Jamel Mankouri

Optineurin Negatively Regulates the Induction of IFNβ in Response to RNA Virus Infection

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.

Enhanced hepatitis C virus genome replication and lipid accumulation mediated by inhibition of AMP-activated protein kinase

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.

Carbon monoxide protects against oxidant-induced apoptosis via inhibition of Kv2.1.

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 Kv2.1 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, Fe2+, 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 Kv2.1. Channel inhibition by CO involves reactive oxygen species and protein kinase G activity. Overexpression of Kv2.1 in HEK293 cells increases their vulnerability to oxidant‐induced apoptosis, and this is reversed by CO. In hippocampal neurons, CO selectively inhibits Kv2.1, 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 Kv2.1. FASEB J. 25, 1519–1530 (2011). www.fasebj.org

Suppression of a pro-apoptotic K+ channel as a mechanism for hepatitis C virus persistence

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.

Hepatitis C virus NS5A protein interacts with beta-catenin and stimulates its transcriptional activity in a phosphoinositide-3 kinase-dependent fashion

Hepatitis C virus (HCV) infection is increasingly associated with the development of hepatocellular carcinoma (HCC). HCV is not thought to be directly oncogenic but, by modulating a range of cellular functions, may predispose patients to the development of liver tumours. However, the molecular mechanisms by which HCV infection might contribute to HCC remain to be characterized. In this regard, we showed previously that the HCV NS5A protein bound to the p85 regulatory subunit of phosphoinositide-3 kinase (PI3K), thereby stimulating the activity of the p110 catalytic subunit of the enzyme. One of the downstream consequences of this was the stabilization of the proto-oncogene, beta-catenin, with a concomitant stimulation of its transcriptional activity. Here, we further analyse the mechanism by which NS5A mediates activation of beta-catenin. Although our previous data were consistent with a role for the PI3K downstream effector kinases, Akt and glycogen synthase kinase-3beta, in NS5A-mediated activation of beta-catenin, we demonstrate here that it is in fact independent of both of these kinases. Truncation analysis revealed that both the N and C termini of NS5A are required for full activation of beta-catenin. Furthermore, we demonstrate that NS5A, either alone or in complex with p85, is able to bind directly to beta-catenin; again both N and C termini contribute to this interaction. We propose that NS5A activates beta-catenin via a novel mechanism that involves a direct interaction between the two proteins and is augmented by PI3K activity. This may contribute to the association between chronic HCV infection and the development of HCC.

Kir6.2 mutations causing neonatal diabetes prevent endocytosis of ATP‐sensitive potassium channels

ATP‐sensitive potassium (KATP) channels couple the metabolic status of a cell to its membrane potential—a property that endows pancreatic β‐cells with the ability to regulate insulin secretion in accordance with changes in blood glucose. The channel comprises four subunits each of Kir6.2 and the sulphonylurea receptor (SUR1). Here, we report that KATP channels undergo rapid internalisation from the plasma membrane by clathrin‐mediated endocytosis. We present several lines of evidence to demonstrate that endocytosis is mediated by a tyrosine based signal (330YSKF333) located in the carboxy‐terminus of Kir6.2 and that SUR1 has no direct role. We show that genetic mutations, Y330C and F333I, which cause permanent neonatal diabetes mellitus, disrupt this motif and abrogate endocytosis of reconstituted mutant channels. The resultant increase in the surface density of KATP channels would predispose β‐cells to hyperpolarise and may account for reduced insulin secretion in these patients. The data imply that endocytosis of KATP channels plays a crucial role in the (patho)‐physiology of insulin secretion.

The Hepatitis C Virus Non-Structural Protein NS5A Alters the Trafficking Profile of the Epidermal Growth Factor Receptor

Hepatitis C virus (HCV) frequently establishes a persistent infection, leading to chronic liver disease. The NS5A protein has been implicated in this process as it modulates a variety of intracellular signalling pathways that control cell survival and proliferation. In particular, NS5A associates with several proteins involved in the endocytosis of the epidermal growth factor receptor (EGFR) and has been previously shown to inhibit epidermal growth factor (EGF)‐stimulated activation of the Ras–Erk pathway by a mechanism that remains unclear. As EGFR signalling involves trafficking to late endosomes, we investigated whether NS5A perturbs EGFR signalling by altering receptor endocytosis. We demonstrate that NS5A partially localizes to early endosomes and, although it has no effect on EGF internalization, it colocalizes with the EGFR and alters its distribution. This redistribution correlates with a decrease in the amount of active EGF–EGFR ligand–receptor complexes present in the late endosomal signalling compartment and also results in a concomitant increase in the total levels of EGFR. These observations suggest that NS5A controls EGFR signalling by diverting the receptor away from late endosomes. This represents a novel mechanism by which a viral protein attenuates cell signalling and suggests that NS5A may perturb trafficking pathways to maintain an optimal environment for HCV persistence.

Viruses and the fuel sensor: the emerging link between AMPK and virus replication.

Adenosine 5′ monophosphate‐activated protein kinase (AMPK) is conserved in all eukaryotic cells and functions as the key regulator of cellular metabolism by responding to the energy status of the cell. It is activated by an increase in the AMP : ATP ratio and then attempts to redress the balance by upregulating catabolic processes, whilst concomitantly inhibiting anabolic processes. Despite its critical importance in the functioning of eukaryotic cells, there has been a paucity of studies investigating the potential for dysregulation of AMPK by viruses. Recently, however, there have been a number of reports that have begun to address this gap in our knowledge. In this article, we will review this emerging field, outlining how a variety of viruses have been shown to either stimulate or inhibit AMPK activity. We will also document the effects of these perturbations on the biology of virus infection, in particular with regard to the ability of viruses to persist or cause cytopathogenesis. Copyright

Serine Phosphorylation of the Hepatitis C Virus NS5A Protein Controls the Establishment of Replication Complexes

ABSTRACT The hepatitis C virus (HCV) nonstructural 5A (NS5A) protein is highly phosphorylated and involved in both virus genome replication and virion assembly. We and others have identified serine 225 in NS5A to be a phosphorylation site, but the function of this posttranslational modification in the virus life cycle remains obscure. Here we describe the phenotype of mutants with mutations at serine 225; this residue was mutated to either alanine (S225A; phosphoablatant) or aspartic acid (S225D; phosphomimetic) in the context of both the JFH-1 cell culture infectious virus and a corresponding subgenomic replicon. The S225A mutant exhibited a 10-fold reduction in genome replication, whereas the S225D mutant replicated like the wild type. By confocal microscopy, we show that, in the case of the S225A mutant, the replication phenotype correlated with an altered subcellular distribution of NS5A. This phenotype was shared by viruses with other mutations in the low-complexity sequence I (LCS I), namely, S229D, S232A, and S235D, but not by viruses with mutations that caused a comparable replication defect that mapped to domain II of NS5A (P315A, L321A). Together with other components of the genome replication complex (NS3, double-stranded RNA, and cellular lipids, including phosphatidylinositol 4-phosphate), the mutation in NS5A was restricted to a perinuclear region. This phenotype was not due to cell confluence or another environmental factor and could be partially transcomplemented by wild-type NS5A. We propose that serine phosphorylation within LCS I may regulate the assembly of an active genome replication complex. IMPORTANCE The mechanisms by which hepatitis C virus replicates its RNA genome remain poorly characterized. We show here that phosphorylation of the viral nonstructural protein NS5A at serine residues is important for the efficient assembly of a complex that is able to replicate the viral genome. This research implicates cellular protein kinases in the control of virus replication and highlights the need to further understand the interplay between the virus and the host cell in order to develop potential avenues for future antiviral therapy.

Molecular cell biology of KATP channels: implications for neonatal diabetes.

ATP-sensitive potassium (KATP) channels play a key role in the regulation of insulin secretion by coupling glucose metabolism to the electrical activity of pancreatic beta-cells. To generate an electric signal of suitable magnitude, the plasma membrane of the beta-cell must contain an appropriate number of channels. An inadequate number of channels can lead to congenital hyperinsulinism, whereas an excess of channels can result in the opposite condition, neonatal diabetes. KATP channels are made up of four subunits each of Kir6.2 and the sulphonylurea receptor (SUR1), encoded by the genes KCNJ11 and ABCC8, respectively. Following synthesis, the subunits must assemble into an octameric complex to be able to exit the endoplasmic reticulum and reach the plasma membrane. While this biosynthetic pathway ensures supply of channels to the cell surface, an opposite pathway, involving clathrin-mediated endocytosis, removes channels back into the cell. The balance between these two processes, perhaps in conjunction with endocytic recycling, would dictate the channel density at the cell membrane. In this review, we discuss the molecular signals that contribute to this balance, and how an imbalance could lead to a disease state such as neonatal diabetes.