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.

We have introduced GFP and photoactivatable GFP into the NS5A coding region of a hepatitis C virus (HCV) subgenomic replicon that gives efficient transient replication. NS5A-GFP, expressed by the replicon, could be detected in cytoplasmic fluorescent foci as early as 4 h after RNA was introduced into cells. The fluorescent foci are likely to be sites where RNA synthesis could occur, although their production was not dependent on prior replication. Photobleaching studies demonstrated that the fluorescent proteins were relatively immobile upon expression from replicon RNAs. By contrast, an NS5A-GFP chimera produced in the absence of other viral proteins was mobile. Hence, interactions in cells expressing HCV replication proteins limit NS5A mobility, and transfer of viral proteins between foci is either slow or does not occur. Thus, the sites of HCV RNA replication possibly have a fixed complement of proteins that may act as discrete factories for producing viral RNA.Subgenomic replicons (SGRs) of hepatitis C virus (HCV), based on sequences from genotype 1a and 1b strains, have been invaluable for examining viral RNA replication (Blight et al., 2000;Lohmann et al., 1999). Recent reports have demonstrated that SGRs possessing sequences from JFH1, an HCV genotype 2a strain, replicate far more efficiently than those from genotype 1b in both colony-forming assays and quantitative transient replication assays (TRAs) using reporters such as luciferase activity (Kato et al., 2003;Miyamoto et al., 2006;Targett-Adams & McLauchlan, 2005;Windisch et al., 2005). Importantly, efficient replication with JFH1 SGRs is not dependent on either highly permissive Huh-7 cells or tissue-culture-adapted mutations that are needed to enhance genotype 1b replicon competence (Krieger et al., 2001;Lohmann et al., 2001). By contrast to genotype 1b SGRs, the JFH1-based replicon exhibits large increases in luciferase activity over the initial 24 h following transfection of Huh-7 cells with SGR RNA (Targett-Adams & McLauchlan, 2005). Hence, this replicon offers unique possibilities to examine the early stages of viral RNA replication.Previous studies have reported stable cell lines that support steady-state replication of SGRs containing genotype 1b sequences that incorporate GFP-tagged NS5A (Liu et al., 2006; McCormick et al., 2006b;Moradpour et al., 2004). However, replication in TRAs with such replicons was barely detectable (Appel et al., 2005b). Here, we describe development of JFH1-based SGRs that incorporate GFP and a photoactivatable variant of GFP (PAGFP) (Patterson & Lippincott-Schwartz, 2002 into the C-terminal region of the NS5A protein.To construct tagged JFH1 SGRs, the GFP and PAGFP open reading frames were inserted into the 39 coding sequence of JFH1-derived NS5A at a site previously shown to be capable of accommodating GFP within genotype 1b-based replicons (Liu et al., 2006; McCormick et al., 2006b;Moradpour et al., 2004). DNA fragments encoding NS5A-GFP/PAGFP fusion proteins were engineered into pSGR-Luc-JFH1 (TargettAdams & McL...

The hepatitis C virus (HCV) non-structural protein NS4B induces morphological changes in the endoplasmic reticulum (ER) membrane that may have a direct role in viral RNA replication. A chimeric GFP-NS4B fusion protein located to the ER membrane and to foci that were attached to the ER. These membrane-associated foci (MAFs) could be related to the membrane alterations observed in cells that replicate HCV RNA. The relationship of MAFs to pre-existing cellular structures is not known. Indirect immunofluorescence analysis demonstrated that they did not contain a cellular marker for vesicles, which have been implicated in the replication of other viruses. From photobleaching studies to examine diffusion of NS4B, the GFP-tagged protein had reduced mobility on MAFs compared with on the ER membrane. This slower mobility suggested that NS4B is likely to form different interactions on MAFs and the ER.Hepatitis C virus (HCV) RNA replication requires a minimum of five viral proteins (NS3, NS4A, NS4B, NS5A and NS5B) (Lohmann et al., 1999;Blight et al., 2000;Ikeda et al., 2002). Viral RNA synthesis is considered to occur in foci, which are novel intracellular structures generated by morphological alteration of the endoplasmic reticulum (ER) membrane (Gosert et al., 2003;Moradpour et al., 2004). The appearance of foci correlates with the presence of a membranous web that is detected by electron microscopy (Egger et al., 2002;Gosert et al., 2003) and it is probable that the foci and membranous web are related, if not identical, structures. Expression of NS4B alone is sufficient for induction of these morphological changes (Egger et al., 2002;Lundin et al., 2003). However, the mechanisms that drive their formation and the nature of the association of NS4B with these structures have not been elucidated. Here, we examined the behaviour of NS4B in live cells to determine the characteristics of the morphological alterations produced by the protein and to analyse its mobility on membranes.To examine NS4B in live cells, the polypeptide was attached to the C terminus of EGFP. A PCR product encoding NS4B (aa 1712-1973 of HCV 1a strain H77) was amplified from pCV-H77C (kindly supplied by Dr J. Bukh; Yanagi et al., 1997) using forward and reverse primers (59-GGGAG-AATTCAGATCTCAGCACTTACCGTACATCGAG-39 and 59-GGGAAGCTTCTAGAGGATCCGCTAGCATGGAGTGG-TACACTCCGAGC-39, respectively) that incorporated restriction enzyme sites for cloning purposes and a stop codon (underlined). The PCR product was introduced into pGEM-1 following digestion with EcoRI and XbaI, to generate plasmid pGEM-1/NS4B. A BglII/XbaI DNA fragment from pGEM-1/NS4B was inserted into pEGFP-C1 (Clontech) to create pGFP-NS4B. pGFP-NS4B was transfected into tissue culture cells estimated to be between 30 and 60 % confluent using Lipofectamine 2000 (Invitrogen). To express untagged protein, an oligonucleotide (59-AATTCTAGGATCCTCATTGATGA-39) that incorporated an initiator methionine codon was introduced between the EcoRI and BglII sites in pGEM-1/NS4B to produce pGEM-1/ atg-NS4B. A BamHI...

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...

The hepatitis C virus NS5A protein has been previously demonstrated to partially attenuate activation of the Ras-Erk signalling pathway, via a conserved class II polyproline motif located towards the C terminus of the protein. However, the role of Ras-Erk signalling in the virus life cycle remains undetermined. To investigate this, levels of RNA replication were measured in genotypes 1 and 2 transient luciferase subgenomic replicon systems in the context of either pharmacological or genetic (dominant-negative) inhibition of MEK1, a kinase in the Ras-Erk signalling cascade. Incubation in the presence of two inhibitors (U0126 and PD184352) resulted in a decrease in the levels of RNA replication, conversely incubation with inhibitor PD98059 resulted in a modest increase in replication. The results obtained with PD98059 could not be explained by an off-target effect on Cox-2, stability of replicon RNA or stimulation of global translation levels, suggesting stimulation by a yet uncharacterized mechanism. To verify data obtained using pharmacological inhibitors the transient replicon RNA was co-electroporated with a dominant-negative mutant of MEK1. This resulted in a reduction in replication, confirming data seen with U0126 and PD184352. Our data are consistent with the hypothesis that a low level Ras-Erk signalling activity is required for RNA replication. However, complete inhibition of Ras-Erk signalling is inhibitory. These results suggest that perturbation of this signalling pathway by NS5A may be a mechanism to regulate levels of genomic RNA replication.