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 hepatitis C virus (HCV) NS5A protein plays a critical role in viral RNA replication and has recently been shown to play a role in particle production in the infectious genotype 2a HCV clone (JFH-1). Here, we show that alanine substitutions of serines 2428/2430 within the C-terminal domain III of NS5A do not affect subgenomic replicon RNA replication but do reduce particle production. In contrast, substitution of serines 2390/2391 had no effect on either RNA replication or particle production. Relative to genotype 1, all genotype 2 HCV isolates contain a 19 residue insertion near the C terminus of domain III which, when deleted (n2408-2426), resulted in a delay to both RNA replication and particle production. None of these mutations affected the ratio of basal to hyperphosphorylated NS5A, suggesting that serines between residues 2390 and 2430 are not phosphorylated. We propose that although domain III is dispensable for RNA replication, it nevertheless influences this process.Hepatitis C virus (HCV) is estimated to infect some 123 million individuals (Shepard et al., 2005). In the majority of cases, the virus establishes a chronic infection which can ultimately result in liver fibrosis, cirrhosis or hepatocellular carcinoma. Combination therapy comprising pegylated alpha interferon (IFN-a) and ribavirin is only successful in approximately 50 % of patients. HCV, a member of the family Flaviviridae, is an enveloped virus with a positive sense RNA genome of 9.6 kb. A single open reading frame is flanked by 59 and 39 untranslated regions (UTRs) containing cis-acting elements required for RNA replication; the 59 UTR also contains an internal ribosome entry site (IRES) which mediates cap-independent translation of the 3000 residue polyprotein. This is cleaved co-and posttranslationally by host cell and viral proteases to release the structural (core, E1, E2 and p7) and non-structural (NS2, NS3, NS4A, NS4B, NS5A and NS5B) proteins.NS5A has been shown to have many functions; foremost, as a component of the RNA replication complex, it is absolutely required for viral RNA replication. Structural analysis has revealed that NS5A comprises three domains separated by short low complexity regions (Tellinghuisen et al., 2004) (Fig. 1a). The structure of domain I has been determined; it coordinates a zinc ion and is postulated to dimerize forming a groove through which RNA is predicted to pass (Tellinghuisen et al., 2005). Domains II and III are less structured and more flexible; domain III appears to be dispensable for RNA replication (Tellinghuisen et al., 2008b) and can accommodate a green fluorescent protein (GFP) insert at the C terminus with no adverse effects (Appel et al., 2005; McCormick et al., 2006b;Moradpour et al., 2004). NS5A is a phosphoprotein existing in both a basally phosphorylated (p56) and a hyperphosphorylated (p58) state. Proline-directed kinases such as casein kinase (CK)II have been implicated in basal phosphorylation (Reed et al., 1997) and CKIa has been implicated in hyperphosphorylation (Quintavall...

One approach to understanding CLL is to investigate the nature of intracellular signals responsible for the development and prolonged survival of the malignant cells. 2 In this regard, signals generated by B-cell receptor (BCR) engagement are known to play an important role. 1 A key mediator of BCR-induced signaling is protein kinase C (PKC). [3][4][5][6][7][8][9] In CLL cells, this class of enzymes has been identified as a possible target of therapeutic intervention based on in vitro studies demonstrating that inhibition of these enzymes induces apoptosis. [10][11][12][13][14][15] Given the role of BCR signals in the survival and clonal expansion of CLL cells and the role of PKC(s) in the signaling pathways induced by BCR engagement, it follows that PKC(s) may play an important role in the BCR-induced survival of CLL cells.The PKC family is divided into 3 subgroups: the classical, which includes PKC␣, ␤I, ␤II, and ␥; the novel, which includes PKC␦, ⑀, , and ; and the atypical, which includes PKC and . These enzymes are activated by the presence of Ca 2ϩ , diacylglycerol, or other activating factors, 16 and they function in an array of cellular processes that can be specific for a particular cell type. In B cells, PKC, 5 PKC␤, 3,4,7,8 PKC␦,6 and PKC⑀ 9 play important roles in regulating signals generated by the BCR. With respect to CLL, active PKC␦ is thought to maintain cell survival downstream of phosphoinositol 3Ј-kinase. 15 Despite the potential of PKCs as therapeutic targets in CLL, [10][11][12][13][14][15] little is known about the relative levels and activities of the different isoforms known to be expressed within the malignant cells of this disease.In the present study, we show that PKC␤II is overexpressed in CLL cells and that the activity of this enzyme inversely correlates with CLL cell response to BCR engagement. Therefore, by regulating BCR signals important for malignant cell survival, PKC␤II may be a key factor in CLL progression. Materials and methods MaterialsMouse monoclonal and rabbit polyclonal anti-PKC␤II, monoclonal anti-PKC␤I, -PLC␥2, and -CD40 antibodies, rabbit anti-PKC␣, -PKC␦, -PKC, Mcl-1 and procaspase-8, and horseradish peroxidase-conjugated antimouse and anti-rabbit immunoglobulin antibodies were purchased from Santa Cruz Biotechnology (Insight Biotechnology, Middlesex, United Kingdom). Monoclonal anti-PKC␦ and anti-PKC⑀ antibodies were purchased from BD Biosciences (Oxford, United Kingdom). F(ab 2 )Ј fragments of goat anti-human IgM were purchased from Jackson ImmunoResearch Laboratories (Stratech, Soham, United Kingdom). Mouse anti-pS 180 -Bruton tyrosine kinase (Btk) and rabbit anti-Btk and anti-pY 759 -PLC␥2 antibodies were purchased from Cell Signaling Technology (New England Biolabs, Hitchin, Herts, United Kingdom). Purified recombinant PKC␣, PKC␤I, PKC␤II, PKC␦, PKC⑀, and PKC proteins and Ro32-0432 were purchased from Merck Biosciences (Nottingham, United Kingdom). Purified recombinant PKC and PKC proteins and mouse anti-ZAP-70 antibody were purchased from Upstate (Milton Ke...

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 ϩ 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

We previously demonstrated that two closely spaced polyproline motifs, with the consensus sequence Pro-X-X-Pro-X-Lys/Arg, located between residues 343 to 356 of NS5A, mediated interactions with cellular SH3 domains. The N-terminal motif (termed PP2.1) is only conserved in genotype 1 isolates, whereas the C-terminal motif (PP2.2) is conserved throughout all hepatitis C virus (HCV) isolates, although this motif was shown to be dispensable for replication of the genotype 1b subgenomic replicon. In order to investigate the potential role of these motifs in the viral life cycle, we have undertaken a detailed mutagenic analysis of these proline residues in the context of both genotype 1b (FK5.1) or 2a subgenomic replicons and the genotype 2a infectious clone, JFH-1. We show that the PP2.2 motif is dispensable for RNA replication of all subgenomic replicons and, furthermore, is not required for virus production in JFH-1. In contrast, the PP2.1 motif is only required for genotype 1b RNA replication. Mutation of proline 346 within PP2.1 to alanine dramatically attenuated genotype 1b replicon replication in three distinct genetic backgrounds, but the corresponding proline 342 was not required for replication of the JFH-1 subgenomic replicon. However, the P342A mutation resulted in both a delay to virus release and a modest (up to 10-fold) reduction in virus production. These data point to critical roles for these proline residues at multiple stages in the HCV life cycle; however, they also caution against extrapolation of data from culture-adapted replicons to infectious virus.Hepatitis C virus (HCV) is an enveloped RNA virus which is estimated to infect some 123 million individuals (24). In the majority of cases the virus establishes a chronic infection that can ultimately result in liver fibrosis, cirrhosis, or hepatocellular carcinoma. Thus, there is great interest in elucidating the mechanisms of viral replication, with a view to developing new chemotherapeutic agents. Since 1999, use of the subgenomic replicon system has led to significant progress in the understanding of the mechanism of viral RNA replication. It has been demonstrated that the five nonstructural proteins-NS3, NS4A, NS4B, NS5A, and NS5B-are necessary and sufficient to replicate an RNA molecule containing the 5Ј and 3Ј untranslated regions (UTRs) of the viral genome. However, apart from the RNA-dependent RNA polymerase (NS5B), the precise details of the roles of each of the nonstructural proteins in the process of RNA replication remain undefined. One problem associated with the subgenomic replicon system is the observation that the replicon RNA undergoes culture adaptation in which, as a result of the error-prone nature of the polymerase, mutations that confer enhanced replicative capacity are selected for in culture. Importantly, it has been shown using the chimpanzee model that, once engineered back into an infectious clone of the virus, such mutations may be attenuating in vivo (5). Recently, the HCV field has been revolutionized by the developmen...