文章

Supporting information Mankouri et al. 10.1073/pnas.0912426107 [AMP]/[ATP] AMPKK M O 25 LBK1STRAD AICAR Metformin AKT HCV β α Active AMPK A769662 β α Ser485 Inactive AMPK β α P Thr172 Energy γ AMPK P Thr172 γ γ P dephosphorylation Energy requiring processes Energy producing pathways ADP ATP ACC ATP ADP ACC Fatty acid & cholesterol biosynthesis P Fatty acid & cholesterol biosynthesis HCV replication P Fig. S1. Schematic of the regulation of AMP-activated protein kinase (AMPK) activity. When activated by ATP consumption or pharmacological activation, AMPK is phosphorylated at threonine 172 on the α subunit by an upstream complex, AMPK kinase. Active AMPK then phosphorylates a number of substrates. Key targets include the two isoforms of acetyl-CoA carboxylase (ACC1/2). Importantly, phosphorylation of ACC1/2 on serine 79/220 by AMPK inhibits enzymatic activity, concomitantly decreasing cellular fatty acid synthesis which could be deleterious to hepatitis C virus (HCV) infection. HCV (most likely via proteins NS4B and NS5A) activates the serine/threonine kinase, AKT, which phosphorylates AMPK on an inhibitory residue (serine 485), leading to activation of ACC1/2 and increased cellular lipid synthesis. Mankouri et al. www.pnas.org/cgi/content/short/0912426107 1 of 4 S E C )A 1.0 SGR-Luc-JFH-1 al is ed L u ci fe ra se ( R L U / S 0.2 0.4 0.6 0.8 ** ** ** N o rm a Control AICAR A769662 Metformin AKTVIII 0.0 0.2 ** B SGR-Luc-JFH-1 E C ) 0.2 0.4 0.6 0.8 1.0 0.2 0.4 0.6 0.8 1.0 0.2 0.4 0.6 0.8 1.0 lis ed L u ci fe ra se ( R L U /S 0.0 0 0.125 0.25 0.5 1 0.0 0 0.125 0.25 0.5 1 0.0 0 12.5 25 50 100 Metformin [mM]AICAR [mM] A769662 [µM] N o rm a DC NS5A BODIPY Merge S G R -L u c- JF H -1 V III C o n tr o l 0.4 0.6 0.8 1.0 1.2 d a b so rb an ce ( 54 0n m ) MTT assay (Huh-7) S + A K T V NS5A BODIPY Merge 0.0 0.2 N o rm al is ed A 76 96 62 (1 00 µ M ) A IC A R (1 m M ) M et fo rm in (! m M ) C o n tr o l Fig. S2. Restoration of AMPK activity using either AMPK agonists or AKTVIII is detrimental to the replication of a genotype 2a (JFH-1) subgenomic replicon (SGR). (A) Huh-7 cells were transfected with in vitro transcripts of a transient luciferase subgenomic replicon (JFH-1: genotype 2a) (1) and left untreated (Control) or treated with the indicated compounds overnight. Luciferase activity measured 24 h posttransfection shows the relative level of genome repli- cation. Error bars indicate mean ± SEM. (B) Huh-7 cells were transfected with in vitro transcripts of a transient luciferase subgenomic replicon (JFH-1: genotype 2a) and treated with the AMPK agonists AICAR, A769662, or metformin at the indicated concentrations overnight. Luciferase activity was measured 24 h posttransfection and shows the relative level of genome replication. Error bars show SEM. (C) Huh-7 cells were left untreated (Control) or treated with AICAR, A769662, or metformin at the indicated concentrations for 24 h before analysis of cell viability by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Error bars indicate mean ± SEM. (D) Distribution and abundance of NS5A or cellular lipids (BODIPY) was evaluated in Huh-7 cells transfected with in vitro transcripts of a transient luciferase subgenomic replicon (JFH-1: genotype 2a) after fixation and permeabilization. Cells were stained for lipid content with BODIPY dye for 1 h after NS5A labeling. Identical settings were maintained for image capturing. Representative confocal images are shown. Cells were left untreated (Control) or treated with AKTVIII for 4 h before processing. Replicon transfected cells are outlined in white. (Scale bars, 10 μm.) 1. Targett-Adams P, McLauchlan J (2005) Development and characterization of a transient-replication assay for the genotype 2a hepatitis C virus subgenomic replicon. J Gen Virol 86: 3075–3080. Mankouri et al. www.pnas.org/cgi/content/short/0912426107 2 of 4 Anti-myc (AMPK) Anti-NS5A Merge A Wildtype B S485D C S485A Fig. S3. Expression of an exogenous AMPK Ser485Ala mutant blocks HCV RNA replication. Huh-7 cells stably harboring an HCV genotype 1b culture-adapted subgenomic replicon (1) were transfected with the indicated Myc-tagged AMPKα constructs and analyzed by immunofluorescence 48 h posttransfection. Representative confocal images are shown. The upper row of images in A–C shows a wide-field image. (Scale bars, 10 μm.) 1. Krieger N, Lohmann V, Bartenschlager R (2001) Enhancement of hepatitis C virus RNA replication by cell culture-adaptive mutations. J Virol 75:4614–4624. Mankouri et al. www.pnas.org/cgi/content/short/0912426107 3 of 4 A JFH-1 B NS5A BODIPY Merge Released virus Intracellular virus 40 60 80 100 % in fe ct iv it y p ar ed t o c o n tr o l) 40 60 80 100 in fe ct iv it y ar ed t o c o n tr o l) 0 20 % (c o m p 0 20 % (c o m p Control AICAR A769662 Metformin Control AICAR A769662 Metformin Fig. S4. Restoration of AMPK activity with AMPK agonists inhibits assembly and release of infectious genotype 2a (JFH-1) virus. (A) Additional images of Huh- 7 cells transfected with full-length in vitro transcripts of JFH-1. Cells were stained for lipid content with BODIPY dye for 1 h after NS5A labeling. Identical settings were maintained for image capture. Representative confocal images are shown. JFH-1 transfected cells are outlined in white; untransfected cells in the same field are outlined in red. (Scale bars, 10 μm.) (B) Huh-7 cells were infected with JFH-1 virus at a multiplicity of infection of 0.5 focus-forming units per cell. At 24 h posttransfection, the indicated AMPK agonists were added. Virus in both supernatant and clarified cell lysates was titered after a further 24-h incubation (n = 3). All error bars indicate mean ± SEM. Mankouri et al. www.pnas.org/cgi/content/short/0912426107 4 of 4