Pegylated IFN-α suppresses hepatitis C virus bypromoting the DAPK-mTOR pathwayWei-Liang Liua,b, Hung-Chih Yangc,d, Ching-Sheng Hsue,f, Chih-Chiang Wanga, Tzu-San Wanga, Jia-Horng Kaoa,c,g,1,and Ding-Shinn Chena,c,g,h,1

aGraduate Institute of Clinical Medicine, National Taiwan University College of Medicine and Hospital, Taipei 10002, Taiwan; bNational Mosquito-BorneDiseases Control Research Center, National Health Research Institutes, Miaoli 35053, Taiwan; cDivision of Gastroenterology and Hepatology, Department ofInternal Medicine, National Taiwan University Hospital, Taipei 10002, Taiwan; dDepartment of Microbiology, National Taiwan University College ofMedicine, Taipei 10051, Taiwan; eLiver Diseases Research Center, Taipei Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Taipei 23142, Taiwan; fPost-Baccalaureate Chinese Medicine, Tzu Chi University School, Hualien 97004, Taiwan; gHepatitis Research Center, National Taiwan University Hospital, Taipei10002, Taiwan; and hGenomics Research Center, Academia Sinica, Nankang, Taipei 11529, Taiwan

Contributed by Ding-Shinn Chen, November 11, 2016 (sent for review August 31, 2016; reviewed by Adrian M. Di Bisceglie and Nobuyuki Enomoto)

Death-associated protein kinase (DAPK) has been found to be inducedby IFN, but its antiviral activity remains elusive. Therefore, weinvestigated whether DAPK plays a role in the pegylated IFN-α(peg-IFN-α)–induced antiviral activity against hepatitis C virus (HCV)replication. Primary human hepatocytes, Huh-7, and infectious HCVcell culture were used to study the relationship between peg-IFN-αand the DAPK-mammalian target of rapamycin (mTOR) pathways. Theactivation of DAPK and signaling pathways were determined usingimmunoblotting. By silencing DAPK and mTOR, we further assessedthe role of DAPK and mTOR in the peg-IFN-α–induced suppression ofHCV replication. Peg-IFN-α up-regulated the expression of DAPK andmTOR, which was associated with the suppression of HCV replication.Overexpression of DAPK enhanced mTOR expression and theninhibited HCV replication. In addition, knockdown of DAPK reducedthe expression of mTOR in peg-IFN-α–treated cells, whereas silencingof mTOR had no effect on DAPK expression, suggestingmTORmay bea downstream effector of DAPK. More importantly, knockdown ofDAPK or mTOR significantly mitigated the inhibitory effects of peg-IFN-α on HCV replication. In conclusion, our data suggest that theDAPK-mTOR pathway is critical for anti-HCV effects of peg-IFN-α.

hepatitis C virus | peg-IFN-α | DAPK | mTOR | PAK-1

Hepatitis C virus (HCV) infection is a major health problemworldwide that may lead to chronic hepatitis, liver cirrhosis,

hepatocellular carcinoma, and liver failure (1). Although the de-velopment of direct antiviral agents (DAAs), such as HCV proteaseand polymerase inhibitors, has revolutionized the treatment of HCVinfection (2, 3), considering the high cost of IFN-free DAAs, com-bination therapy with pegylated IFN-α (peg-IFN-α) and ribavirin(RBV) may remain for a while, especially in countries with limitedmedical resources (4–6). However, many HCV patients fail combi-nation therapy of peg-IFN-α and RBV, and determinants of theresponsiveness to IFN have not been fully understood. Therefore, itis scientifically important to identify biomarkers or mechanisms forprediction of the IFN response in patients with hepatitis C.Type I IFNs are a family of pleiotropic cytokines with known

antiviral, antiproliferative, and immunomodulatory functions exertedon a wide range of cell types. IFNs bind to its receptor and lead toactivation of the Janus kinase–signal transducer and activator oftranscription (STAT) pathway, resulting in the association of phos-phorylated STATs with IFN regulatory factor-9 to form a tran-scriptional activator complex, which subsequently translocates to thenucleus and activates an array of IFN-stimulated genes, such asdsRNA-dependent protein kinase (PKR), 2′,5′-oligoadenylatesyn-thesase, p56, and myxovirus resistance protein A (MxA) (7).Through the action of these IFN-induced genes, IFN-α confers anantiviral state on hepatocytes and exerts its anti-HCV activity inpatients with chronic hepatitis C. However, the full function of theIFN-induced genes in suppression of HCV replication has not yetbeen comprehensively evaluated. Recently, death-associated proteinkinase (DAPK) has been identified as an IFN-induced gene that

contributes to cell death (8), but the role of DAPK in the antiviralaction remains elusive.DAPK is a calmodulin (CaM)-regulated serine/threonine kinase

and possesses multiple structural and functional domains (9).Several lines of evidence indicate that DAPK functions as a pos-itive mediator of apoptosis induced by a variety of stimuli, in-cluding TNF-α, Fas, TGF-β, detachment from extracellular matrix,and c-Myc (10–12). In addition, DAPK also plays an importantrole in autophagy and tumor suppression (13). DAPK activity canbe modulated by posttranslational modifications. Autophosphor-ylation of DAPK at Ser308 interferes with CaM binding and in-hibits DAPK catalytic activity, and this phosphorylation is down-regulated under certain death conditions (14, 15). Phosphorylationof DAPK by MAPK/ERK at Ser735 also enhances its kinase ac-tivity- and death-promoting effects (16). Recently, DAPK has beendemonstrated to contribute to the regulation of immune responses(17). However, little is known about how DAPK itself regulates ormodifies the functions and signal transduction pathways in im-mune responses at the molecular level.Mammalian target of rapamycin (mTOR) has been identified as

a downstream kinase of DAPK and shown to play a critical role inprotein synthesis and antiviral effects (18–21). Kaur et al. (22) re-cently reported that the IFN-activated mTOR pathways mediatesthe IFN antiviral responses against encephalomyocarditis virus.Zhou et al. (23) also found that the PI3K-PKB-mTOR signalingpathway restricts the replication of hepatitis E virus via 4E-bindingprotein 1 (4E-BP1). Moreover, several previous studies demon-strated the antiviral activity of mTOR in inhibition of HCV repli-cation. IFN combined with insulin had an additive anti-HCVeffect by the PI3K-mTOR pathway (24). Ishida et al. (25) showedthat p21-activated kinase 1 (PAK-1) is activated through themTOR/p70 S6 kinase pathway and suppresses HCV replication.

Significance

Death-associated protein kinase (DAPK) regulates several impor-tant biological functions through a diverse range of signaltransduction pathways, including cell growth, the immune re-sponse, apoptosis, and autophagy, but its antiviral activity hasnot been explored. Our data provide evidence that DAPK is oneof the major regulators for the pegylated IFN-α–induced antiviralactivity against hepatitis C virus, suggesting that DAPK may serveas a novel antiviral target and also a new biomarker to predict thetherapeutic efficacy of pegylated IFN-α and other antiviral agents.

Author contributions: W.-L.L., J.-H.K., and D.-S.C. designed research; W.-L.L., C.-S.H., C.-C.W.,and T.-S.W. performed research; W.-L.L., H.-C.Y., C.-S.H., J.-H.K., and D.-S.C. analyzed data;and W.-L.L., H.-C.Y., J.-H.K., and D.-S.C. wrote the paper.

Reviewers: A.M.D.B., Saint Louis University; and N.E., University of Yamanashi.

The authors declare no conflict of interest.1To whom correspondence may be addressed. Email: kaojh@ntu.edu.tw or chends@ntu.edu.tw.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1618517114/-/DCSupplemental.

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In addition, our earlier study found that RBV enhances the IFNantiviral signaling through stimulation of mTOR and p53 activ-ities to reduce HCV replication (26).To the best of our knowledge, no study has demonstrated the

antiviral activity of DAPK. Whether DAPK could exhibit antiviraleffects by regulation of mTOR activity also remains unclear. In thepresent study, we therefore examined whether DAPK played a

critical role in peg-IFN-α–induced antiviral activity against HCVreplication and the relationship between the DAPK and mTORsignaling in antiviral responses.

ResultsThe Activity of DAPKWas Induced by Peg-IFN-α and Inversely Associatedwith the Replication of HCV. We demonstrated that peg-IFN-α up-regulated the levels of total and phosphorylated DAPK proteins inboth primary hepatocytes (27) and infectious HCV cell culture(HCVcc) (Huh-7.5.1/HCV-1b: Huh7.5.1 cells were infected withJFH1-based genotype 1b) replication cells (28) in a dose-dependentmanner (Fig. 1 A and B). We also observed the up-regulation oftotal and phosphorylated DAPK proteins by peg-IFN-α in Huh-7,HepG2, and HCVcc (Huh-7.5.1/HCV-2a) replication cells (Fig.S1). However, peg-IFN-α did not increase the mRNA level ofDAPK (Fig. S2). To study whether DAPK plays a role in sup-pression of HCV replication by peg-IFN-α, we measured the levelsof HCV NS3/NS5A and phosphorylated DAPK proteins in thepeg-IFN-α–treated HCVcc (Huh-7.5.1/HCV-1b) replication cellsby immunoblots and antibodies are listed in Table S1. Along withthe increase of the peg-IFN-α dose, the reduction of HCVNS3 andNS5A proteins and HCV RNA was inversely correlated with theincrease of total and phosphorylated DAPK levels (Fig. 1B).Treatment of HCVcc replication cells with 10, 50, and 100 ng/mLof peg-IFN-α resulted in a dose-dependent reduction of HCVRNA levels by 5, 18, and 45%, respectively (Fig. 1C). The associ-ation of DAPK activation with inhibition of the HCV replication bypeg-IFN-α treatment implies that DAPK may be involved in theantiviral activity of peg-IFN-α. In addition, in HCVcc (Huh-7.5.1/HCV-1b) replication cells, RBV slightly increased theexpression of DAPK, but RBV plus peg-IFN-α exhibited agreater effect on up-regulation of DAPK activity and inhibition ofHCV replication than that observed with either peg-IFN-α orRBV alone (Fig. 1D).To exclude the possibility that the DAPK activity observed above

was induced by cell death, we measured the cytotoxic effects ofpeg-IFN-α in Huh-7.5.1 and HepG2 cells by lactate dehydrogenase(LDH) release, MTT assay, and annexin-V/propidium iodide la-beling (Fig. S3). The results showed that peg-IFN-α in these dosesdid not exert cytotoxic effects.

Knockdown of DAPK Reduced the Antiviral Activity of peg-IFN-α on HCVReplication. To confirm the critical role of DAPK in suppression ofHCV replication, we used DAPK-specific siRNA to silence theDAPK expression in the HCVcc (Huh-7.5.1/HCV-1b) replicationcells. Although peg-IFN-α suppressed the expression of NS3 andNS5A proteins by 70% and 55%, respectively, in scrambled siRNA-treated Huh-7.5.1/HCV-1b replication cells (Fig. 2A, lane 2 vs. lane 1),knockdown of DAPK by DAPK-specific siRNA significantlymitigated the suppressive effect of peg-IFN-α on HCV replication(Fig. 2A, lane 4 vs. lane 2). Actually, knockdown of DAPK almostrestored the NS3 and NS5A protein levels in peg-IFN-α–treatedcells (Fig. 2A, lane 4 vs. lane 1). Besides, peg-IFN-α only suppressedNS3 and NS5A proteins by 35% and 25%, respectively, in DAPK-silenced cells (Fig. 2A, lane 4 vs. lane 3). Likewise, peg-IFN-αreduced the HCV RNA level by 60% (Fig. 2B, lane 2 vs. lane 1,mean ± SD: 40.8 ± 10.1% vs. 100%, P < 0.01), but compared withthe scrambled siRNA treatment, silencing of DAPK expressioncould increase the HCV RNA levels by 55% in peg-IFN-α–treatedcells (Fig. 2B, lane 4 vs. lane 2, mean ± SD: 94.8 ± 5.1% vs. 40.8 ±10.1%, P < 0.01). With silencing of DAPK expression, peg-IFN-αonly inhibited the HCV RNA levels by 35% (Fig. 2B, lane 4 vs.lane 3, mean ± SD: 129.5 ± 10.6% vs. 94.8 ± 5.1%, P < 0.05).Taken together, these data indicate that knockdown of DAPKmitigated the antiviral effect of peg-IFN-α on HCV replication,suggesting its critical role in the antiviral activity of peg-IFN-α.

mTOR Plays a Critical Role in Downstream Signaling of DAPK forSuppression of HCV Replication. To investigate the antiviral activityof DAPK, we overexpressed DAPK and mutant DAPK(DAPK42A, a kinase-defective mutant) (16) as a control in HCVcc

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Fig. 1. Induction of DAPK activity by Peg-IFN-α with/without RBV was asso-ciated with inhibition of HCV replication. Peg-IFN-α–induced activity of DAPKwas associated with inhibition of HCV replication. (A) Primary human hepa-tocytes and (B) HCVcc (Huh-7.5.1/HCV-1b) replication cells were treated for24 h with the incremental doses of peg-IFN-α. The cell extracts were subjectedto immunoblotting analysis (Left). The expression levels of total DAPK, phos-phorylated DAPK (DAPK∼s735pi), NS3, and NS5 were quantified by phosphorimaging analysis and are shown in the bar graph (Right). (C) HCVcc replicationcells were treated for 24 h with incremental doses of peg-IFN-α and the in-tracellular HCV RNA levels were determined by quantitative RT-PCR. (D) RBVcombined with peg-IFN-α exerts a greater effect on increasing DAPK andinhibiting HCV viral gene expression. The HCVcc were treated with peg-IFN-α(100 ng/mL), RBV (30 μg/mL), or peg-IFN-α combined with RBV (100 ng/mL and30 μg/mL) for 2 d, and total cell lysates and RNA were extracted. The expres-sion of DAPK, phosphorylated DAPK, and HCV NS3 viral protein was analyzedby immunoblots. HCV RNA replication was detected by quantitative RT-PCRassay. Each result represents means ± SD of at least five independent experi-ments and was considered statistically significant at *P < 0.05, **P < 0.01.Control cells were not treated with either RBV or peg-IFN-α.

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(Huh-7.5.1/HCV-1b) replication cells. We showed that over-expression of WT DAPK, but not mutant DAPK, could signifi-cantly suppress HCV replication, supporting that DAPK had theantiviral activity (Fig. 3, Left, panel 3). Moreover, overexpressionof WT DAPK also inhibited the influenza virus nucleoprotein ex-pression in Madin–Darby canine kidney (MDCK) cells (Fig. S4).All these results suggested that DAPK exhibited broad antiviralactivity against both HCV and influenza virus.Previous studies showed that DAPK activated several signaling

pathways, including mitogen-activated protein kinase family (ERK,c-Jun NH2-terminal kinase, and p38), NF-κB, p53, caspase, andmTOR, to regulate its biological functions (29–31). Therefore, weexamined which signaling pathway could be activated by DAPK andresponsible for suppression of HCV replication. We found that WTDAPK enhanced the expression of mTOR but not other signalingpathways tested here (Fig. 3). However, mutant DAPK42A failedto induce the expression of mTOR and inhibit HCV replication.The antiviral activity of mTOR has been demonstrated by

previous studies. Therefore, we further investigated whetherpeg-IFN-α inhibits the replication of HCV by promoting the activityof the DAPK-mTOR pathway. We found that peg-IFN-α up-reg-ulated not only DAPK but also mTOR and its downstream sig-naling molecule PAK-1, including both total and phosphorylatedPAK-1 proteins, in HCVcc (Huh-7.5.1/HCV-1b) replication cells(Fig. 4A, lane 2 vs. lane 1). Similar results were also observed inHuh-7, HepG2, and HCVcc (Huh-7.5.1/HCV-2a) replication cellsas well (Fig. S1). Moreover, knockdown of DAPK reduced theexpression of mTOR and total and phosphorylated PAK-1 inpeg-IFN-α–treated cells, whereas knockdown of mTOR did notaffect the expression of DAPK (Fig. 4A, lanes 6 and 7 vs. lane 2).These results suggested that peg-IFN-α promoted the expression

of DAPK, which in turn promoted the downstream mTOR ac-tivity. In cells without peg-IFN-α treatment, silencing of DAPK,mTOR, or DAPK plus mTOR increased HCV NS3 proteinexpression by 30, 30, and 45%, respectively (Fig. 4A, lanes 3, 4,and 5 vs. lane 1, and Fig. 4B). Moreover, although peg-IFN-αsuppressed HCV NS3 by 65% (Fig. 4A, lane 2 vs. 1, and Fig. 4B),silencing of DAPK, mTOR, or DAPK plus mTOR significantlymitigated the antiviral effect of peg-IFN-α on HCV replication(Fig. 4A, lanes 6, 7, and 8 vs. lane 2, and Fig. 4B). We also mea-sured the effect of DAPK and mTOR on HCV RNA levels andobtained results similar to those above. Although HCVRNA levelswere reduced by 70% in cells treated with peg-IFN-α comparedwith untreated cells (Fig. 4C, lane 2 vs. lane 1, mean ± SD: 30.7 ±11.1% vs. 100%, P < 0.01), silencing of DAPK, mTOR, or DAPKplus mTOR significantly enhanced the HCV RNA levels in peg-IFN-α–treated cells (Fig. 4C, lanes 6, 7, and 8 vs. lane 2). Theseresults indicate that mTOR is critical for the antiviral activityof DAPK.

Rictor Is a Downstream Effector Molecule of DAPK for Suppression ofHCV Replication. mTOR forms two functionally and structurallydistinct complexes termed mTOR complex1 (mTORC1) andmTORC2 that differ in their upstream and downstream signalingpathways. To further explore how mTOR regulated the antiviralactivity of DAPK, we investigated the roles of the two mTORC1and mTORC2 essential components raptor and rictor, respectively,in HCV RNA replication. Whereas overexpression of rictor sig-nificantly reduced HCV replication, overexpression of raptorslightly enhanced HCV replication (Fig. 5A). Moreover, over-expression of DAPK significantly up-regulated the expression ofrictor but only slightly increased the expression of raptor (Fig. 5B,lane 2). In addition, knockdown of rictor, but not raptor, mitigatedthe antiviral activity of DAPK overexpression (Fig. 5 B and C).Taken together, these results suggested that rictor is a criticaldownstream effector molecule of DAPK for suppression ofHCV replication.

The mTOR-Independent Pathways Might Be Involved in the AntiviralActivity of DAPK. Interestingly, we found that, in cells withoverexpression of WT DAPK, silencing of mTOR could reducethe antiviral activity of DAPK but did not completely abolish it,indicating that DAPK might suppress HCV replication throughthe mTOR-independent pathways (Fig. 6). A previous studyreported that reduced eukaryotic initiation factor 2 alphasubunit (eIF2α) phosphorylation was associated with increasein HCV protein synthetic rates and viral RNA replication (32).In addition, DAPK was reported to regulate eIF2-α (33). Ourdata also confirmed that DAPK, but not mutant DAPK, up-regulated the level of phosphorylated eIF2α in the HCVcc(Huh-7.5.1/HCV-1b) replication cells. However, knockdownof mTOR did not affect the level of phosphorylated eIF2α,

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Fig. 3. The effect of DAPK overexpression on signal transduction pathwaysand HCV replication. HCVcc (Huh7.5.1/HCV-1b) replication cells were trans-fected with WT (Flag-WT-DAPK) and mutant DAPK (Flag-mt-dapk, a kinase-defective mutant). The expression levels of the candidate DAPK-regulatedgenes, including mTOR, p38, p38∼Thr180/Tyr182∼pi, p44/42, p44/42∼Thr202/Tyr204∼pi, JNK, JNK∼Thr183/Tyr185∼pi, NFκB, NFκB∼Ser536∼pi, p53,p53∼Ser15∼pi, and caspase-3, were determined by immunoblot analysis. Eachresult represents the mean ± SD of four independent determinations.

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Fig. 2. The effect of DAPK silencing on HCV replication in HCVcc (Huh7.5.1/HCV-1b) replication cells. Replication cells were transfected with DAPK siRNA(100 nM) and scrambled siRNA for 48 h and treated with peg-IFN-α (100 ng/mL)for 24 h. Total RNA and protein were extracted and analyzed. (A) The expressionof total DAPK, phosphorylated DAPK (DAPK∼s735pi), mTOR, and HCV NS3/NS5Aproteins was analyzed by immunoblots (Left) and quantified by phosphor im-aging analysis and shown in the bar graph (Right). (B) HCV RNA levels weredetermined by quantitative RT-PCR and are shown in the bar graph. Each resultrepresents the mean ± SD of five independent determinations. Statistical sig-nificance was evaluated using the Student’s t test for comparison the DAPKsiRNA-transfected cells and scrambled siRNA-transfected cells after peg-IFN-αtreatment. Statistical significance was considered at *P < 0.05, **P < 0.01.

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indicating that DAPK-induced activation of eIF2α was in-dependent of mTOR (Fig. 6). Altogether, these findings sug-gested that DAPK might suppress HCV replication partly viathe mTOR-independent pathway, probably through eIF-2αactivation.

DiscussionLittle is known about the antiviral activity of DAPK. In thisstudy, we showed that DAPK was induced by peg-IFN-α andsubsequently activated mTOR in primary human hepatocytes andHCVcc. Silencing DAPK reduced the expression of mTOR and itsdownstream effector PAK-1 and mitigated the antiviral activity ofpeg-IFN-α. Moreover, overexpression of DAPK significantlypromoted mTOR expression and then inhibited HCV replication.Silencing rictor, an essential component of mTORC2, significantlymitigated the antiviral activity of DAPK overexpression. Takentogether, these results provided convincing evidence that the

DAPK-mTOR pathway is critical for the antiviral activity of peg-IFN-α against HCV replication.RBV is a guanosine analog that has demonstrated several

effective antiviral mechanisms. Although RBV alone onlyinduces a small and early transient decrease of the HCV viralload, combination of RBV with peg-IFN-α provides a markedlysynergistic anti-HCV effect. However, the mode of action ofRBV remains mysterious. In these studies, we demonstrated thecritical role of DAPK in the suppression of HCV replication bypeg-IFN-α but also found that RBV plus peg-IFN-α exhibitsa greater effect on both the up-regulation of DAPK and thesuppression of HCV-RNA replication (Fig. 1D). The detailedmechanisms regarding how RBV up-regulates DAPK activity arecurrently under active investigation.Prior studies revealed that IFN-α induces several signaling

pathways for the suppression of HCV replication, including PKR,p56, and p53, that are independent of DAPK activation (34, 35).Consistent with this, in DAPK-knockdown cells, peg-IFN-α couldstill inhibit HCV RNA replication by 35% (Fig. 2B, lane 4 vs. lane3, mean ± SD: 94.8 ± 5.1% vs. 129.5 ± 10.6%; P < 0.05). Thisresult suggests that a DAPK-independent pathway might be in-volved in the suppression of HCV RNA expression. In HCVcc

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Fig. 4. The effect of knockdown of DAPK and mTOR alone or in combi-nation on the activity of DAPK-mTOR pathway and HCV replication. HCVcc(Huh7.5.1/HCV-1b) replication cells with or without peg-IFN-α treatmentwere transfected with 100 nM of scrambled siRNA, DAPK siRNA, mTORsiRNA, or both DAPK siRNA and mTOR siRNA for 48 h. (A) The expression ofDAPK, mTOR, PAK-1, PAK-1- phosphor-Thr-423, and HCV NS3/NS5A viralprotein was analyzed by immunoblots. (B) The levels of NS3 and NS5A in Awere quantified by phosphor imaging analysis and shown in the bar graph.(C) HCV RNA levels were determined by quantitative RT-PCR. Each resultrepresents the mean ± SD of five independent measurements and wasconsidered statistically significant at *P < 0.05, **P < 0.01.

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Fig. 5. Altered expression of rictor and raptor affected HCV replication. (A) TheWT raptor and WT rictor overexpressing plasmids were transiently transfectedinto HCVcc (Huh7.5.1/HCV-1b) replication cells. (B and C) HCVcc replication cellswere initially infected with lentivirus vectors expressing a scramble shRNA orshRNAs targeting raptor and rictor (150 nM). After infection and selection, cellswere then transfected with the WT-DAPK-expression plasmid. In A and B, theexpression of rictor, raptor, and HCV NS3 proteins was analyzed by immunoblots(Left) and quantified by phosphor imaging analysis and shown in the bar graph(Right). In C, HCV RNA levels were determined by quantitative RT-PCR andshown in the bar graph. Each result represents the mean ± SD of four in-dependent determinations. Statistical significance was evaluated using the Stu-dent’s t test and *P < 0.05, **P < 0.01.

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replication cells without knockdown of DAPK, peg-IFN-α caused60% suppression of HCV RNA replication (Fig. 2B, lane 2 vs.lane 1, mean ± SD: 40.8 ± 10.1% vs. 100%; P < 0.05), probablydue to both DAPK-dependent and DAPK-independent mecha-nisms. Therefore, this 25% difference (60% vs. 35%) in suppres-sion was attributable to DAPK’s function in the suppression ofHCV RNA replication. The DAPK-independent antiviral path-ways deserve further investigation.We clearly showed that peg-IFN-α activated the DAPK-mTOR

pathways, which contributed to the suppression of HCV replication.However, silencing of mTOR failed to completely eliminate theantiviral activity of DAPK in DAPK-overexpressing HCVcc repli-cation cells, although it significantly enhanced HCV replication.This suggests that, in addition to mTOR, DAPK may stimulateother kinases or cellular factors that lead to activation of mTOR-independent antiviral pathways (Figs. 4 and 6). A previous studyhas shown that a reduced level of eIF2α phosphorylation was as-sociated with increases in HCV replication. In addition, DAPK wasreported to involve the up-regulation of eIF2-α expression (33). Inthis study, overexpression of WT DAPK in the HCVcc caused theup-regulation of mTOR and phosphorylated eIF2α levels and re-pressed HCV replication. Knockdown of mTOR did not affect theexpression of phosphorylated eIF2-α in DAPK-overexpressing cells,suggesting that the eIF2-α signaling pathway probably involves themTOR-independent antiviral action of DAPK.The potential roles of DAPK, mTOR, PAK-1, and the eIF2-α

kinase pathway, as defined in the present study, are depicted in Fig.7. Peg-IFN-α has the capacity to activate DAPK, which can inducethe expression of mTOR/PAK-1 and phosphorylated eIF2-α andthus in turn promote an antiviral state of host cells. During theantiviral response, DAPK can suppress HCV replication throughboth the phosphorylation of eIF2α and the mTOR/PAK-1 pathway(Figs. 4 and 6). Thus, our data revealed signal transductionpathways responsible for the anti-HCV activity of peg-IFN-α.Whether these pathways work for the inhibition of HCV replicationby IFN-free DAAs awaits further studies.mTOR complexes contain two distinct protein complexes and

regulate different branches of the mTOR network. mTORC1,which contains mTOR, GβL, and raptor, directly phosphorylatesribosomal S6 kinase and eukaryotic translation initiation factor 4E-BP1 to regulate cell growth and protein synthesis (36). mTORC2contains mTOR, GβL, and rictor and directly phosphorylates theprosurvival kinase Akt/PKB, but its cellular function is just begin-ning to be understood (37). Recently, Stöhr et al. (38) reportedthat the host cell mTORC1/raptor is required for HCV replication,rather than suppression of HCV replication. To clarify the seem-ingly contradictory roles of mTOR in their and our studies, we

dissected the antiviral effects of raptor (mTORC1) and rictor(mTORC2) downstream of DAPK. Consistent with their find-ings, overexpression of raptor slightly enhanced HCV NS3 pro-tein expression (Fig. 5A). In cells with overexpression of the WTDAPK, knockdown of raptor in HCVcc cells further decreasedHCV RNA replication. In contrast, overexpression of DAPKsignificantly increased the expression of rictor, but not raptor(Fig. 5B). Knockdown of rictor mitigated the antiviral activityof DAPK overexpression. In line with our findings, several studiesindicated that rictor can phosphorylate Akt and subsequently ac-tivate PAK-1 (37, 39, 40). Taken together, these results indicatethat rictor is the critical effector molecule of mTOR complexes indownstream signaling of DAPK for suppression of HCV replica-tion. How rictor, but not raptor, involves the suppression of HCVreplication requires further investigation.In summary, our results indicate that DAPK is up-regulated by

peg-IFN-α and subsequently activates the mTOR-rictor and themTOR-independent pathways to suppress the HCV replication.Herein, our data provide evidence that DAPK is one of the majorregulators for the antiviral activity of peg-IFN-α against HCVreplication. Understanding the antiviral role of DAPKmay improvethe effectiveness of IFN-based therapy against chronic viral infec-tions, including the treatment of hepatitis B virus (41, 42).

Materials and MethodsSee SI Materials and Methods for more information.

Cell Lines and Viral Propagation and Titration. HCVcc replication cells, JFH1genotype 2a virus, and JFH1-based intergenotypic recombinants genotype 1bstrain to infect Huh7.5.1 cells (a gift from T. Jake Liang, National Institutes ofHealth, Bethesda) were maintained in DMEM containing 10% FBS and peni-cillin, streptomycin, and glutamine. HCV JFH-1b and -2a strains were propa-gated and infectivity titrated as previously described (28). HCV infection wasapplied to cell culture experiments at a multiplicity of infection (MOI) of 0.5,and assays were typically performed at 48 h postinfection. Peg-IFN-α2b(Schering Plough) and RBV (Sigma-Aldrich) were used for the experiments.

Plasmid and Transient Transfection. pRK5- and pBabepuro-based expressionvectors for WT DAPK and mutant-DAPK42A (mt-DAPK; a kinase-defectivemutant) were gifts from Ruey-Hwa Chen, Institute of Biological Chemistry,Academia Sinica, Taipei, Taiwan(16). The WT raptor and WT rictor plasmids

0

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120A

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Fig. 6. eIF2-α is activated by overexpression of DAPK. The effects of over-expression of WT DAPK (Flag-WT-DAPK) and mutant-dapk (Flag-mt-dapk)and knockdown mTOR on eIF2-α and HCV replication were analyzed inHCVcc (HCV-1b/Huh7.5.1) replication cells. (A) Replication of HCV, mTOR,eIF2-α, and phospho-eIF2-α were determined by immunoblotting. (B) HCVRNA levels were measured by quantitative RT-PCR. Each result represents themean ± SD of five independent measurements and is considered statisticallysignificant at *P < 0.05, **P < 0.01.

peg-IFN-α

HCV replication

DAPK-dependent

mTOR

PAK-1

eIF-2α

DAPK-independent (PKR, p56, Mx, …)

Fig. 7. Schematic diagram of the DAPK signal transduction pathways in sup-pression of HCV replication by peg-IFN-α. Our results demonstrate that DAPK isone of the important regulators mediating the antiviral activity of peg-IFN-α.During the antiviral response, DAPK can function in parallel to suppress HCVreplication through activation of mTOR/PAK-1 and phosphorylation of eIF2α.Additionally, peg-IFN-α induces several signaling pathways for suppression ofHCV replication, including PKR, p56, and p53 that are independent of DAPKactivation. We propose that the antiviral response of peg-IFN-α against HCV isby promoting the DAPK-dependent and -independent pathway.

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were obtained from Addgene. Plasmids were transiently transfected into cellsusing the GenJet in vitro DNA transfection reagent kit (SignaGen) according tothe manufacturer’s instructions.

siRNA and Transfection. For the knockdown of DAPK and mTOR gene ex-pression, cells were transfected with DAPK and mTOR siRNA (Dharmacon) andscrambled control siRNAusing the siIMPORTERTMkit (Upstate) according to themanufacturer’s instructions.

Lentiviral Vector-Mediated shRNA Knockdown of Rictor and Raptor. Plasmidsencoding lentiviruses that express either scramble sequence or shRNAagainst rictor (plasmids 1853; Addgene) and raptor (plasmids 1857;

Addgene) have been described (43). Lentiviruses were prepared in 293FTcells following the established protocols. HCVcc (Huh7.5.1/HCV-1b) wereinfected twice with lentivirus. Recombinant lentivirus infected cells wereenriched by puromycin selection for 5–7 d.

Statistical Analysis. Data were expressed as means ± SEMs. The differencebetween the control and peg-IFN-α–treated cells was evaluated using a Stu-dent’s t test and a P value less than 0.05 was considered statistically significant.

ACKNOWLEDGMENTS. This work was supported by National Science CouncilGrant 103-2314-B-002-51-MY3, the Jin-Kang Hepatology Research Fund, andNational Taiwan University College of Medicine.

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