A Caspase 9-Dependent Manner Hepatocytes to TRAIL-Induced

The Journal of Immunology

Hepatitis C Virus Infection Sensitizes Human Hepatocytes to TRAIL-Induced Apoptosis in a Caspase 9-Dependent Manner1

Lin Lan,2*† Sebastian Gorke,2*‡ Sibylle J. Rau,*‡ Mirjam B. Zeisel,§¶ Eberhard Hildt,*ʈ Kiyoshi Himmelsbach,*‡ʈ Monica Carvajal-Yepes,*‡ʈ Roman Huber,* Takaji Wakita,# Annette Schmitt-Graeff,** Cathy Royer,§¶ Hubert E. Blum,* Richard Fischer,3,4* and Thomas F. Baumert3§¶††

Apoptosis of infected cells represents a key host defense mechanism against viral infections. The impact of apoptosis on the elimination of hepatitis C virus (HCV)-infected cells is poorly understood. The TRAIL has been implicated in the death of liver cells in hepatitis-infected but not in normal liver cells. To determine the impact of TRAIL on apoptosis of virus-infected host cells, we studied TRAIL-induced apoptosis in a tissue culture model system for HCV infection. We demonstrated that HCV infection sensitizes primary human hepatocytes and Huh7.5 hepatoma cells to TRAIL induced apoptosis in a dose- and time-dependent manner. Mapping studies identiﬁed the HCV nonstructural proteins as key mediators of sensitization to TRAIL. Using a panel of inhibitors targeting different apoptosis pathways, we demonstrate that sensitization to TRAIL is caspase-9 dependent and mediated in part via the mitochondrial pathway. Sensitization of hepatocytes to TRAIL-induced apoptosis by HCV infection represents a novel antiviral host defense mechanism that may have important implications for the pathogenesis of HCV infection and may contribute to the elimination of virus-infected hepatocytes. The Journal of Immunology, 2008, 181: 4926–4935.

epatitis C virus (HCV)5 infection is a leading cause of ribosomal entry site which directs cap-independent translation. liver cirrhosis and hepatocellular carcinoma (1). HCV Posttranslational cleavage of the polyprotein yields in structural H belongs to the Flaviviridae family and has an enveloped, and nonstructual proteins including core, E1, E2, p7, NS2, NS3, positive-stranded RNA genome of 9.6 kb length containing one NS4A, NS4B, NS5A, and NS5B (2, 3). open reading frame translated into a single polyprotein. A highly Histopathological studies demonstrated that enhanced hepato- conserved, untranslated region at the 5Ј site serves as an internal cyte apoptosis is a common feature in the HCV-infected liver (4, 5). The close physical proximity of apoptotic hepatocytes and in- filtrating lymphocytes seen in HCV infection suggests that apoptosis is initiated by an interaction between effector cells of the *Department of Medicine II, University of Freiburg, Germany; †Institute of Infectious host immune system and hepatoyctes (4). Effector cells of the in- Diseases, Southwestern Hospital, Third Military Medical University, Chongqing, ‡ § nate and acquired immune system are able to kill target cells China; Faculty of Biology, University of Freiburg, Freiburg, Germany; Institut Na- ␣ tional de la Sante´ et de la Recherche Me´dicale, U748, Strasbourg, France; ¶Universite´ through ligands of the death receptor family. In contrast to TNF- Louis Pasteur, Strasbourg, France; ʈInstitute for Infection Medicine, Molecular Med- and CD95/Fas, TRAIL induces apoptosis only in infected or trans- # ical Virology, University of Kiel, Kiel, Germany; Department of Virology II, Na- formed tumor cells (6). In line with these observations, TRAIL tional Institute of Infectious Diseases, Tokyo, Japan; **Institute of Pathology, Uni- versity of Freiburg, Freiburg, Germany; and ††Service d’He´patogastroente´rologie, does not induce apoptosis in noninfected healthy hepatocytes in Hoˆpitaux Universitaires de Strasbourg, Strasbourg, France vivo (5, 7). For all three death ligands an up-regulation of expres- Received for publication December 21, 2007. Accepted for publication July 23, 2008. sion has been described in chronic HCV infection (4, 5, 8). TRAIL The costs of publication of this article were defrayed in part by the payment of page binding induces the formation of a death-inducing signaling com- charges. This article must therefore be hereby marked advertisement in accordance plex, resulting in the activation of caspase-8. Active caspase-8 can with 18 U.S.C. Section 1734 solely to indicate this fact. trigger two signaling pathways, the first involving direct activation 1 This study was supported by the Fritz Thyssen Stiftung (to R.F.), Germany, the Faculty of Medicine, University of Freiburg, Germany (to R.F. and A.S.-G.), and by of caspase-3, the second involving cleavage of Bid, followed by grants of Institut National de la Sante´ et de la Recherche Me´dicale, France, the Eu- mitochondria-dependent activation of caspase-9 via cytochrome C ropean Union (LSHM-CT-2004–503359 “VIRGIL”; to T.F.B.), Belgium, the chair of release and Apaf-1 activation (9). Hepatocytes most likely repre- excellence program of the Agence Nationale de la Recherche (ANR-05-CEXC-008; to T.F.B.), France, the Agence Nationale de la Recherche sur le SIDA et les He´patites sent so-called type-II cells, for which external activation of the Virales (ANRS 06221; to T.F.B.), France, the Deutsche Forschungsgemeinschaft death signaling pathway often is insufficient to induce apoptosis (Ba1417/11–2; to T.F.B.), Germany, France and the Else Kro¨ner-Fresenius Founda- and requires in addition amplification by the mitochondrial path- tion, Bad Homburg, Germany (P17/07//A83/06; T. F. B.). M.B.Z. was supported by the Inserm Poste Vert program in the framework of Institut National de la Santeétde way (intrinsic apoptosis pathway). The latter is affected by oxida- la Recherche Me´dicale European Associated Laboratory Freiburg-Strasbourg. T.W. is tive stress, DNA damage, and viral proteins (10, 11). Mitochon- supported by a grant-in-aid for Scientific Research from the Japan Society for the Promotion of Science, from the Ministry of Health, Labour and Welfare of Japan and dria-dependent apoptosis is amplified by proapoptotic Bax, Bad, from the Ministry of Education, Culture, Sports, Science and Technology, and by the Research on Health Sciences Focusing on Drug Innovation from the Japan Health Sciences Foundation. 2 L.L. and S.G. contributed equally to this study. 5 Abbreviations used in this paper: HCV, hepatitis C virus; PHH, primary human 3 hepatocyte; MOI, multiplicity of infection; JFH1, Japanese fulminant hepatitis 1 iso- R.F. and T.F.B. contributed equally to this study. late; HCVcc, cell culture-derived HCV; PARP, poly (ADP-ribose) polymerase; HBV, 4 Address correspondence and reprint requests to Dr. Richard Fischer, Department of hepatitis B virus. Medicine II, University of Freiburg, Hugstetter Strasse 55, D-79106 Freiburg, Germany. E-mail address: richard.fi[email protected] Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 www.jimmunol.org The Journal of Immunology 4927

Bak, and other proteins, while Bcl-2 or Bcl-xL act anti-apoptotic (for review see Ref.12). The molecular mechanisms of hepatocyte apoptosis in HCV infection are poorly understood. For both HCV structural and nonstructural proteins, pro- and antiapoptotic properties have been described (13). Viral RNA has been shown to be able to induce apoptosis via activation of protein kinase R and the retinoic acid inducible gene-I-Cardif pathway (14–16). However, the signifi- cance of these interactions for productive viral infection and pathogenesis of HCV-induced liver disease remains unknown. Studies on HCV-host interactions had been hampered for a long time by the lack of an efficient tissue culture model for HCV infection. Thus, alternative model systems, such as recombinant proteins, pseudotype particles, or subgenomic replicons, had been developed for the study of defined aspects of the HCV life cycle (for review see Ref. 17). A major breakthrough for the study of HCV- host cell interaction was the recent establishment of an efficient cell culture system for HCV (18–20). This model system now allows the study of HCV-host interactions and apoptosis in the context of the complete viral life cycle in human hepatocyte-derived target cells. To determine the impact of TRAIL on apoptosis of virus-infected host cells, we studied mechanisms of TRAIL-induced apoptosis in a tissue culture model system for HCV infection. FIGURE 1. HCV constructs and viral protein expression. A, Constructs: Materials and Methods HCV-pJFH1 resulting in the production of infectious viral particles, pJFH1/⌬E1E2 containing a deletion of the HCV envelope protein coding Cells region preventing production of infectious viral particles, pJFH1/GND Primary human hepatocytes (PHH) were isolated and cultured as described containing a mutation in NS5B protein preventing viral replication, and (21). Human hepatoma cells Huh7.5 have been described (18, 22). subgenomic replicon pSGR-JFH1 (lacking the coding region for HCV structural proteins and NS2, respectively). B, Protein expression from rep- Constructs lication-competent constructs. Seventy-two hours following electropora- Plasmids pJFH1, pJFH1/⌬E1E2, pJFH1/GND, pSGR-JFH1, pSGR-JFH1/ tion with HCV RNA transcribed from constructs depicted in A, cells were GND, and pFK-Jc1 have been described (19, 23, 24) and are depicted in lysed and subjected to immunoblot analysis using Abs against HCV core, Fig. 1. E2, NS3, and NS5A proteins as described in Materials and Methods. Electroporation of HCV RNA, cell culture-derived HCV (HCVcc) production, infection of Huh7.5 cells, and PHH Electroporation of RNA derived from plasmids pJFH1, pJFH1/⌬E1E2, Cells were washed with PBS and incubated in cell culture medium with pJFH1/GND, pSGR-JFH1, pSGR-JFH1/GND, and pFK-Jc1 was per- recombinant TRAIL that includes the extra cellular domain of human formed as described (19, 23, 24). Culture supernatants from HCV JFH16, TRAIL (aa 95–281) fused at the N terminus to a His-tag and a linker HCV Jc1, and HCV JFH1/⌬E1E2 RNA transfected cells were harvested, peptide (SuperKillerTRAIL, Axxora). Alternatively, cells were prein- concentrated and used to infect naive Huh7.5 cells (22) as well as primary cubated in the presence or absence of caspase inhibitors (caspase-3 human hepatocytes. Culture supernatants used to infect naive Huh7.5 cells inhibitor (2-(4-Methyl-8-(morpholin-4-ylsulfonyl)-1,3-dioxo Ϫ1,3-di- 4 5 6 hydro-2H-pyrrolo [3,4-c]quinolin-2-yl)-ethyl-acetate), caspase-8 inhibi- had a TCID50 of 10 /ml for JFH1 and 10 -10 /ml for Jc1. tor: Z-IETD-FMK, caspase-10 inhibitor: Z-AEVD-FMK, caspase-9 inhib-

Immunoblot analysis of viral and cellular proteins itor: Z-LEHDFMK; all from R&D Systems), Bcl-2/Bcl-xL inhibitors (dimethoxy-dihydro-dibenzodiazocine dioxide and dimethoxy-dinitroso- Cell lysis, protein determination, Western blot analysis, and densitometric benzyl; Calbiochem), or Bcl-xL mimetic peptide (Calbiochem) containing analysis were performed as recently described (25). Cytosolic and mito- the human conserved N-terminal homology domain of Bcl-xL (aa 4–23), chondrial protein fractions were obtained using a ProteoExtract Subcellular linked to a carrier peptide. Subsequently, cells were washed with PBS and Proteome Extraction Kit according to the manufacturer’s protocol (Calbio- harvested in lysis buffer for Western blot analysis or fixed for immunoflu- chem). Relative OD was calculated by correlation of the given protein to orescence staining. ␤-actin; maximal intensity was arbitrarily set to 100. At least three independent experiments were performed; the mean relative OD and the cor- Immunofluorescence staining of viral proteins in infected responding SEM is given. Protein loading was controlled by staining the host cells membranes with Coomassie-blue and blotting of ␤-actin. Immunoblot analysis of HCV core, E2, and NS5A protein was performed using mono- Fixation and immunostaining of Huh7.5 cells was performed using monoclonal mouse anti-core (28), anti-E2 (26), and polyclonal anti-NS5A Abs clonal anticore Abs (C1/C2) as described recently (25, 28, 29). Cells were (27). mAbs directed against NS3 (ViroStat), caspase-3 (3G2, Cell Signal- embedded in fluorescence-mounting medium containing DAPI (4–6-dai- ing Technology), caspase-8 (1C12, Cell Signaling Technology), caspase-9 midino-2-phenylindole; Vector Laboratories) for cell nuclei staining. Mi- (C9, Cell Signaling Technology), ␤-actin (Sigma-Aldrich), cleaved poly croscopy was performed using a fluorescent microscope (Leica). For de- (ADP-ribose) polymerase (PARP) (Asp214, Cell Signaling Technology), tection of DNA fragmentation, a TUNEL assay was used as previously

TRAIL-R1, -R2, and TRAIL (R&D Systems), Bcl-2 and Bcl-xL (Santa described (30). The percentage of apoptotic cells was determined by count- Cruz Biotechnology), cytochrome C (Biovision), and HRP-linked species- ing the number of TUNEL-positive cells. At least 300 cells were counted specific Abs (Amersham Biosciences) were used for immunoblot analysis out of five microscopic fields at ϫ400 magnification for each experiment. of the respective proteins. Statistical analysis Induction of apoptosis by TRAIL Data are expressed as means Ϯ SEM (n ϭ number of cell preparations). Naive and HCV RNA transfected Huh7.5 cells were seeded at a density of SEM was omitted in the figures when smaller than symbol size. Each 2–4 ϫ 105 cells/well in 6-well plates 72 h before apoptosis experiments. experiment was performed at least in three independent cell preparations. 4928 TRAIL-INDUCED APOPTOSIS IN HCV INFECTION

caspase-9, and PARP declined during apoptosis. This decline most likely reflects degradation of these proteins due to apoptosis-activated proteases as described in many model systems for apoptosis (34–36). Caspase activation is a marker of the susceptibility of the cell to undergo apoptosis, but not equivalent to apoptosis (for review, see Ref. 9). To confirm that Huh7.5 cells indeed underwent irreversible apoptosis following TRAIL incubation, we analyzed PARP cleavage, a marker of irreversible cell death (37). After incubating Huh7.5 cells with TRAIL at a concentration of 50 ng/ml for 2 h, cleavage of PARP was detectable (Fig. 2). These data demonstrate that TRAIL induces apoptosis in Huh7.5 hepatoma cells in a dose-dependent manner. Next, we aimed to study the mechanism of TRAIL-induced apoptosis in hepatocyte-derived cell lines. Using defined inhibitors targeting key mediators of distinct apoptotic pathways, we studied

the functional impact of Bcl-2, Bcl-xL, and caspases for TRAIL- induced cell death. Proteins belonging to the Bcl-family are important modulators of mitochondrial apoptosis (38). As shown in Fig. 2B speciﬁc inhibition of anti-apoptotic effectors Bcl-2 and

Bcl-xL with dimethoxy-dihydro-dibenzodiazocine dioxide and dimethoxy-dinitrosobenzyl did not result in spontaneous apoptosis in Huh7.5 cells, but enhanced TRAIL-induced apoptosis. These data indicate that TRAIL-induced apoptosis is suppressed by Bcl-2 and

Bcl-xL, but not completely inhibited. Hepatocytes belong to the so-called type-II cells that need mitochondrial ampliﬁcation and consecutive caspase-9 activation for apoptosis induction via death ligands (39). In Huh7.5 cells, inhibition of caspase-9 with Z- FIGURE 2. TRAIL-induced apoptosis in human Huh7.5 hepatoma cells LEHDFMK largely abolished TRAIL-induced apoptosis, as deter-

is caspase and Bcl-2/Bcl-xL dependent. Following incubation with TRAIL, mined by cleaved PARP fragments (Fig. 2C, lane 3). Inhibition of Huh7.5 cells were lysed and protein lysates were subjected to SDS-PAGE the initiator caspases-8 and -10 with Z-IETD-FMK and Z-AEVD- and Western blot analysis using anti-human PARP and caspase-specific FMK, completely prevented TRAIL-induced apoptosis (lanes 2 Abs. A, left panel, Time course of TRAIL-induced apoptosis with PARP-, and 4). In contrast, inhibition of caspase-3 only marginally altered caspase-3, caspase-8, and caspase-9 cleavage following 1–24 h incubation TRAIL-induced apoptosis (see Fig. 8). These data demonstrate with TRAIL at a concentration of 50 ng/ml. Right panel, Dose-dependent that TRAIL-induced apoptosis in Huh7.5 cells, similar to hepato- induction of TRAIL-dependent apoptosis at TRAIL concentrations ranging cytes, largely depends on mitochondrial amplification. Further- from 1 to 200 ng/ml (incubation time 2 h). B, Mechanism of TRAIL- induced apoptosis. Huh7.5 cells were incubated with TRAIL following a more, our data indicate that TRAIL-induced apoptosis is blocked if downstream signaling of the receptor-activated death-inducing 30-min preincubation with Bcl-2-, Bcl-xL-, and caspase inhibitors. Cells were incubated at a TRAIL concentration of 50 ng/ml (left panel), or for signaling complex via caspase-8/-10 is inhibited. 2 h with different TRAIL concentrations (right panel). Inhibition of anti- Replicating infectious HCV sensitizes Huh7.5 cells and primary apoptotic Bcl-2 and Bcl-xL using dimethoxy-dihydro-dibenzodiazocine dioxide and dimethoxy-dinitrosobenzyl (50 ␮mol/l) showed a marked induc- human hepatocytes to TRAIL-induced apoptosis tion of TRAIL-induced apoptosis. C, After 10 min preincubation time with Because many viruses including cytomegalovirus (40), influenza ␮ caspase-8 and -10 inhibitors Z-IETD-FMK (20 mol/l) and Z-AEVD- virus (41), and adenovirus (42) have been shown to sensitize in- ␮ FMK (20 mol/l), cells were incubated for2hataTRAIL concentration fected cells to TRAIL-induced apoptosis, we aimed to study of 50 ng/ml. The inhibition of proapoptotic caspases-8 and -10 completely whether HCV infection sensitizes Huh7.5 cells to TRAIL-induced abolished TRAIL induced apoptosis (lanes 2 and 4), whereas inhibition of caspase-9 by inhibitor Z-LEHDFMK (20 ␮mol/l) markedly reduced apo- apoptosis. To address this issue, we first exposed Huh7.5 cells ptosis (lane 3). harboring replicating infectious HCV to TRAIL. Compared with control transfected cells, replicating infectious HCV RNA markedly and significantly enhanced TRAIL-induced apoptosis, as Results were compared using the Student’s t test; p Ͻ 0.05 was considered shown by cleaved PARP (Fig. 3, A–D) and cleaved caspase-8 and statistically significant. -9 (Fig. 3, E and F). At the single-cell level, the TUNEL assay showed a statistically significant enhancement of TRAIL-induced Results apoptosis in cells harboring replicating HCV RNA, but not in the TRAIL induces apoptosis in Huh7.5 hepatoma cells, the target control cells harboring replication-deficient control RNA (Fig. 3, cell line for HCV infection G and H). These results demonstrate that HCV sensitizes its host Sensitivity of human hepatoma cells to induction of apoptosis has cells to TRAIL-induced apoptosis. been shown to differ among various cell lines. This is illustrated by Next, we aimed to confirm whether sensitization to TRAIL- the fact that for Huh7 cells, the parental cell line of Huh7.5, apo- induced apoptosis was also present in HCV-infected cells. Thus, ptosis induction as well as resistance following interaction with we incubated Huh7.5 cells with HCVcc or noninfectious control TRAIL have been reported (31–33). Thus, we studied mechanisms supernatants derived from cells transfected with JFH1/⌬E1E2 of TRAIL-induced apoptosis in Huh7.5 cells, a target cell line for RNA unable to produce infectious viral particles. As shown in Fig. infectious recombinant HCVcc. As shown in Fig. 2, TRAIL rap- 4, exposure of HCV-infected Huh7.5 cells to TRAIL resulted in idly and dose-dependently induced cleavage of caspase-8, -9, and enhanced apoptosis compared to control cells incubated with con- -3 (Fig. 2). The amounts of cleaved and uncleaved caspase-8, trol supernatants, as shown by cleaved PARP (Fig. 4A). Compared The Journal of Immunology 4929

FIGURE 3. HCV replication sensitizes Huh7.5 cells to TRAIL-induced apoptosis. Huh7.5 cells were transfected with JFH1 RNA or replication-deficient control RNA (GND). Seventy-two hours later, TRAIL was added and cells were incubated for an additional time period as indicated. Following cell lysis, apoptosis was assessed by immunoblot analysis of cleaved PARP, caspase-8, -9, and TUNEL assay. A and C, Analysis of apoptosis of Huh7.5 cells by immunoblot analysis of cleaved PARP. In the experiment depicted in A, cells were incubated with TRAIL (50 ng/ml) for 0–24 h. In the experiment depicted in C, cells were incubated for 6 h with increasing concentrations of TRAIL (0–200 ng/ml). B and D, Quantification of apoptosis by optical densitometric analysis of cleaved PARP levels relative to ␤-actin levels. Means Ϯ SEM from three independent experiments are shown. E and F, Analysis of apoptosis of Huh7.5 cells incubated with TRAIL (50 ng/ml) for6hbyimmunoblot analysis of cleaved caspase-8 (E) and caspase-9 (F) as described in Fig. 2. G, Analysis of apoptosis of Huh7.5 cells by TUNEL assay as described in Materials and Methods. Immunostaining of HCV core protein (red, Cy3) and TUNEL reaction (green, FITC) were performed as described in Materials and Methods. H, Quantification of TUNEL-positive cells in HCV RNA- transfected Huh7.5 cells. Data are shown as mean percentage of TUNEL-positive cells divided by all cells (n ϭ 500 corresponding to 100%); error bars .(statistically significant vs JFH1 (Student’s t test, p Ͻ 0.05 ,ء ;represent SEM of three independent experiments to viral genome delivery by electroporation, HCV-dependent en- Huh7.5 cells are derived from transformed hepatoma cells and hancement of TRAIL-induced apoptosis appeared to be lower in therefore only represent a surrogate model for the natural target HCV JFH1-infected hepatoma cells (as shown by the lower level cell for HCV infection, the PHH. To confirm the relevance of our of cleaved PARP when comparing Fig. 4A to 3C). This difference findings for the natural target cell of HCV infection, we repro- 4 was most likely due to low levels of MOI (TCID50 of 10 /ml for duced key findings in HCV-infected primary human hepatocytes in HCVcc derived from HCV-JFH1 strain) resulting in less efficient contrast to Huh7.5 hepatoma cells have been shown to be resistant delivery of the viral genome in infection compared with transfec- to TRAIL-induced apoptosis (5–7). Indeed, incubation of PHH tion experiments. To address this issue, we repeated infection ex- with TRAIL did not yield in cleavage of PARP (Fig. 4D, lane 4). periments using infectious particles of viral strain Jc1 (J6-JFH1). Whereas incubation of PHH with TRAIL did not result in apopto- In contrast to the JFH1 strain, the Jc1 isolate has been shown to sis (Fig. 4D, lane 5), HCV JFH1 infection rendered PHH sensitive allow production of virus with markedly enhanced infectivity (24). to TRAIL-induced apoptosis (Fig. 4D, lane 2). Infection of PHH Indeed, infection of Huh7.5 cells at higher MOIs with HCV virions with HCV JFH1 was confirmed by detection of HCV NS5A pro- derived from the HCV Jc1 strain (TCID50 of 105–106/ml) resulted tein expression in infected cells using immunoblot (Fig. 4D, lanes in similar enhancement of TRAIL-induced apoptosis as seen in 2 and 3). These results confirm the relevance of HCV-dependent transfection experiments (compare Fig. 4C with 3C). sensitization to TRAIL-induced apoptosis for PHH. 4930 TRAIL-INDUCED APOPTOSIS IN HCV INFECTION

FIGURE 5. Enhancement of TRAIL-induced apoptosis during HCV infection is independent of HCV structural protein expression and viral RNA. A, Huh7.5 cells were transfected with HCV JFH1 RNA derived from constructs described in Fig. 1. Seventy-two hours following transfection, cells were exposed to TRAIL at a concentration of 50 ng/ml for 2 h and apoptosis was analyzed as described in Fig. 3. B, Densitometric analysis of cleaved PARP (see Fig. 3) from three independent experiments indicated a similar enhancement of TRAIL-apoptosis by JFH1 full-length and subgenomic replicons.

TRAIL in cells harboring replicating HCV (compare lanes 1 and 5 in Fig. 4A; lanes 2 and 4 in Fig. 3, E and F; lanes 2 and 4 see in Fig. 8B).

Enhancement of TRAIL-induced apoptosis is mediated by HCV nonstructural proteins To identify the viral factor(s) enhancing TRAIL-induced apoptosis, we determined whether HCV structural protein expression is required for HCV-TRAIL interaction. To address this question, we transfected Huh7.5 cells with a JFH1 variant (JFH1/⌬E1E2) con- FIGURE 4. Enhancement of TRAIL-induced apoptosis during HCV in- taining a deletion in the HCV envelope coding region preventing fection. Infection of PHH and Huh7.5 cells with HCV JFH1 TCID50 of synthesis of infectious viral particles. Because the phenotype of 104/ml and HCV Jc1 TCID of 106/ml was performed by incubation of 50 full-length JFH1 RNA and JFH1/⌬E1E2 was indistinguishable in cells for 3 h with tissue culture supernatants of JFH1, Jc1, or JFH1/⌬E1E2 regard to enhancement of TRAIL-induced apoptosis (Fig. 5), we transfected Huh7.5 cells as described previously (19, 22). Seventy-two hours postinfection, TRAIL was added at the concentrations indicated. Six conclude that HCV-TRAIL interaction is independent of produc- hours following the addition of TRAIL, apoptosis was analyzed by immu- tion of infectious viral particles. To determine whether structural noblot analysis of cleaved PARP as described in Fig. 3. HCV infection was proteins are required for HCV-enhanced TRAIL-induced apopto- confirmed by immunoblot analysis of HCV NS5A expression. A, Immu- sis, we compared the full-length JFH1 RNA with a JFH1 sub- noblot analysis of cleaved PARP in JFH1 infected Huh7.5 cells vs JFH1/ genomic replicon lacking the HCV core-NS2 region. Because the ⌬E1E2 control cells. B, Densitometric analysis of cleaved PARP from JFH1 subgenomic replicon exhibited a similar apoptosis pheno- three independent experiments (see Fig. 3) showed that JFH1 infection type as full-length JFH1 RNA (Fig. 5), we conclude that the ex- significantly enhanced TRAIL-induced apoptosis as compared with incu- pression of HCV structural proteins and NS2 is not required for ⌬ bation with noninfectious control supernatants (JFH1/ E1E2). C, Immu- enhancement of TRAIL induced apoptosis and sensitization to noblot analysis of cleaved PARP in Jc1-infected Huh7.5 cells vs JFH1/ TRAIL-induced apoptosis is mediated predominantly by the HCV ⌬E1E2 control cells. Cells were incubated in presence or absence of 100 ng/ml TRAIL for 2 h. D, Immunoblot analysis of cleaved PARP in JFH1- nonstructural proteins. infected PHH and Huh7.5 vs JFH1/⌬E1E2 control cells. TRAIL (50 ng/ml, HCV JFH1-dependent enhancement of TRAIL-induced apoptosis 6 h) induced cleavage of PARP only in HCV infected PHH (lane 2), but not Statistically significant using Student’s is caspase-9 dependent ,ء .(in JFH1/⌬E1E2 control (lane 4 t test (p Ͻ 0.05). Following mapping the viral factors required for HCV-dependent enhancement of TRAIL-induced apoptosis, we investigated the cellular factors mediating this HCV-host interaction. Several stud- Interestingly, we observed that transfection of Huh7.5 cells with ies pointed to an involvement of TRAIL in apoptosis of virus- replication-competent full-length HCV RNA itself can result in infected hepatocytes by an autocrine loop (43, 44). To address this apoptosis of Huh7.5 cells as indicated by an increase in the number question, we studied the impact of HCV replication and protein of TUNEL-positive cells compared with control cells (Fig. 3H) expression on the expression of TRAIL and TRAIL receptor R1 in and a low-level induction of cleaved PARP using immunoblots Huh7.5 cells. As shown in Fig. 6, cells harboring replicating in- with long exposure times (see Fig. 8B, lane 2 and data not shown). fectious HCV RNA showed decreased expression levels of TRAIL However, the induction of apoptosis by the virus itself was less and unchanged levels of TRAIL receptor R1 expression when pronounced when compared with apoptosis after exposure to compared with cells transfected with replication-deficient viral The Journal of Immunology 4931

FIGURE 7. HCV JFH1 replication suppresses antiapoptotic Bcl-2 and

Bcl-xL expression. Huh7.5 cells and PHH were transfected/infected with HCV RNA/virions derived from constructs depicted in Fig. 1. Seventy-two

hours post transfection, Bcl-2 (A and C), Bcl-xL (B and C) expression and cytochrome C levels in cellular subfractions (D) were analyzed by Western blot analysis. Suppression of antiapoptotic Bcl-2 (A) and antiapoptotic

Bcl-xL (B) protein expression in JFH1-transfected Huh7.5 cells compared with Huh7.5 cells transfected with replication-deﬁcient HCV GND RNA

was detectable. C, Bcl-2 and Bcl-xL protein expression in PHH infected with HCV JFH1. PHH were infected with HCV JFH1 as described in Fig.

4 and Bcl-2 and Bcl-xL protein expression was detected 72 h following infection as described above. D, Cytochrome C levels in subcellular fractions (cytosolic and organelle fractions containing mitochondria) of cells with replicating virus incubated with or without TRAIL. Cells were transfected with JFH1 RNA or replication-deficient control RNA (GND). Sev- enty-two hours post transfection TRAIL was added (100 ng/ml) for 2 h. Following lysis and cellular fractionation of cell lysates as described in FIGURE 6. JFH1-induced apoptosis in primary human hepatocytes and Materials and Methods, cytochrome C levels in cellular subfractions were Huh7.5 cells is independent of TRAIL and TRAIL-R1/-R2 expression. analyzed by immunoblot using anti-cytochrome C specific Ab. Cyt, cyto- PHH and Huh7.5 cells were infected/transfected with virions/RNA derived solic subfraction; Org, organelle subfraction containing mitochondria. from constructs depicted in Fig. 1. Seventy-two hours postinfection/transfection, TRAIL, TRAIL-receptor R1, and TRAIL-receptor R2 expression taining replicating HCV in PHH. Incubation of Huh7.5 with a were analyzed by Western blot analysis using specific Abs directed against human TRAIL, TRAIL-R1, and TRAIL-R2. A, Transfection of Huh7.5 Bcl-xL mimetic peptide resulted in a small but reproducible reduc- cells with JFH1 RNA resulted in suppression of TRAIL expression, as tion (three of three experiments) of TRAIL-induced apoptosis in compared with Huh7.5 cells transfected with replication-deficient GND JFH1 replicating cells (Fig. 8D). Mitochondrial-dependent apopto- RNA. B and C, Absent modulation of TRAIL-R1 expression and lacking sis is characterized by release of mitochondrial cytochrome C into TRAIL R2 expression in HCV JFH1 transfected Huh7.5 cells. Immunoblot the cytoplasm (11, 12). We therefore studied cytochrome C levels analyses of naive PHH are shown as positive controls for TRAIL-R1 and in cytosolic fractions and fractions containing the mitochondria of TRAIL-R2 expression (lane 1). D, Down-regulation of TRAIL expression HCV JFH1-replicating Huh7.5 cells incubated with TRAIL. As in PHH infected with HCV JFH1. Expression of TRAIL-R1 and shown in Fig. 7D, hepatoma cells with replicating virus and treated TRAIL-R2 in PHH infected with HCV JFH1. with TRAIL demonstrated an increase of cytochrome C levels in cytosolic fractions compared with control-transfected cells incubated with TRAIL. These data suggest that cytochrome C appears control RNA. In contrast to PHH, TRAIL receptor R2 was not to be released from the mitochondria in TRAIL-treated cells with expressed in Huh7.5 cells (Fig. 6C). Similar to Huh7.5 cells, in- replicating HCV and suggests that the mitochondrial pathway con- fection of PHH with HCV JFH1 neither increased TRAIL expres- tributes to HCV-induced enhancement of TRAIL-induced apopto- sion, nor enhanced protein expression of TRAIL receptors R1, sis. Interestingly, HCV replication alone also resulted in release of respectively (Fig. 6D). These data suggest that a TRAIL autocrine cytochrome C into the cytosol (Fig. 7D) consistent with low level loop does not play a major role for TRAIL-induced sensitization in induction of apoptosis by JFH1 itself (see Fig. 3H). this HCV infectious tissue culture system. Next, we aimed to study the impact of caspases for HCV-me- The mitochondrial apoptosis pathway has been shown to play an diated enhancement of TRAIL-induced apoptosis. Several forms important role in virus-induced modulations of host cell apoptosis of apoptosis do not involve caspase activation. We therefore in- (10). As shown in Fig. 2, we have already demonstrated that hibited caspases with the pan-caspase inhibitor Z-VAD-FMK. In- TRAIL-induced apoptosis in Huh7.5 cells is enhanced following terestingly, inhibition of caspases completely abolished JFH1-de- the inhibition of Bcl-2/Bcl-xL (Fig. 2B). We therefore analyzed pendent enhancement of TRAIL-induced apoptosis (Fig. 8A, lane Bcl-2 and Bcl-xL protein expression in JFH1-transfected Huh7.5 3 and Fig. 9C, lane 3). These findings indicate an essential role cells and JFH1 infected PHH. Interestingly, expression of Bcl-2 of caspase activation for TRAIL-induced apoptosis of Huh7.5 and Bcl-xL was suppressed in JFH1-transfected Huh7.5 cells (Fig. cells harboring replicating HCV. To determine the impact of 7, A and B). These results were confirmed in PHH, where infection mitochondrial apoptosis in HCV infection, we inhibited the mi- with JFH1 appeared to reduce Bcl-2 and Bcl-xL protein expression tochondrial-activated caspase-9. Inhibition of caspase-9 with Z- (Fig. 7C). The less pronounced reduction in Bcl-2/Bcl-xL protein LEHD-FMK largely blocked JFH1-dependent enhancement of expression observed in PHH (Fig. 7C) compared with Huh7.5 cells TRAIL-induced apoptosis (Fig. 8B, lane 5), suggesting an es- (Fig. 7, A and B) may be due to the lower number of cells con- sential contribution of the mitochondrial apoptotic pathway for 4932 TRAIL-INDUCED APOPTOSIS IN HCV INFECTION

FIGURE 9. TRAIL-induced apoptosis in HCV-replicating cells induces FIGURE 8. JFH1-dependent enhancement of TRAIL-induced apoptosis down-regulation of viral protein expression. Huh7.5 cells were transfected is caspase-9 dependent. Huh7.5 cells were transfected with HCV JFH1 or with HCV JFH1 or control GND RNA as described in Fig. 2. Seventy-two control GND RNA as described in Fig. 3. 72 h following transfection, cells hours following transfection, cells were exposed to TRAIL in the presence were exposed to TRAIL in the presence or absence of caspase-inhibitors or absence of the pan-caspase inhibitor Z-VAD-FMK (Fig. 2) and apopto- (see Fig. 2) and apoptosis was analyzed as described in Fig. 2. A, JFH1- sis was analyzed as described in Fig. 2. HCV core and E2 protein expres- dependent enhancement of TRAIL-induced apoptosis was completely in- sion were analyzed by immunoblot. A, TRAIL (50 ng/ml) induces apopto- hibited by preincubation with pan-caspase inhibitor Z-VAD-FMK (Casp- sis as shown by cleaved PARP and results in down-regulation of HCV core Inh., 20 ␮M). B, JFH1-dependent enhancement of TRAIL-induced and E2 protein expression. TRAIL incubation time is indicated above the apoptosis was blocked by caspase-9 inhibitor Z-LEHD-FMK (Casp-9 Inh., lanes. B, Densitometric analysis of HCV core and E2 levels demonstrated 20 ␮M; lane 5), whereas JFH1-dependent enhancement of TRAIL-induced statistically significant reduction of HCV core and E2 protein expression 6 apoptosis (lane 4) was reduced by caspase-9 inhibitor to the level of JFH1 and 24 h following incubation with TRAIL, respectively. C and D, Inhi- induced apoptosis (lane 2). C, Inhibition of caspase-3 using a caspase 3 bition of TRAIL-induced apoptosis by preincubation with pan-caspase- inhibitor (2-(4-Methyl-8-(morpholin-4-ylsulfonyl)-1,3-dioxo-1,3-dihydro- inhibitor Z-VAD-FMK (20 ␮mol/l) restores HCV E2 and core protein 2H-pyrrolo[3,4-c]quinolin-2-yl)ethyl- acetate; 10 ␮M) only marginally expression as shown by immunoblot (C) and densitometric analysis of modified JFH1/TRAIL induced apoptosis as determined by analysis of HCV core and E2 protein levels (D). Data are presented as mean values of Statistically significant using ,ء .cleaved PARP fragment. D, Preincubation of JFH-1 transfected Huh7.5 three independent experiments Ϯ SEM Ͻ cells with Bcl-xL mimetic peptide (100 nM, 60 min) resulted in a minor Student’s t test, p 0.05. E, Analysis of HCV protein expression in ap- reduction of TRAIL-dependent induction of apoptosis, as determined by optotic cells by immunofluorescence. Huh7.5 cells were transfected with analysis of cleaved PARP fragment. JFH1 RNA as described in Fig. 1 stained with a monoclonal anti-HCV core Ab (red) and counterstained with DAPI (blue) for visualization of cell nuclei. Apoptosis was determined by TUNEL assay (green) as described in HCV-mediated sensitization of TRAIL-induced apoptosis. In- Materials and Methods. Single cell analysis using confocal laser scanning microscopy revealed reduced HCV core expression levels in apoptotic terestingly, inhibition of caspase-3 only marginally affected Huh7.5 cells compared with nonapoptotic cells expressing HCV proteins. TRAIL-induced apoptosis in JFH1 replicating Huh7.5 cells Cells with replicating HCV without apoptosis are indicated by long, thin (Fig. 8C) indicating that caspase-3 does not play a major role in arrows, apoptotic (TUNEL positive) cells with replicating HCV are indi- execution of TRAIL/HCV apoptosis. cated by short, thick arrows. TRAIL-induced apoptosis in HCV target cells results in down-regulation of viral protein expression transfected Huh7.5 cells with TRAIL induced a significant reduc- If HCV replication enhances TRAIL-induced apoptosis, HCV-in- tion of HCV core and E2 protein expression (Fig. 9, A and B). As fected cells should preferentially be eliminated. To address this shown in immunofluorescence analyses of individual cells, apo- question, we studied the impact of TRAIL on HCV protein ex- ptotic Huh7.5 cells with replicating HCV exhibited a marked re- pression in Huh7.5 cells and PHH harboring replicating infectious duction of HCV core protein expression compared with nonapop- HCV. In line with this hypothesis, incubation of HCV JFH1 RNA- totic cells (Fig. 9E). These results were further confirmed by The Journal of Immunology 4933 quantification of viral protein expression using immunofluores- enhancement of TRAIL-induced apoptosis in HCV-infected hu- cence analyses (data not shown). Similar to findings in Huh7.5 man hepatoma cells. hepatoma cells, incubation with TRAIL appeared to result in sup- Interestingly, proapoptotic effects of HCV nonstructural pro- pression of HCV protein expression in HCV JFH1-infected PHH teins NS3/NS4A (46, 47) and NS5A (48) have been described (Fig. 4D, lane 2, compared with lane 3). The TRAIL-dependent recently. NS3 complexed with NS4A is known to translocate to the reduction of HCV protein expression was dependent on apoptosis mitochondrial membrane where direct apoptosis induction, inde- induction, since preincubation of Huh7.5 cells with pan-caspase- pendent from caspase-8 activation can occur (47). Conversely, inhibitor Z-VAD-FMK completely restored expression of the NS3 has been described to cleave Cardif and thereby act antiapop- HCV proteins core and E2, respectively (Fig. 9, C and D). These totically (49). NS5A has recently been shown to directly inhibit findings confirm the impact of TRAIL-induced apoptosis for proapoptotic Bin1, a tumor suppressor protein with a SH3 domain, HCV-host interactions and suggest that sensitization to TRAIL- thereby facilitating apoptosis induction in hepatoma cells (48). By induced apoptosis in cells harboring replicating HCV may con- contrast, NS5A has sequence homologies with Bcl-2 and binds to tribute to control of HCV infection. FKBP38, thereby augmenting the antiapoptotic effect of Bcl-2 (50) and inhibiting the proapoptotic action of Bax in hepatoma cells Discussion (51). Although we cannot exclude a functional relevance of antiapoptotic properties of NS5A and NS3, our data suggest that these In this study, we demonstrate that HCV infection sensitizes the effects are not able to override the proapoptotic effects of the HCV host cell to TRAIL-induced apoptosis. Detailed analyses indicate nonstructural proteins mediating sensitization to TRAIL. that this sensitization depends on the expression of HCV nonstructural proteins NS3, NS4, and NS5. The interaction between HCV Mechanism of HCV-induced apoptosis: impact of host cell and the host cell apoptosis machinery involves caspase-9 and the factors mitochondrial but not the autocrine TRAIL-mediated pathway. The proapoptotic effects of both NS3/NS4A (47) and NS5A (48) Furthermore, we show that induction of apoptosis by TRAIL re- have been described to converge at the level of the mitochondria sults in suppression of HCV protein expression, suggesting that and may explain the proapoptotic properties of HCV infection. In this mechanism may contribute to elimination of HCV-infected this study, we demonstrate evidence that HCV-induced enhance- hepatocytes. ment of Huh7.5 cell apoptosis depends on the mitochondrial pathway, because HCV replication induced cytochrome C release into Viral factors required for sensitization to TRAIL-induced the cytosol (Fig. 7D) and inhibition of caspase-9 markedly abol- apoptosis ished HCV-dependent TRAIL-induced apoptosis (Fig. 8). The cy- For virtually all HCV proteins, interference with apoptosis has tochrome C release may be partly due to the HCV-induced down- been reported. However, most studies assessing the effect of HCV regulation of Bcl-2 and Bcl-xL expression (Fig. 7, A–C). infection on cellular apoptosis were performed in surrogate models Furthermore, preincubation with a Bcl-xL mimetic peptide resulted of HCV infection, e.g., stably transfected cell lines or recombinant in a small but reproducible (three of three experiments) reduction proteins expressed in heterologous systems. Thus, the relevance of of HCV-dependent apoptosis as shown in Fig. 8D. Bcl-2 and these observations for HCV infection remained uncertain. Limita- Bcl-xL are known to be essential in inhibiting mitochondrial apo- tions of surrogate models of HCV infection are, among others, ptosis (12). In line with these results, a recent study demonstrated artificially elevated levels of expressed HCV proteins or the lack of down-regulation of antiapoptotic Bcl-2 and up-regulation of pro- genetic elements in subgenomic replicons (13). To overcome these apoptotic PUMA and Bax in freshly prepared liver sections of limitations and to study the mechanism of apoptosis in a model HCV-infected patients (5). HCV nonstructural proteins have been system closer to HCV infection in vivo, we used the HCVcc model shown to induce ER stress (52), and Bcl-2-/Bcl-xL-dependent system allowing us to study HCV-host interactions in the context transmission of ER-stress into a mitochondrial apoptotic signal has of the complete viral life cycle (18–20). recently been demonstrated (38). Nevertheless, down-regulation of

In contrast to our observations having identified the HCV non- Bcl-2 and Bcl-xL alone seems not sufficient to explain proapoptotic structural proteins as key players for sensitization of TRAIL-in- properties of HCV JFH1, as inhibition of both proteins did not duced apoptosis, a recent study had demonstrated an important role enhance TRAIL-induced apoptosis to the extent of the HCV-de- of core for TRAIL-induced apoptosis (31). Isolate-specific factors pendent apoptosis (Fig. 2), and incubation of cells with a Bcl-xL (genotype 1 vs 2), different core protein levels in the infectious cell mimetic peptide was only partially modulating TRAIL-induced culture system (this study), and transfection of cDNA (31), as well apoptosis in Huh7.5 cells (Fig. 8D). as a dominant effect of nonstructural proteins in the infectious Several authors have postulated an autocrine TRAIL-dependent tissue culture system, may explain the different findings. Using a apoptosis of hepatocytes. Mundt et al. (43) demonstrated TRAIL- full-length replicon expressing all viral proteins, Lee et al. showed dependent apoptosis in hepatocytes using an adenoviral vector ex- that HCV envelope glycoprotein E2 can antagonize the proapop- pressing TRAIL, and Volkmann et al. (5) described an up-regula- totic effects of HCV core protein (32). In our study, we did not tion of TRAIL receptors in HCV-infected human liver sections. observe a markedly altered induction of apoptosis in cells trans- Most recently, HCV-dependent up-regulation of TRAIL and apo- fected with HCV RNA containing a deletion in the HCV envelope ptosis induction in a novel hepatoma cell line has been described proteins (JFH1/⌬E1E2) compared with infectious HCV RNA (Fig. (44). In the latter study, apoptosis of hepatoma cells was dependent 5). Because subgenomic replicons still showed sensitization to on autologous TRAIL expression and HCV-dependent apoptosis TRAIL-induced apoptosis (Fig. 5), our findings clearly demon- resulted in death of all infected cells. In contrast, in PHH and strate that the HCV structural proteins may not represent the major Huh7.5 cells harboring replicating HCV, we observed a decreased factors that determine sensitization to TRAIL, although we cannot expression of TRAIL and unchanged TRAIL receptor expression exclude a modulatory role of these proteins. Because subgenomic levels (Fig. 6). Moreover, immunohistochemistry of TRAIL ex- replicons lack a functional NS2 protein (Fig. 1), our data also pression in the HCV-infected liver revealed that nonparenchymal suggest that the recently described antiapoptotic effect of NS2 in a mononuclear cells, but not hepatocytes, appear to be predominant transgenic mouse model (45) may not play a major role in the producers of TRAIL (data not shown). Taken together, these data 4934 TRAIL-INDUCED APOPTOSIS IN HCV INFECTION suggest that a TRAIL autocrine loop does not play a major role for Disclosures TRAIL-induced sensitization. The authors have no financial conflict of interest. Although HCV replication resulted in detectable induction of apoptosis (Fig. 3, A–H) and cytochrome C release (Fig. 7D), this References induction was significantly less efficient than sensitization to 1. Lauer, G. M., and B. D. Walker. 2001. Hepatitis C virus infection. N. Engl. TRAIL-induced apoptosis (see side-by-side experiments depicted J. Med. 345: 41–52. 2. Bartenschlager, R. 2006. Hepatitis C virus molecular clones: from cDNA to in- in Fig. 3, A–H and Fig. 8). Thus, it seems unlikely that induction fectious virus particles in cell culture. Curr. Opin. Microbiol. 9: 416–422. of endogenous TRAIL production is responsible for the identified 3. Dustin, L. B., and C. M. Rice. 2007. 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