Hepatitis C Virus-Linked Mitochondrial Dysfunction PromotesHypoxia-Inducible Factor 1�-Mediated Glycolytic Adaptation�†
Maria Ripoli,1 Annamaria D’Aprile,1 Giovanni Quarato,1 Magdalena Sarasin-Filipowicz,2
Jerome Gouttenoire,3 Rosella Scrima,1 Olga Cela,1 Domenico Boffoli,1 Markus H. Heim,2Darius Moradpour,3 Nazzareno Capitanio,1 and Claudia Piccoli1*
Department of Biomedical Sciences, University of Foggia, Foggia, Italy1; Division of Gastroenterology and Hepatology,University Hospital Basel, Basel, Switzerland2; and Division of Gastroenterology and Hepatology,
Centre Hospitalier Universitaire Vaudois, University of Lausanne, Lausanne, Switzerland3
Received 16 April 2009/Accepted 9 October 2009
Hepatitis C virus (HCV) infection induces a state of oxidative stress by affecting mitochondrial-respiratory-chain activity. By using cell lines inducibly expressing different HCV constructs, we showed previously thatviral-protein expression leads to severe impairment of mitochondrial oxidative phosphorylation and to majorreliance on nonoxidative glucose metabolism. However, the bioenergetic competence of the induced cells wasnot compromised, indicating an efficient prosurvival adaptive response. Here, we show that HCV proteinexpression activates hypoxia-inducible factor 1 (HIF-1) by normoxic stabilization of its � subunit. In conse-quence, expression of HIF-controlled genes, including those coding for glycolytic enzymes, was significantlyupregulated. Similar expression of HIF-controlled genes was observed in cell lines inducibly expressingsubgenomic HCV constructs encoding either structural or nonstructural viral proteins. Stabilization andtranscriptional activation of HIF-1� was confirmed in Huh-7.5 cells harboring cell culture-derived infectiousHCV and in liver biopsy specimens from patients with chronic hepatitis C. The HCV-related HIF-1� stabili-zation was insensitive to antioxidant treatment. Mimicking an impairment of mitochondrial oxidative phos-phorylation by treatment of inducible cell lines with oligomycin resulted in stabilization of HIF-1�. Similarresults were obtained by treatment with pyruvate, indicating that accumulation of intermediate metabolites issufficient to stabilize HIF-1�. These observations provide new insights into the pathogenesis of chronichepatitis C and, possibly, the HCV-related development of hepatocellular carcinoma.
Hepatitis C virus (HCV) infection is a major cause ofchronic liver disease worldwide (43). Its progression to cirrho-sis and hepatocellular carcinoma (HCC) may take decades, butthe precise pathogenic mechanisms are unknown (20). HCV isa positive-strand RNA virus with a 9.6-kb genome encoding alarge polyprotein (2). This is translated on the endoplasmicreticulum (ER) and processed by viral and host proteases into10 individual membrane-associated proteins comprising struc-tural (core, E1, and E2) and nonstructural (p7 and NS2 toNS5B) proteins (28). Evidence derived from experimental sys-tems, as well as from liver biopsy specimens from patients withchronic hepatitis C (CHC), point to HCV-related dysfunctionof mitochondria (32, 33, 47).
Warburg first proposed that the prime cause of cancer wasimpaired energy metabolism (50), which involved irreversibleinjury to cellular respiration followed by a gradual dependenceon fermentation energy to compensate for the lost energy frommitochondrial respiration. Indeed, elevated glycolysis is themetabolic hallmark of nearly all tumors (23). Although War-burg’s hypothesis has been overshadowed by support for themutational theory of carcinogenesis, his original observations
still have merit, even though the exact mechanisms have yet tobe clarified.
In mammalian cells, an essential control element of themetabolic state is hypoxia-inducible factor 1 (HIF-1) (25). Awell-defined oxygen-sensitive pathway regulates the activity ofHIF-1 by posttranslational prolyl hydroxylation within the �subunit (38). Since the HIF prolyl hydroxylases (PHDs) havean absolute requirement for molecular oxygen, this process issuppressed in hypoxia, allowing HIF-1� to escape its degrada-tion route (46) and to activate transcription. However, non-hypoxia-related factors have been shown to contribute to theactivation of HIF-1, providing additional interfaces that maybe important in regulating cellular stress adaptation (6). Al-most all the enzymes of the glycolytic pathway are encoded bygenes whose expression is under the control of HIF-1 (40). Incancer, the metabolic phenotype is activated by a variety ofgenetic and environmental mechanisms, most strikingly by sta-bilization of HIF-1 (34), which accounts for the classical tu-mor-associated properties of deregulated glycolysis and angio-genesis (34, 40).
In a recent report (29), it was shown that HCV infectionelicits oxidant-mediated HIF-1� stabilization, leading to ex-pression of vascular endothelial growth factor (VEGF). In thepresent study, we show that long-term HCV protein expressioncauses depression of mitochondrial oxidative phosphorylation(OXPHOS). Cell survival, however, is preserved by enhancednonoxidative glucose utilization. Consistent with the results ofMoradpour et al. (28), this adaptive response to HCV-induced
* Corresponding author. Mailing address: Department of BiomedicalSciences, University of Foggia, Via L. Pinto 1-OO.RR.-71100 Foggia,Italy. Phone: 39-0881-711148. Fax: 39-0881-714745. E-mail: [email protected].
† Supplemental material for this article may be found at http://jvi.asm.org/.
� Published ahead of print on 21 October 2009.
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mitochondrial injury proved to be mediated by stabilization ofHIF-1� and, in consequence, upregulation of glycolytic en-zymes. Comparable results were obtained in two different cellculture systems and, more importantly, in liver biopsy speci-mens from HCV-infected patients. Further, our study suggeststhe involvement of intermediate-metabolite-mediated inhibi-tion of HIF-1 �-prolyl hydroxylation as a cause of the observedHCV-related alterations, and it provides novel insights into thepathogenesis of HCC.
MATERIALS AND METHODS
Cell lines. UHCVcon-57.3 is a U-2 OS human osteosarcoma-derived tetracy-cline-regulated cell line inducibly expressing the HCV polyprotein (37). Cell linesUCp7con-9.10 and UNS3-5Bcon-27 inducibly express the HCV structural pro-teins and p7 or the nonstructural proteins 3 to 5B, respectively, derived from theHCV H consensus clone (15; D. Moradpour, unpublished data). As a control, theUGFP-9.22 cell line inducibly expressing the nonrelevant green fluorescent pro-tein (GFP) was used (37). Inducible cell lines were cultured in complete Dul-becco’s modified Eagle’s medium (DMEM) containing 5.5 mM glucose or, when
indicated, 10 mM galactose. Huh-7.5 human HCC cells (3) (kindly provided byCharles M. Rice, The Rockefeller University, New York, NY) were transfectedby microelectroporation (Digital Bio Technology, Seoul, Korea) with a J6-JFH-1(Jc1) chimeric full-length HCV genome harboring a GFP insertion in domain IIIof NS5A, as described previously (36) (kindly provided by R. Bartenschlager,University of Heidelberg, Heidelberg, Germany).
Jc1 infection of Huh-7.5 cells. Huh-7.5 cells were electroporated with invitro-transcribed Jc1 RNA. Supernatants were collected 48 h postelectropora-tion, and 50% tissue culture infectious doses were determined as describedpreviously (19). The Huh-7.5 cells were infected with Jc1 virus at a multiplicity ofinfection of about 7.5. Four days postinfection, the cells were lysed in a buffercontaining 150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% sodium dodecylsulfate (SDS), 50 mM Tris-Cl, pH 8.0, and protease inhibitors.
Subcellular fractionation. For mitoplast (i.e., an isolated mitochondrion de-void of the outer mitochondrial membrane) preparation, UHCVcon-57.3 cellswere harvested with 0.05% trypsin and 0.02% EDTA and washed in phosphate-buffered saline (PBS), pH 7.4, with 5% calf serum. The cells in PBS were exposedfor 10 min on ice to 0.2 mg of digitonin/mg cellular protein. The mitoplasts werepelleted at 14,000 � g and resuspended in PBS. For the mitochondrion-enrichedfraction, the harvested UHCVcon-57.3 cells were subjected to nitrogen cavita-tion in a cell disruption bomb (Parr Instrument Company, Moline, IL; model4639). Briefly, 20 � 106 to 30 � 106 cells were suspended in 1 to 2 ml of 250 mMsucrose, 1 mM EGTA, 5 mM HEPES, 3 mM MgCl2, 40 �l of protease inhibitor
FIG. 1. Effect of long-term HCV protein expression on the mitochondrial OXPHOS system in UHCVcon-57.3 cells. (A) Respiratory activityof intact UHCVcon-57.3 cells. Measurements of oxygen consumption were performed by high-resolution respirometry, as described in Materialsand Methods. Repression of HCV protein expression is indicated as � Tet (i.e., medium supplemented with 1 �g/ml tetracycline); derepressionof HCV proteins is indicated as � Tet (i.e., medium without tetracycline). The incubation time is also shown. End. resp., endogenous respiration;End. resp. � oligom., respiratory activity in the presence of oligomycin. The values represent averages of six independent cell preparations plusstandard errors of the mean (SEM); the P values reported are versus the non-HCV-induced condition. (B) Mitochondrial OXPHOS complexactivities. Specific activities of complex I (CI), CIII, CIV, and CV were measured by spectrophotometrical assays as specified in Materials andMethods. Empty bars, noninduced cells; light-gray and dark-gray bars, cells induced for 2 and 5 days, respectively. The activities of the complexesfrom induced cells were normalized to the values of control cells. The absolute activities were 3.60 nmol NADH/min/106 cells for CI; 3.43 nmolcytochrome c reduced/min/106 cells for CIII, 8.46 nmol cytochrome c oxidized/min/106 cells for CIV, and 6.09 nmol ATP/min/106 cells for CV. Thevalues represent averages of four independent experiments plus SEM. The P value is also shown where significant. (C) Western blotting of HCVproteins in total cell lysates and mitochondrion-enriched fractions. Fifty micrograms of proteins was loaded on the gels in all cases. �-Actin, the�-subunit of the FoF1-ATP synthase (�-CV), and the Ca2� pump ATPase (SERCA) were taken as cytosol, mitochondrial, and ER markers,respectively. The results are representative of two experiments. (D) Cell growth analysis. Light microscopy images of noninduced (� Tet) cells andcells induced (� Tet) for 1, 2, and 5 days. Cell numbers are shown. The results are averages of five independent assays � SEM; the statisticalsignificance versus control cells is shown.
648 RIPOLI ET AL. J. VIROL.
cocktail (Roche), pH 7.4, and equilibrated for 20 min in the bomb at a nitrogenpressure of 700 lb/in2. Following rapid decompression, the ejected disrupted cellswere centrifuged at 400 � g for 5 min. The pelleted undisrupted cells wereresuspended and subjected to a second cycle of nitrogen compression-decom-pression. The pooled supernatants were centrifuged at 600 � g for 5 min toremove nuclei and cell debris and further centrifuged at 12,000 � g for 15 min.The pelleted mitochondrion-enriched fraction was resuspended in 50 to 100 �l of250 mM sucrose, 0.2 mM EDTA, 10 mM Tris, pH 7.8. The protein contents ofthe cell suspension and subcellular fractions were assayed by the Bradfordmethod using albumin as a standard.
Patients. From March 2007 to October 2007, patients with CHC referred tothe outpatient liver clinic of the University Hospital Basel were asked for per-mission to use part of their diagnostic liver biopsy specimens for research pur-poses. Patients’ biopsy specimens with no histological signs of cirrhosis (or otherinflammatory alterations) were selected. All of the patients were Caucasians. Theprotocol was approved by the Ethics Committee of the University Hospital Basel.Written informed consent was obtained from all patients. As non-CHC controls,14 patients who underwent ultrasound-guided liver biopsies of focal lesions gaveinformed consent for a biopsy from the normal liver tissue outside the focallesion. These control patients had normal liver values, and the absence of liverpathology was histologically confirmed.
Measurements of cell respiration, respiratory complexes, and FoF1-ATPaseactivity. Cells were trypsinized, washed in PBS, and immediately assessed for O2
consumption by high-resolution respirometry (Oroboros Instruments) as de-scribed previously (33). The activities of complexes I, III, and IV were assayed asdescribed previously (33). Complex V activity was measured spectrophotometri-cally by a coupled assay of the mitoplast fraction of ultrasound-treated cells in 50mM Tris, 5 mg/ml serum albumin, 20 mM MgCl2, 50 mM KCl, pH 8.0, supple-mented with 0.75 �M antimycin A, 1 �M carbonyl cyanide m-chlorophenylhy-drazone, 1.5 mM phosphoenolpyruvate, 2.5 units/ml of lactate dehydrogenase(LDH), 1.12 units/ml of pyruvate kinase, 350 �M ATP. The reaction was startedby adding 50 �M NADH, whose oxidation was recorded from the absorbancedecrease at 340 nm.
Measurement of metabolites. Cellular ATP was extracted by a one-step pro-cedure in boiling water and assayed by bioluminescence using a luciferin-lucif-erase reaction system as described previously (33). Lactate was assayed spectro-photometrically in culture medium and quantified by comparison with acalibration curve using standard titrated lactate according to the manufacturer’sinstructions (Vinci-Biochem). The values reported were normalized to the cellnumber.
Measurement of reactive oxygen species (ROS) production. Huh-7.5 cells weresuspended at 0.25 � 106 cells/ml in DMEM plus 10% fetal bovine serum andplaced in the spectrofluorometric cuvette with stirring. The instrument settingswere as follows: �ex 485 nm and �em 535 nm. To remove the greenfluorescence background of the transfected Huh-7.5 cells harboring the GFPinsert, the fluorescence was instrumentally zeroed, and then 10 �M 2,7-dichloro-fluorescein diacetate (DCF-DA) was added. DCF-DA is not fluores-cent, but following its entry into the cell, it is hydrolyzed by intracellular esteraseto generate DCF, which becomes fluorescent following reaction with hydrogenperoxides. The fluorescence changes were recorded for 30 min.
Immunoblotting. Cells were resuspended in lysis buffer (20 mM HEPES, pH7.2, 150 mM NaCl, 1 mM EGTA, 10% glycerol, 1% Triton X-100, 1.5 mMMgCl2, 2 mM sodium phosphate, and protease inhibitor cocktail). The lysateswere run on a 10% SDS-polyacrylamide gel electrophoresis gel, and Westernblotting was performed by standard transferring procedures. The polyvinylidenedifluoride membranes were incubated with monoclonal antibody (MAb) 5B-3B1against HCV NS5B (26), MAb C7-50 against HCV core (27), MAb 11H againstHCV NS5A (4), MAb 1B6 against HCV NS3 (51), 1:500 rabbit anti-HIF-1�antibody (Santa Cruz Biotechnology), 1:10,000 mouse anti-�-actin (Sigma), and1:300 rabbit anti-SERCA (Santa Cruz Biotechnology) overnight at 4°C andfinally treated with 1:8,000 horseradish peroxidase-conjugated secondary anti-body (Santa Cruz Biotechnology) before analysis with a chemiluminescence kit(Pierce) using the VersaDoc Imaging System (Bio-Rad).
Immunocytochemistry and LSCM analysis. U-2 OS-derived inducible celllines or transfected Huh-7.5 cells were seeded onto fibronectin-coated glassbottom dishes. Then, samples were fixed, permeabilized, blocked, and incubatedwith 1:100-diluted rabbit anti-human HIF-1� (Acris) overnight at 4°C. Afterbeing washed in PBS-bovine serum albumin, the samples were incubated with 8�g/ml of fluorescein isothiocyanate-labeled goat anti-rabbit immunoglobulin Gor rhodamine-labeled goat anti-mouse immunoglobulin G (Santa Cruz Biotech-nology). ROS production was evaluated by treating the cells with 10 �MDCF-DA as described previously (33). After being washed in PBS, the cells wereimmediately analyzed. Imaging of labeled cells was performed with a Nikon TE
2000 microscope (the images were collected using a 60� objective, 1.4 numericalaperture) coupled to a Radiance 2100 laser scanning confocal microscopy(LSCM) system (Bio-Rad). Acquisition, storage, and analysis of data were doneusing LaserSharp and LaserPix software from Bio-Rad or ImageJ version 1.37.
RNA extraction, RT, and qRT-PCR. Total cellular RNA was isolated from cellcultures with an Absolutely RNA miniprep kit (Stratagene) with an on-columnDNase treatment. First-strand cDNA synthesis was carried out using 300 ng ofrandom hexamer primers (Invitrogen) by Accuscipt High Fidelity Reverse Trans-criptase (Stratagene) and Ribolock RNase Inhibitor (Fermentas), starting from1 �g RNA. Reverse transcription (RT) quantification was performed with 1.5 �lcDNA using Brilliant SYBR green QPCR Master Mix (Stratagene) in a 25-�lreaction volume on Mx3000P (Stratagene) with 300 nM primers (see Table S1 inthe supplemental material). The quantification of transcript abundance in dere-pressed cells was done using the ��CT method, with the repressed cells as thereference sample (calibrator) and �-actin as the internal control. For liver biopsyspecimens from CHC and control patients, samples were stored at �75°C afterhaving been stabilized in RNAlater solution (Ambion/Applied Biosystems, Rot-kreuz, Switzerland) at 4°C overnight. Total RNA was extracted from liver sam-ples using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s in-structions. RNA was aliquoted and stored at �75°C. The RNA was reversetranscribed with Moloney murine leukemia virus reverse transcriptase (PromegaBiosciences, Inc., Wallisellen, Switzerland) in the presence of random hexamers(Promega) and deoxynucleotide triphosphates. The reaction mixtures were in-cubated for 5 min at 70°C and then for 1 h at 37°C and were stopped by heatingthem at 95°C for 5 min. The SYBR PCRs were performed using the SYBR greenPCR master mix (Applied Biosystems) and primers (data not shown), Thedifference in the cycle threshold (�CT) value was derived by subtracting the CT
value for GAPDH (glyceraldehyde 3-phosphate dehydrogenase), serving as aninternal control, from the CT values for the transcripts of interest. All reactions
FIG. 2. Effect of long-term HCV protein expression on cell bioen-ergetics. (A) Intracellular ATP content. The ATP contents of celllysates were measured as described in Materials and Methods. Whereindicated, glucose or galactose was the main carbon source in theDMEM base culture medium. Noninduced cells, � Tet (gray bars);5-day-induced cells, � Tet (black bars). Each bar represents the aver-age of five independent measurements plus the standard error of themean (SEM) normalized to the ATP content measured in noninducedcells. The absolute mean values of ATP in noninduced cells were 5.9and 8.4 nmol/mg protein in cells grown in glucose and galactose media,respectively. The statistical differences in ATP content between in-duced and noninduced cells are also shown. (B) Measurements oflactate in noninduced (gray bar) and 5-day-induced (black bar) UHCVcon-57.3 cells. The metabolite content was evaluated spectrophotometri-cally on cell culture media as described in Materials and Methods. Thevalues reported in the histogram are the averages of three independentexperiments plus SEM, along with the statistical difference.
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were run in duplicate, using an Applied Biosystems Prism 7000 Sequence De-tection System. mRNA expression levels were calculated relative to GAPDHfrom the �CT values, using the formula 2��CT. The primer pairs used forquantitative RT (qRT)-PCR were for HIF-1�, LDH-A, hexokinase 1 (HK1),VEGF, VHL, and PHD2; �-actin or GAPDH amplicons were used as internalcontrols.
Statistical analyses. The two-tailed Student’s t test was applied, with a P valueof �0.05, to evaluate the statistical significance of differences measured through-out the data sets from cell culture experiments. For statistical analyses of themRNA levels in human liver biopsy specimens, the two-tailed Mann-Whitneytest was used.
RESULTS
Effect of long-term HCV protein expression on the mito-chondrial OXPHOS system. Previously, we reported the im-pact of 2 days of induction of HCV protein expression on
mitochondrial-respiratory-chain (RC) activity and OXPHOSefficiency (33). In this study, we extended these observations toevaluate longer-term effects. Figure 1A shows the results of arespirometric analysis carried out on stably transfected intactU-2 OS-derived cell lines. Induction of HCV proteins for 2 and5 days resulted in the following similar outcomes: (i) 40%inhibition of the resting O2 uptake, (ii) a slight increase inrespiratory activity in the presence of oligomycin, and (iii) amarked reduction of the respiratory control ratio (RCR) from5 to 2.
The mitochondrial membrane potential (mt� ) is known tocontrol the rate of O2 consumption at the level of the RC.Thus, in the presence of oligomycin, which inhibits mt� -consuming H�-ATP synthase, a rise in the mt� and, conse-quently, a reduction of the respiratory rate are expected. The
FIG. 3. Effect of HCV protein expression on HIF-1� in U-2 OS-derived inducible cell lines. (A) HIF-1� protein expression. The three imagesat the top left are representative images (from four independent experiments) of immunofluorescence confocal microscopy analysis to detectHIF-1�. Noninduced UHCVcon-57.3 cells, �Tet; 5-day-induced cells, �Tet; noninduced cells treated with 10 mM desferroxiamine for 2 h, �Tet �DFX. The secondary fluorescein isothiocyanate-conjugated antibody (2nd-Ab-FITC), when added directly to the cell sample without the primaryanti-HIF-1�, did not result in a detectable fluorescence signal (not shown). The inset in the image relative to induced cells shows a doubleimmunodetection, in a parallel sample, of HIF-1� and the HCV NS5B protein. The latter was immunodetected using a secondary rhodamine-conjugated antibody (red fluorescence). Bars, 10 �m. A representative immunoblot (Western blot [WB]) analysis of HIF-1� and HCV NS5Aprotein expression on cell lysates under identical conditions is also shown. The histogram on the right displays the quantitative analysis of theimmunofluorescence intensity (gray bars) assessed by averaging 20 to 30 single cells from at least 10 different optical fields; the mean values fromfour different cell preparations for each condition are shown, along with statistical analysis. The histogram also shows the result of densitometricanalysis (black bars) of the WB HIF-1� band normalized to the internal �-actin control. The average value plus standard error of the mean (SEM)of three independent immunoblot assays is shown, along with statistical analysis. (B) Transcript levels of HIF-1-controlled genes. The transcriptlevels of HIF-1�, VHL, LDH-A, HK1, and VEGF genes were quantified by real-time RT-PCR as described in Materials and Methods. The analysiswas carried out on U-2 OS-derived cell lines cultured for 5 days in the absence of tetracycline to express the entire HCV polyprotein(UHCVcon-57.3); the structural proteins, as well as p7 (UCp7con-9.10); or the nonstructural proteins 3 to 5B (UNS3-5Bcon-27). The expressionlevel of each gene is shown as a percentage of that attained in noninduced cells and represents the average of three independent experiments plusSEM, along with statistical analysis.
650 RIPOLI ET AL. J. VIROL.
RCR (attained by dividing the resting rate of respiration bythat in the presence of oligomycin) is an index of the OXPHOSefficiency. In HCV-induced U-2 OS-derived cells, the occur-rence of a combined effect contributed by inhibition of the RCactivity and by a dissipatory pathway for mt� was observed.The consequent depression of the RCR indicated a severeimpairment of the OXPHOS yield.
Analysis by blue native/SDS bidimensional polyacrylamidegel electrophoresis of the protein profile of the mitoplastsshowed no significant change either in the amount or in thecomposition of the OXPHOS complexes between noninducedand induced cells (data not shown). The measurement of thespecific activities of the OXPHOS complexes revealed 40%and 50% inhibition of complex I and FoF1-ATPase, respec-tively, at both 2 and 5 days of HCV protein induction, whereasthe activities of cytochrome c reductase and cytochrome coxidase remained unchanged (Fig. 1B). Immunoblots of theHCV core, NS3, NS5A, and NS5B proteins verified their ex-pression following the 5-day induction protocol and displayed,in addition, a significant accumulation of HCV core and NS5A,and to a lesser extent of NS3 and NS5B, in mitochondria asassessed by comparing their relative amounts in cell lysates andorganelle-enriched fractions (Fig. 1C). The effect of HCV pro-tein expression on cell growth was evaluated by cell densityanalyses (Fig. 1D), demonstrating significant changes from thesecond day of induction compared to noninduced cells. How-ever, cell growth was not as severely depressed as expectedfrom the extent of the mitochondrial OXPHOS crisis.
Effect of long-term HCV protein expression on cell bioener-getics. The effect of HCV protein expression on cell bioener-getics was assessed by measuring the steady-state intracellularlevel of ATP. Figure 2A shows that 5 days of HCV protein
induction resulted in an 80% increase in ATP relative to non-induced cells. This effect depended on the energy substratepresent in the culture medium. Indeed, replacing glucose withgalactose (a condition forcing cells to rely largely on mitochon-drial OXPHOS [35]) resulted in a significant 30% decrease inthe ATP content in HCV-induced cells. Consistently, the growthof HCV-induced cells in galactose-based medium was largelyinhibited compared to that of cells grown in glucose (data notshown). Measurement of the amount of lactate released into aglucose-based medium following 5 days of HCV protein induc-tion resulted in an almost threefold increase relative to non-induced cells (Fig. 2B). This observation indicates an HCVprotein-induced metabolic shift toward glycolysis.
HCV proteins induce HIF-1� stabilization under normoxicconditions. To explain the glycolytic phenotype in the HCVprotein-expressing cells, we postulated an upregulation of theglycolytic enzymes, whose expression is largely controlled byHIF-1. Therefore, we analyzed by LSCM the level and subcel-lular localization of the HIF-1� subunit. As shown in Fig. 3A,5 days of HCV protein induction resulted in a 2.4-fold en-hancement of the HIF-1�-related immunofluorescence, whichlargely localized to the nuclear compartment. Immunoblot anal-yses of cell lysates confirmed the HCV protein-dependent up-regulation of HIF-1�. Treating noninduced cells with the hy-poxia-mimetic desferroxiamine (or CoCl2 [not shown]) resultedin comparable outcomes. Of note, the HIF-1� stabilization ininduced cells occurred under normal oxygen tension, thus sug-gesting a hypoxia-independent mechanism of HIF-1� stabili-zation. After 2 days of HCV protein induction, no significantincrease in HIF-1� was observed. However, it became clearlydetectable after 4 days of induction (data not shown). This
FIG. 4. Effects of GFP expression in U-2 OS-derived inducible cell lines. UGFP-9.22 cells were cultured in the presence (�) or absence (�)of tetracycline (Tet) for 5 days. (A) Confocal microscopy analysis showing GFP expression and mt� generation in mitochondria assessed by theprobe TMRE. Bars, 10 �m. (B) Western blot (WB) of HIF-1�. The blot is representative of three independent experiments. (C) Transcript levelsof HIF-1-controlled genes. The transcript levels of HIF-1�, VHL, LDH-A, HK1, and VEGF genes were quantified by real-time RT-PCR asdescribed in Materials and Methods. The expression level of each gene is shown as a percentage of that attained in noninduced cells and representsthe average of three independent experiments plus the standard error of the mean (P � 0.05 for all the genes).
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indicated the need for a progressive setting of conditions forHIF-1� stabilization.
HCV proteins induce upregulation of HIF-controlled genes.To assess whether the HCV protein-linked HIF-1� stabiliza-tion was functional, we measured the transcriptional level ofknown HIF-controlled genes by qRT-PCR. Figure 3B shows asignificant 2- to 2.5-fold increase relative to noninduced cells ofthe glycolytic enzymes LDH-A and HK1, as well as of VEGF,encoded by a well-characterized HIF target gene (25). Ofnote, the transcript level of HIF-1� itself and that of theVHL protein, which controls HIF-1� stability (46), were un-affected by the viral-protein expression, suggesting HCV-linkedregulation of HIF-1 at the posttranslational level.
To obtain insights into the possible involvement of a specificHCV protein in HIF activation, we examined U-2 OS-derived
cell lines inducibly expressing either the structural or the non-structural proteins. No significant difference in the induction ofthe HIF-controlled genes between the UHCVcon-57.3,UCp7con-9.10, and UNS3-5Bcon-27 cell lines was observed(Fig. 3B). Hence, these results did not allow us to attribute theobserved effect to either the structural or nonstructural region.
To confirm the specificity of our observations, we used a U-2OS-derived cell line inducibly expressing the GFP as a nonrel-evant control (Fig. 4A). After 5 days of GFP induction, thelevels of HIF-1� protein and of the related gene expressionwere comparable in cells cultured in the presence and absenceof tetracycline, as shown in Fig. 4B and C. Moreover, theexpression of the GFP did not cause any mitochondrial dys-function, as assessed by probing the mt� (Fig. 4A).
To verify that the observed HCV protein-dependent activa-
FIG. 5. Effect of HCV protein expression on the activity of HIF-1 in transiently transfected Huh-7.5 cells. (A) HIF-1� immunofluorescenceconfocal microscopy analysis. Representative images of control and Jc1-GFP RNA-transfected Huh-7.5 cells are shown illustrating the merge fromthe rhodamine (R)-related HIF-1� immunodetection and the GFP-related fluorescence signal; the latter was used to track HCV-transfected cells(�95% of the cell population). Control Huh-7.5 cells were subjected to the transfection protocol but without HCV RNA. The time point aftertransfection was 72 h. see Materials and Methods for further details. Bars, 10 �m. Enlargements with false-color imaging of the original picturesare also shown to better visualize the nuclear localization of HIF-1�. On the right, a histogram of the HIF-1�-related fluorescence in GFP-positivetransfected cells compared to control cells is shown. The fluorescence intensity was assessed by averaging 10 to 20 cells from each of at least 10optical fields. The average of four independent experiments plus the standard error of the mean (SEM), along with statistical analysis, is shown.A density profile of the HIF-1�-related immunofluorescence signal is representatively shown along two lines crossing two selected control andtransfected cells. A representative Western blot (WB) of HIF-1� on cell lysates of parallel samples is also shown. A.U., arbitrary units. (B) Effectof HCV protein expression on the transcript level of HIF-1-controlled genes in transfected Huh-7.5 cells. The transcript levels of HIF-1�, VHL,PHD2, LDH-A, HK1, and VEGF genes were quantified by real-time RT-PCR as described in Materials and Methods. The expression level of eachgene is shown as a percentage of that attained in nontransfected control cells and represents the average of three independent experiments plusthe standard error of the mean, along with statistical analysis.
652 RIPOLI ET AL. J. VIROL.
tion of HIF-1 was not limited to a specific in vitro cell system,we extended the analyses to Huh-7.5 human HCC cells elec-troporated with a GFP-tagged HCV genome allowing the fullreplication cycle and production of infectious virus (37, 3). Asshown in Fig. 5A, Huh-7.5 cells harboring HCV displayed bothstabilization and nuclear localization of HIF-1�, as assessed byWestern blotting and immunofluorescence microscopy, respec-tively. qRT-PCR analyses confirmed a significant upregulationof HK1 and VEGF, but not of LDH-A (Fig. 5B). Of note, thetranscript levels of HIF-1�, PHD2 (isoform 2 of PHD), andVHL were all significantly enhanced compared to nontrans-fected cells. Furthermore, normoxic stabilization of HIF-1�was also verified in Huh-7.5 cells infected with cell culture-derived HCV, resulting in a 4.5-fold increase in the proteinlevel assayed 4 days after infection (Fig. 6).
HCV-linked activation of HIF-1 is independent of the redoxstate of the cell. Among the nonhypoxic factors known tostabilize HIF-1� are ROS (6). Since expression of HCV pro-teins was shown to cause enhanced mitochondrial ROS pro-duction, we tested their involvement in the upregulation ofHIF-1�. The results shown in Fig. 7 ruled out this possibility.Indeed, in spite of the clear antioxidant effects exerted byN-acetyl-cysteine (NAC) or by Tiron on the HCV-dependentredox alteration (Fig. 7A), no significant change in the HIF-1aprotein or in the expression of HK1, LDH-A, and VEGF wasobserved in induced cells treated with both ROS scavengers(Fig. 7B). HCV protein expression is linked to deregulation ofthe calcium fluxes between ER and mitochondrial stores (21,33). Increased mitochondrial calcium influx is known to acti-vate a mitochondrial isoform of nitric oxide synthase and therebyto cause nitric oxide release (33), which has been reported tostabilize HIF-1 (5). Furthermore, activation of mitogen-acti-vated protein kinase and phosphatidylinositol 3-kinase (PI3K)
signaling pathways was shown to induce HIF-1 (9). However,treatment of UHCVcon-57.3 cells with inhibitors of the ERCa2� channel (dantrolene), mitochondrial Ca2� uniporter (ru-thenium red), nitric oxide synthase (N-methyl-L-arginine [L-NNMA]), and the PI3K/Akt pathway (wortmannin) were in-effective in modifying the upregulated expression profile ofHIF-1 target genes (Fig. 7C). Importantly, the inefficiency ofROS scavenging in preventing the HCV protein-related up-regulation of HIF was also fully confirmed in Huh-7.5 cellstransfected with the GFP-tagged genome, as shown in Fig. 8Ato C. Under conditions that prevented ROS generation intransfected Huh-7.5 cells (measured by probe-assisted fluorim-etry), both NAC and Tiron had no significant effect on thestabilization of HIF-1� protein and on the transcription of theHIF-controlled VEGF gene.
Stabilization of HIF-1� is elicited by mimicking the HCV-induced deregulation of the mitochondrial OXPHOS system.To clarify the cause-effect relationship between the observednormoxic stabilization of HIF-1� and the metabolic changecaused by HCV protein expression, we mimicked an impair-ment of the mitochondrial OXPHOS system in noninducedUHCVcon-57.3 cells and assessed its impact on the HIF sys-tem. As shown in Fig. 9, normoxic treatment of UHCVcon-57.3 cells with a subcytotoxic concentration of oligomycin, aspecific inhibitor of the mitochondrial ATP synthase, resultedin a remarkable stabilization and nuclear localization of HIF-1�. As inhibition of the terminal catabolic flux is expected tocause accumulation of intermediate metabolites, we tested theeffect of pyruvate on noninduced UHCVcon-57.3 cells. Figure9 shows that incubation with a concentration of pyruvate, in arange compatible with its physiologic accumulation in prolif-erating glycolytic cells (49), caused stabilization of HIF-1� asobserved in cells expressing HCV proteins. The effect of oli-gomycin or pyruvate treatment was also tested in Huh-7.5 cells,confirming their capability to induce stabilization of HIF-1� tolevels comparable to that attained following HCV transfection(Fig. 10A and B).
Upregulation of HIF-1�, VEGF, HK-1, and VHL, but notLDH-A, in liver biopsy specimens from patients with CHC. Tovalidate the HCV-linked upregulation of HIF-1 observed in invitro cell culture systems, we extended our analyses to humanliver biopsy specimens. A cohort of 19 patients affected by CHCwas selected and compared with 14 control patients. Figure 11shows the comparative results of qRT-PCR analyses. It showsthat the levels of HIF-1�, VHL, HK1, and VEGF transcriptswere significantly higher in the liver biopsy specimens fromHCV-infected patients than in the controls. Conversely, nosignificant difference was observed for the LDH transcripts.Interestingly, the differential expression profile observed in theHCV-infected patients closely resembled that described abovein Huh-7.5 cells harboring cell culture-derived HCV (Fig. 5B).Importantly, a statistical analysis comparing the HIF-1� ex-pression levels and the inflammatory grade (using the Metavirgrading/staging system) allowed us to exclude any correlationbetween them (data not shown).
DISCUSSION
HCV causes impairment of mitochondrial OXPHOS andinduces a compensatory bioenergetic adaptation. HCV infec-
FIG. 6. Stabilization of HIF-1� in HCV-infected Huh-7.5 cells.Huh-7.5 cells were infected with Jc1 HCV as described in Materialsand Methods and analyzed 4 days after infection. (Top) Micrographsof cells stained for HCV core protein, as revealed by MAb C7-50 anda horseradish peroxidase-conjugated secondary antibody. (Bottom)Representative (out of three independent preparations) Western blots(WB) of cell lysates for HCV core, HIF-1�, and �-actin. Relativedensitometric values for HIF-1� normalized to �-actin are indicated.
VOL. 84, 2010 HCV INFECTION STABILIZES HIF-1� 653
tion is associated with mitochondrial stress. In a previousstudy, we showed that the expression of the HCV proteinselicits a sequence of events primed by enhanced uptake ofCa2� into mitochondria (33). The increased intramitochon-drial concentration of Ca2� was found to stimulate nitric oxideproduction and to inhibit RC complex I. This led to a progres-sive inhibition of the RC activity and of the mt� , accompa-nied by production of ROS. Interestingly, the ATP content ofHCV protein-expressing cells was found to be increased de-spite these profound alterations.
To obtain insights into this cell adaptive response, we extendedour studies to cells expressing HCV proteins for a longer time.The results obtained confirmed the remarkable depression of theOXPHOS system, further characterized by reduced oligomycin-sensitive FoF1 ATPase activity (Fig. 1A and B). No compen-satory upregulation of the biogenesis of the OXPHOS com-plexes was observed. In this study, we confirmed previousreports showing partial localization of some HCV proteins on
mitochondria (Fig. 1C) (37, 39, 47), thus supporting the evi-dence that a direct physical interaction was causally related tothe HCV-linked mitochondrial alterations. However, cellbioenergetics was not compromised even after longer-term in-duction of HCV protein expression because of an enhancedglycolytic catabolic flux, resulting in an increased level/produc-tion of intracellular ATP (Fig. 2). This paradoxical overpro-duction of ATP, hardly explainable by an allosteric regulationof glycolysis, was a clue arguing in favor of changes in theglycolytic-gene expression profile.
The upregulation of glycolytic enzymes in HCV-infectedcells is mediated by activation of HIF-1. HIF-1 is the majortranscription factor controlling the expression of practically allthe enzymes of the glycolytic pathway, as well as of the induc-ible glucose transporters (25, 41). It is activated under condi-tions of low O2 tissue tension and provides a first adaptivemetabolic response at the cellular level. If the hypoxic insultpersists, HIF-1 upregulates the expression of factors involved
FIG. 7. Effects of antioxidants and inhibitors on HCV-induced HIF-1 activation. (A) LSCM analysis of intracellular ROS production. Imagingof intracellular H2O2 was evaluated by DCF as specified in Materials and Methods. UHCVcon-57.3 cells were induced for 5 days. Where indicated,induced cells were treated for the entire induction time with either 20 mM NAC or 0.5 mM Tiron. Bars, 10 �m. The quantitative analysis of theDCF fluorescence from three different experiments is displayed as bars superimposed on the images; the fluorescence mean � standard error ofthe mean (SEM), averaging the pixel intensities of 10 to 15 cell from at least 10 optical fields, is shown for each condition, along with statisticalanalysis. A representative Western blot (WB) of HIF-1� and HCV core on cell lysates of parallel samples is also shown. A.U., arbitrary units.(B) qRT-PCR assay of HIF-1�, VHL, LDH, HK1, and VEGF-A transcript levels. For experimental details, see Materials and Methods and thelegend to Fig. 5. The light-gray and dark-gray bars refer to the effects of NAC and Tiron, respectively, under the experimental conditions of panelA. The level of each transcript was normalized to that of the noninduced cells, as in Fig. 5. The values shown are the ratios of the NAC- andTiron-treated cells with respect to the induced untreated cells and represent the average of three independent experiments plus SEM. (C) Effectsof ruthenium red (RR), dantrolene (Dantr.), wortmannin (Wortm.), and L-NNMA on the RT-PCR-assayed transcript levels of HIF-1�, VHL,LDH, HK1, and VEGF-A. UHCVcon-57.3 cells were induced for 5 days in the presence of each inhibitor at the following concentrations: 5 �MRR, 10 �M Dantr., 100 nM Wortm., and 1 mM L-NNMA. The transcript levels are expressed as in panel B and were averaged from twoindependent experiments.
654 RIPOLI ET AL. J. VIROL.
in angiogenesis and erythropoiesis, thereby allowing an adap-tive response at the systemic level (25). In addition to theseearly-recognized tasks, the involvement of HIF-1 in controllinga wide variety of cell functions has been unveiled over time.Importantly, conditions other than hypoxia proved to regulatethe basal activity of HIF-1, which provides prosurvival adapta-tion to a variety of cells (6).
In this study, we showed that induction of HCV proteinexpression enhances the amount and the nuclear localizationof the HIF-1� subunit and the transcriptional level of glycolyticenzymes, as well as of VEGF (Fig. 3). Comparable results, ona qualitative basis, were obtained in a completely different cellculture system fully competent in replicating the HCV genomeand producing infectious virus (Fig. 5 and 6A) and, moreimportantly, in liver biopsy specimens from patients affected byHCV infection (Fig. 11). This last observation, which to ourknowledge has not previously been reported in HCV-infectedindividuals, might be linked to the results of a study showinghypoxia-independent overexpression of HIF-1� as an earlychange in a murine model of chemically induced hepatocarci-nogenesis (45).
Some differences were observed among the HCV protein-
expressing cell samples examined in this study. In particular,the transfected Huh-7.5 cells and liver biopsy specimens showedsignificant upregulation of HIF-1� and VHL at the transcrip-tional level, whereas the level of LDH-A did not change (Fig.5B and 11). In contrast, the UHCVcon-57.3 cells expressingthe entire HCV polyprotein displayed significant transcriptionalupregulation of LDH-A, HK1, and VEGF, whereas HIF-1�and VHL remained unaltered (Fig. 3B). A diverse phenotypicbackground may reasonably account for the observed differ-ences. Concerning the difference in LDH-A transcript levels,the possibility that the control of its expression is not exclu-sively dependent on HIF-1 must be considered. Indeed, itsinteraction with a cyclic AMP response element on the LDH-Apromoter is needed to achieve maximal trans-activation effi-ciency (6). Although HIF-1� was initially reported to be con-stitutively expressed, recent evidence indicates that its mRNAtranscription is under the control of the PI3K/Akt pathway (9),as well as of NF-�B (48) and the chromatin-remodeling com-plex SWI/SNF (14). It is therefore plausible that in Huh-7.5cells and liver, HCV infection may tissue specifically foster thePI3K/Akt and/or NF-�B signaling pathway, leading to upregu-lation of HIF-1�. The increased levels of the VHL and PHD2
FIG. 8. Effects of antioxidants in transiently HCV-transfected Huh-7.5 cells. (A, left) ROS production in intact Huh-7.5 cells assessed byfluorimetry as described in Materials and Methods. The experimental traces refer to control and Jc1-GFP RNA-transfected Huh-7.5 cells (timepoint, 3 days after transfection) and were corrected point by point over the same time course for the recordings attained under identical conditionswith cells treated with 20 mM NAC (comparable outcomes were obtained when corrections were made for cells treated with 0.5 mM Tiron). Therates of ROS generation in control and transfected Huh-7.5 cells without/with NAC or Tiron treatment are shown on the right. The values werecalculated from the initial increase of the fluorescence following the addition of DCF-DA and are the average plus the standard error of the mean(SEM), along with statistical analysis, of recordings carried out in triplicate for each condition from three independent experiments. (B) Westernblot of HIF-1� and HCV core on cell lysates of control and HCV-transfected Huh-7.5 cells, the latter without/with NAC or Tiron treatment. theresults are representative of three independent experiments. (C) qRT-PCR assay of HIF-1� and VEGF-A transcript levels. For experimentaldetails, see Materials and Methods and the legend to Fig. 5. The dark-gray and light-gray bars refer to the effects of NAC and Tiron, respectively,under the experimental conditions of panel A in transfected Huh-7.5 cells. The level of each transcript was normalized to that of the noninducedcells as in Fig. 5 and represents the average of three independent experiments plus SEM, along with statistical analysis.
VOL. 84, 2010 HCV INFECTION STABILIZES HIF-1� 655
mRNAs might be consistent with a described feedback mech-anism whereby HIF-1� positively controls the expression ofVHL and PHD2 by binding to their promoters (8, 13). Thismechanism, which under normal conditions negatively self-controls the basal activation of HIF, failed in HCV protein-expressing cells. The last argument, together with the evidenceprovided here that activation of HIF was also observed inanother cell type inducibly expressing HCV proteins in theabsence of change in the HIF-1� transcript level, strongly arguesin favor of HIF stabilization resulting from impaired posttrans-lationally linked degradation rather than from enhanced tran-scription.
HCV-linked HIF-1 activation is mediated by intermediate-metabolite accumulation causing inhibition of PHDs. The ab-sence of differences in the regulation of the transcription ofHIF-controlled genes between cells expressing either the en-tire HCV polyprotein or subgenomic constructs comprisingeither the structural or the nonstructural proteins excludesdirect interaction of components of the HIF system with asingle specific HCV protein (Fig. 3B). On the other hand,overexpression of an unrelated HCV protein in the induciblecell system did not cause effects on either HIF or mitochondria(Fig. 4). All these observations argue that the HCV-mediatedactivation of HIF-1 via stabilization of its subunit HIF-1� oc-curs via different HCV proteins or an indirect mechanism.
In a recent report, it was shown that HCV-infected Huh-7cells release angiogenic factors as a consequence of normoxic
HIF-1� stabilization (29). It was further demonstrated that thestabilization of HIF-1� was mediated by oxidative stress in-duced by HCV gene expression. In the present study, however,the treatment of both HCV-inducible and -transfected celllines with antioxidants proved to be inefficient in preventingthe overexpression of HIF-controlled genes (Fig. 7 and 8). Thereasons for this discrepancy are not clear but may rely on thedifferent tumor-derived cell hosts used (U-2 OS and Huh-7.5in this study versus Huh-7 [29]) and/or on the features of the invitro HCV protein/RNA expression/replication (HCV-induc-ible and -transfected cells in this study versus HCV-infectedcells [29]). Therefore, in our cell culture systems, factors otherthan ROS apparently cause stabilization of HIF-1�. Weshowed that pharmacological inhibition of the FoF1 ATP syn-thase induces HIF-1� stabilization and that treatment of bothUHCVcon-57.3 cells and transfected Huh-7.5 cells with anexcess of pyruvate, even in the absence of inhibitors of theOXPHOS, is in itself sufficient to stabilize HIF-1� (Fig. 9 and10). The last evidence strongly suggests that the accumulationof metabolic intermediates, as a consequence of deregulationof the mitochondrial terminal metabolism, is likely to mediateactivation of HIF-1.
PHDs play fundamental roles in the mechanism of HIFactivation (37). They are nonheme Fe(II)- and 2-oxoglutarate(2-OG)-dependent dioxygenases. During catalysis, the splittingof molecular oxygen is coupled to hydroxylation of HIF-1� andoxidative decarboxylation of 2-OG to yield succinate and CO2
FIG. 9. Effects of oligomycin and pyruvate on HIF-1� stabilization in U-2 OS cells. (A, left) Immunofluorescence analysis of HIF-1� innoninduced UHCVcon-57.3 cells. For experimental details, see Materials and Methods and the legend to Fig. 3. Where indicated, cells weretreated with 1 �M oligomycin or 2 mM pyruvate for 4 h. Bars, 10 �m. False-color images of the original pictures are shown to better visualize thenuclear localization of HIF-1�. The average fluorescence intensities from three independent experiments plus standard errors of the mean, alongwith statistical analysis, are represented in the histogram on the right. (B) Western blot (WB) of HIF-1� on lysates of cell samples treated as inpanel A; the results are representative of three independent experiments. Oligo., oligomycin; Pyr., pyruvate.
656 RIPOLI ET AL. J. VIROL.
(10). It has been reported that carboxylic acids and 2-oxo acids,like succinate, oxalacetate, and pyruvate, are efficient compet-itors of 2-OG with Kis comparable to their physiological con-centrations (15, 16, 24). This led us to propose for these Krebscycle and glycolytic intermediates a role of modulators of PHDactivity, thus establishing a link between mitochondrial dys-
function involving the OXPHOS system and activation of HIF-1.It is worth noting that in the case of the 2-oxo acids a mech-anism of inhibition that is not simply competitive has beendescribed, in which inactivation of the dioxygenases is accom-plished by oxidation of the active metal center (15). Rereduc-tion of the Fe(III) center (and consequent recovery of the
FIG. 10. Effects of oligomycin and pyruvate on HIF-1� stabilization in Huh-7.5 cells. (A, left) Immunofluorescence analysis of HIF-1� inHuh-7.5 cells. For experimental details, see Materials and Methods and the legend to Fig. 3. Where indicated, cells were treated with 1 �Moligomycin or 2 mM pyruvate for 4 h. Bars, 10 �m. The average fluorescence intensities from three independent experiments plus standard errorsof the mean, along with statistical analysis, are represented in the histogram on the right. (B) Western blot (WB) of HIF-1� on lysates of cellsamples treated as in panel A; the results are representative of three independent experiments. Oligo., oligomycin; Pyr., pyruvate.
FIG. 11. Expression of HIF-1�-controlled genes in liver biopsy specimens from HCV-infected patients. Shown is qRT-PCR analysis of HIF-1�,VHL, LDH-A, HK1, and VEGF mRNAs in 14 healthy control liver biopsy samples (Controls) and 19 samples derived from patients with CHC(HCV). P values were determined by two-tailed Mann-Whitney tests.
VOL. 84, 2010 HCV INFECTION STABILIZES HIF-1� 657
PHD activity) specifically requires ascorbate (42). The main-tenance of a reduced pool of ascorbate needs reduced gluta-thione (22), which is the main intracellular redox buffer. De-pletion of glutathione to counteract a chronic oxidative insult,such as that induced by HCV protein expression (1, 33), isexpected to affect the recovery of PHD activity.
A pathogenic model for HCV-related disease. Figure 12 il-lustrates a plausible pathogenic mechanism integrating the re-sults presented here with evidence reported in the literature. Itshows that the HCV-related calcium-mediated depression ofmitochondrial OXPHOS causes accumulation of Krebs cycleand glycolytic intermediate metabolites. These may inhibitPHDs, allowing HIF-1� to escape degradation. Inactivated/oxidized PHDs can recover full activity by ascorbate-depen-dent rereduction of their catalytic metal center. In a pro-oxi-dant condition, like that set by HCV infection, the availabilityof ascorbate decreases, and this progressively enhances the lifespan of the PHD inactivation. The accumulating HIF-1� trans-locates into the nucleus, where, upon binding to its cognate,HIF-1�, it forms the active heterodimer transcription factor.This positively controls the transcription of genes coding forthe glycolytic enzymes, which promote aerobic glycolysis, therebyproducing pyruvate. Accumulation of pyruvate might be alsofavored by a limited or unaffected overexpression of LDH.Thus, a positive feed-forward mechanism is, in turn, fueled.
The model foresees that ROS may indirectly upregulate theexpression of HIF-1� itself by activating a PI3K/Akt- and/orNF-�B-linked signaling pathway(s) (18, 30).
HIF controls the expression of a variety of antiapoptotic andcell growth factors, thereby establishing a prosurvival condi-tion. This sequence of events may turn out to be favorable toHCV, which can proceed through its replicative and viral-particle assembly phases in a bioenergetically competent cellhost. The importance of providing ATP buffering to sustainviral replication has recently been shown (11). In this study,interaction of creatine kinase B, a key ATP-generating enzymethat regulates ATP in subcellular compartments of nonmusclecells, with NS3-4A was found to be important for efficientreplication of the HCV genome and propagation of infectiousvirus. Noticeably, it was reported that the hepatitis B virus Xprotein enhances the transcriptional activity of HIF-1� throughactivation of the mitogen-activated protein kinase pathway (52).Thus, upregulation of HIF might be part of a more general viralstrategy established during infection. On the other hand, thegrowth and proliferative advantage acquired by the infected cellmay facilitate its clonogenic expansion, which, under the muta-genic pressure of ROS, may eventually lead to carcinogenic celltransformation (12, 31, 44).
Manipulation of HIF-1 activity by genetic or pharmacolog-ical means has been shown to markedly affect tumor growth
FIG. 12. Proposed mechanism for the HCV-linked normoxic stabilization of HIF-1 and its pathogenic implications. The impairment of themitochondrial OXPHOS system caused by HCV proteins (1) is suggested to induce a metabolic shift toward glycolysis. This persistent metabolicsetting would cause accumulation of pyruvate and Krebs cycle intermediates (2). These are proposed to inhibit the PHDs (3), thereby stabilizingHIF-1� (4). Nuclear translocation of HIF-1� and transactivation of hypoxia-responding genes would upregulate the expression of glycoliticenzymes (5). Therefore, a positive feed-forward mechanism is activated (6). Moreover, as a “side effect,” other HIF-dependent angiogenetic andprosurvival factors are upregulated (7 and 8). These events, in combination with HCV protein expression-dependent ROS overproduction (9), mayeventually lead to carcinogenic transformation (10 and 11). See the text for further details. OAA, oxalacetate.
658 RIPOLI ET AL. J. VIROL.
(42). Accordingly, the results of the present study suggest thatdirect or indirect inhibitors of HIF-1 might represent promis-ing components of novel combination therapies to prevent ortreat HCC in patients with CHC.
ACKNOWLEDGMENTS
We are grateful to Ralf Bartenschlager and Charles M. Rice forkindly providing the Jc1-GFP construct and Huh-7.5 cells, respectively.
The work was supported by the University of Foggia (Local Re-search Funds 2007–2008), Fondazione Banca del Monte DomenicoSiniscalco Ceci-Foggia-Italy, the Swiss National Science Foundation(3100AO-122447), the Swiss Cancer League (OCS-01762-08-2005),and the Leenaards Foundation.
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