antagonists and five oestrogen receptor modulators identified. As such, it was reasoned that these drugs exert their inhibitory effects on EBOV through the blockade of TPCs, subsequently confirmed through EBOV virus-like particle (VLP) assays. Using single-molecule FRET (smFRET)-imaging to further characterised the role of Ca²⁺ in EBOV entry. It was shown that Ca²⁺ and pH synergistically induce a conformational change in the EBOV glycoprotein GP2 to form a reversible intermediate state primed for NPC1 binding. NPC1 binding then further promotes the conformational transition into a fusion-ready "primed" state of invading EBOV virions.

Fig. 2 Ion channels implicated in viral entry since 2017.

• K Channels and Viral Entry
  The involvement of K channels in viral entry has been extensively characterised for the model bunyaviruses Bunyamwera orthobunyavirus (BUNV); and Hazara orthononairovirus (HAZV), a model for Crimean Congo haemorrhagic fever virus, which causes severe viral haemorrhagic fever outbreaks, with a case fatality rate of up to 40%. Initial work using known K channel pharmacology suggested that the blockade of two-pore K channels (K⁺⁺⁺_2P) inhibited the early stages of the BUNV lifecycle. Subsequent work identified both acidic pH and K in endosomes as crucial biochemical cues for the endosomal escape of BUNV. Similar studies in HAZV highlighted a dependence on K channels for infection, and that K primarily accumulated in cholesterol-rich endosomes. The K dependence of HAZV involves the glycoprotein spikes; a change in K concentration triggers conformational changes in the glycoproteins, as revealed through cryo-electron tomography of HAZV virions incubated with K that "primed" them for insertion into target membranes (Figure 2A). Moreover, it was shown that both BUNV and HAZV could be "primed" in vitro in buffers containing high [K], which expedited entry and subsequent viral gene expression.

Fig. 3 Predicted mechanisms of ion channel dependence for two enveloped viruses.

• Cl⁻ Channels and Viral Entry
  BK polyomavirus (BKPyV) is a potentially fatal pathogen in patients undergoing solid organ transplantation. Researches demonstrated that the pharmacological and genetic disruption of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ channel could reduce BKPyV infection in primary kidney cell models. Time of addition assays using the CFTR inhibitors CFTR172 and glibenclamide, combined with the assessment of exposure of VP2/VP3 minor capsid proteins, indicated a role for CFTR in the trafficking of BKPyV to the ER.

Ion Channels in Viral Replication

Once viral genomes are released inside the host cell, replication can commence. Recent evidence suggests that this process is partially controlled by ion concentrations and can therefore be targeted by ion channel drugs.

• Ca²⁺ Channels and Viral Replication
  Flaviviruses establish replication complexes in modified intracellular membranes, often derived from the ER. The ER stores the majority of intracellular Ca²⁺, and so it is perhaps unsurprising that an array of flaviviruses depend on intracellular Ca²⁺ ion channels for their replication. Japanese encephalitis virus (JEV) is an arthropod-borne virus linked to acute encephalitis. A study screening FDA-approved drugs to assess in vitro activity against JEV showed that three of the five most potent inhibitors were blockers of voltage-gated Ca²⁺ channels (VGCCs), including manidipine, cilnidipine, and benidipine hydrochloride. Time of addition assays suggested that these three drugs did not act during viral entry, nor were they virucidal, but specifically inhibited virus replication. The potential of these drugs as broad-acting anti-flavivirus treatments was further assessed and each led to a concentration-dependent inhibition of Dengue virus (DENV), West Nile virus, and Zika virus (ZIKV) replication. Yellow fever virus (YFV) was, however, insensitive to manidipine.

Fig. 4 Overview of ion channels implicated in viral replication.

• Cl⁻ Channels or Other Anions and Vials Replication
  Chikungunya virus (CHIKV) is a re-emerging arbovirus associated with long-term complications and high morbidity. Using siRNA silencing of the Cl⁻ intracellular channels (CLIC) 1 and 4, Müller et al. demonstrated a requirement for both channels during the replication of a CHIKV sub-genomic replicon in mammalian and invertebrate cells. The voltage-dependent anion channel 1 (VDAC1) is also implicated in viral replication; it is upregulated by infectious bursal disease virus (IBDV).

Viruses as Causative Agents of Acquired Channelopathies

Viral pathologies are becoming increasingly linked to the dysregulation of host ion channels. This reveals an interesting and new avenue for ion channel drugs, as the pharmacological manipulation of virus-targeted channels may not only impair viral infection at the cellular level, but may circumvent virus-induced channelopathies.

Fig. 5 Overview of ion channels implicated in virus-mediated disease.

• Viral Channelopathies and Ca²⁺ Channels
  Recent studies have linked viral infection to neuronal pathologies through the dysregulation of Ca²⁺ signalling. The FDA-approved Alzheimer’s drug memantine protected against neuronal cell death induced by ZIKV infection. Furthermore, the reactivation of herpes simplex virus 1 (HSV-1) can lead to cranial nerve disorders and severe pain. Rotavirus (RV) dysregulates cellular Ca²⁺ homeostasis through the depletion of ER stores.
• Viral Channelopathies Associated with K Channels⁺
  Coxsackie virus B3 (CVB3), amongst other enteroviruses, is associated with cardiomyopathies and sudden cardiac death. Numerous data suggest that CVB3 re-programmes ion channel expression in cardiac tissue, leading to an increased risk of arrhythmia. This highlights these channels as therapeutic targets to prevent the sudden cardiac death that results from CVB3 infection.
• Viral Channelopathies Associated with Na Channels⁺
  A number of viruses that primarily infect the airway system have been shown to dysregulate airway epithelial Na transport. Human respiratory syncytial virus (HRSV) primarily infects airway epithelial cells, and dysregulates epithelial Na channels (ENaC) to disrupt Na flux in the airways. ENaCs are a critical mediator of osmotic fluid absorption across airway epithelia, through the selective transport of Na. Electrochemical balance is maintained through apical Cl⁺⁺⁺⁺⁻ channels, which include CFTR. Clinical studies of infants diagnosed with HRSV showed a negative correlation between ENaC mRNA expression and the severity of HRSV bronchiolitis.
• Viral Channelopathies Associated with Cl⁻ Channels
  Studies identifying changes in the host channelome in response to MCPyV small tumor antigen (ST) overexpression by proteomic approaches have shown that two CLIC proteins (CLIC1 and CLIC4) are involved in the metastatic progression of specific tumour types, through switching of cellular localisation and function to integral transmembrane proteins as active anion channels and signal transducers.

Further Perspectives

It is now clear that host cell ion channels play an important role during viral infection at the cellular level, and as causative factors of disease states in infected tissues. Ion channels have been linked to multiple stages of viral lifecycles, in which viruses are either passively dependent upon or actively modulate channel functionality. Given this knowledge, evidence is emerging that ion channel inhibitors represent a new antiviral strategy. Whilst toxicity profiles for ion channel inhibitors are only available in the context of those used to treat hereditary channelopathies, in vivo evidence is emerging that these drugs can be efficacious against viruses. Moreover, overlapping mechanisms of acquired and hereditary channelopathies may underpin the efficacy of ion channel modulators in treating virally-induced pathophysiologies. The manipulation of host ion homeostasis presents an attractive target for the treatment of many clinically important viruses and their associated pathologies, whilst circumventing the risks of resistance associated with direct-acting antiviral drugs. As such, continued studies of host-virus interactions may guide future antiviral approaches.