Hepatitis C virus (HCV) infection is associated with dysregulation of both lipid and glucose metabolism. As well as contributing to viral replication, these perturbations influence the pathogenesis associated with the virus, including steatosis, insulin resistance, and type 2 diabetes. AMP-activated protein kinase (AMPK) plays a key role in regulation of both lipid and glucose metabolism. We show here that, in cells either infected with HCV or harboring an HCV subgenomic replicon, phosphorylation of AMPK at threonine 172 and concomitant AMPK activity are dramatically reduced. We demonstrate that this effect is mediated by activation of the serine/threonine kinase, protein kinase B, which inhibits AMPK by phosphorylating serine 485. The physiological significance of this inhibition is demonstrated by the observation that pharmacological restoration of AMPK activity not only abrogates the lipid accumulation observed in virus-infected and subgenomic replicon-harboring cells but also efficiently inhibits viral replication. These data demonstrate that inhibition of AMPK is required for HCV replication and that the restoration of AMPK activity may present a target for much needed anti-HCV therapies.

The innate immune response provides a critical defense against microbial infections, including viruses. These are recognised by pattern recognition receptors including Toll-like receptors (TLRs) and RIG-I like helicases (RLHs). Detection of virus triggers signalling cascades that induce transcription of type I interferons including IFNβ, which are pivotal for the initiation of an anti-viral state. Despite the essential role of IFNβ in the anti-viral response, there is an incomplete understanding of the negative regulation of IFNβ induction. Here we provide evidence that expression of the Nemo-related protein, optineurin (NRP/FIP2), has a role in the inhibition of virus-triggered IFNβ induction. Over-expression of optineurin inhibited Sendai-virus (SeV) and dsRNA triggered induction of IFNβ, whereas depletion of optineurin with siRNA promoted virus-induced IFNβ production and decreased RNA virus replication. Immunoprecipitation and immunofluorescence studies identified optineurin in a protein complex containing the antiviral protein kinase TBK1 and the ubiquitin ligase TRAF3. Furthermore, mutagenesis studies determined that binding of ubiquitin was essential for both the correct sub-cellular localisation and the inhibitory function of optineurin. This work identifies optineurin as a critical regulator of antiviral signalling and potential target for future antiviral therapy. Viral infection stimulates the innate immune response to produce various cytokines and chemokines to induce an anti-viral state within the host. The best studied of these are the type I interferons (IFNα/β), which are essential for an effective anti-viral response. Our understanding of how IFNβ is regulated is not well understood. This study demonstrates that the Nemo-related protein optineurin helps to regulate the levels of IFNβ in response to virus infection. We expressed optineurin in cells and found that the cells failed to express IFNβ when infected with various RNA viruses. Using biochemical experiments we showed that optineurin interacts with the protein kinase TBK1 and the ubiquitin ligase TRAF3. Furthermore, a mutation in optineurin that prevents the interaction with the small protein modifier ubiquitin (D474N) ablated the negative regulatory function of optineurin. Our findings provide a first example of a role for optineurin in anti-viral signalling and aid in our understanding of the negative regulation of IFNβ.

Oxidative stress induces neuronal apoptosis and is implicated in cerebral ischemia, head trauma, and age‐related neurodegenerative diseases. An early step in this process is the loss of intracellular K ⁺ via channels, and evidence indicates that K _v 2.1 is of particular importance in this regard, being rapidly inserted into the plasma membrane in response to apoptotic stimuli. An additional feature of neuronal oxidative stress is the up‐regulation of the inducible enzyme heme oxygenase‐1 (HO‐1), which catabolizes heme to generate biliverdin, Fe ²⁺ , and carbon monoxide (CO). CO provides neuronal protection against stresses such as stroke and excitotoxicity, although the underlying mechanisms are not yet elucidated. Here, we demonstrate that CO reversibly inhibits K _v 2.1. Channel inhibition by CO involves reactive oxygen species and protein kinase G activity. Overexpression of K _v 2.1 in HEK293 cells increases their vulnerability to oxidant‐induced apoptosis, and this is reversed by CO. In hippocampal neurons, CO selectively inhibits K _v 2.1, reverses the dramatic oxidant‐induced increase in k ⁺ current density, and provides marked protection against oxidant‐induced apoptosis. Our results provide a novel mechanism to account for the neuroprotective effects of CO against oxidative apoptosis, which has potential for therapeutic exploitation to provide neuronal protection in situations of oxidative stress.—Dallas, M. L., Boyle, J. P., Milligan, C. J., Sayer, R., Kerrigan, T. L., McKinstry, C., Lu, P., Mankouri, J., Harris, M., Scragg, J. L., Pearson, H. A., Peers, C. Carbon monoxide protects against oxidant‐induced apoptosis via inhibition of K _v 2.1. FASEB J. 25, 1519–1530 (2011). www.fasebj.org

An estimated 3% of the global population are infected with hepatitis C virus (HCV), and the majority of these individuals will develop chronic liver disease. As with other chronic viruses, establishment of persistent infection requires that HCV-infected cells must be refractory to a range of pro-apoptotic stimuli. In response to oxidative stress, amplification of an outward K⁺current mediated by the Kv2.1 channel, precedes the onset of apoptosis. We show here that in human hepatoma cells either infected with HCV or harboring an HCV subgenomic replicon, oxidative stress failed to initiate apoptosis via Kv2.1. The HCV NS5A protein mediated this effect by inhibiting oxidative stress-induced p38 MAPK phosphorylation of Kv2.1. The inhibition of a host cell K⁺channel by a viral protein is a hitherto undescribed viral anti-apoptotic mechanism and represents a potential target for antiviral therapy.

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.