The autophagy machinery is required to initiatehepatitis C virus replicationMarlene Dreux, Pablo Gastaminza, Stefan F. Wieland, and Francis V. Chisari1

Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA 92037

Contributed by Francis V. Chisari, July 2, 2009 (sent for review April 10, 2009)

In addition to its cellular homeostasis function, autophagy isemerging as a central component of antimicrobial host defenseagainst diverse infections. To counteract this mechanism, manypathogens have evolved to evade, subvert, or exploit autophagy.Here, we report that autophagy proteins (i.e., Beclin-1, Atg4B,Atg5, and Atg12) are proviral factors required for translation ofincoming hepatitis C virus (HCV) RNA and, thereby, for initiation ofHCV replication, but they are not required once infection is estab-lished. These results illustrate a previously unappreciated role forautophagy in the establishment of a viral infection and theysuggest that different host factors regulate the translation ofincoming viral genome and translation of progeny HCV RNA oncereplication is established.

flaviviridae � autophagosome � LC3 � ATG � translation

HCV infection is a leading cause of chronic liver diseaseworld-wide. With 180 million persistently infected people,

chronic hepatitis C infection, which induces end stage liverdiseases, such as liver cirrhosis and hepatocellular carcinoma(HCC), represents a major public health problem of highsocioeconomic impact (1). However, treatment options forchronic hepatitis C are limited and a vaccine against HCV is notavailable. Thus, efforts to elucidate the details of the host-virusrelationship during HCV infection are needed to develop effi-cient therapeutic strategies against this major human pathogen.

HCV is an enveloped, positive strand RNA virus in theFlaviviridae family. The HCV genome is approximately 9.6 kb inlength and consists of a single ORF flanked by untranslatedregions (UTR) (2). Translation of the single ORF is driven by aninternal ribosomal entry site (IRES) sequence present within the5�UTR. The resulting polyprotein is processed by cellular andviral proteases into the structural proteins (core, E1, and E2) andthe nonstructural proteins (p7, NS2, NS3, NS4A, NS4B, NS5A,and NS5B) (2). HCV NS3 to NS5B proteins are necessary andsufficient to establish membrane-bound replication complexesthat catalyze RNA replication (2).

Autophagy is an intracellular degradation process in whichcytoplasmic components, including organelles, are directed tothe lysosome by a membrane-mediated process (3). More than31 autophagy-related genes (ATG) whose products regulateautophagy have been identified, primarily through yeast genet-ics, and several have mammalian counterparts (3). They regulatedistinct aspects of autophagic vesicle formation and maturation,including the assembly of an isolation membrane around aportion of cytoplasm, completion of a double-membrane-surrounded autophagosome, and lysosomal fusion with degra-dation of its contents (3). Importantly, many studies demonstratethat autophagy is activated upon viral or bacterial infection (4).In Sindbis virus, tobacco mosaic virus, herpes simplex virus type1 (HSV-1) and several bacterial infections, autophagy may havea protective function by restricting intracellular pathogen repli-cation or by ensuring the survival of infected and/or uninfectedcells (4). In this regard, autophagy serves as an innate hostdefense mechanism, and some viruses and bacteria producevirulence factors that counteract these antimicrobial processes(4–7). Conversely, certain viruses, for example, mouse hepatitis

virus, poliovirus, coxsackievirus and dengue virus, actually ex-ploit the elements of the autophagy system for their replication(8–12).

In this study, by manipulating the expression and function ofkey autophagy regulators, we show that these proteins arerequired for productive HCV infection. Moreover, by dissectingthe individual steps of HCV infection, we demonstrate thatautophagy is required for translation and/or delivery of incomingviral RNA to the translation apparatus.

ResultsEnhanced Autophagic Vesicle Content in HCV-Infected Cells. A hall-mark of autophagy induction is lipidation of the microtubule-associate protein 1 light chain 3 (LC3) (13). A series of covalenttransfers leads to phosphatidylethanolamine (PE) conjugation toLC3, termed LC3-II, which becomes membrane-associated andparticipates in autophagosome formation (3, 14, 15). To deter-mine whether JFH-1-HCV infection regulates autophagy, weexamined the conversion of endogenous LC3 to LC3 II. Theintensity of LC3-II band was increased in HCV infected cellsrelative to uninfected (mock) cells (Fig. 1A). In addition, co-migration of LC3-II in infected cells and in cells treated withautophagy inducers, for example, the proteasome inhibitoracetyl-L-leucyl-L-leucyl-L-norleucinal (ALLN) and the ER stressinducer thapsigargin (Tg) (16, 17), confirmed that the banddetected was authentic LC3-II. Another hallmark of autophagyis the redistribution of LC3 from a diffuse cytoplasmic localiza-tion to a characteristic punctate cytoplasmic pattern reflectingthe recruitment of LC3 to autophagic vesicles (13, 14). Indeed,in cells transfected with a GFP-tagged LC3 expression vector(LC3-GFP), LC3-GFP redistribution into discrete dots wasmarkedly increased by HCV infection (Fig. 1B and C), furtherconfirming the accumulation of autophagic vesicles upon HCVinfection.

The Autophagy Machinery Is Required for the HCV Life Cycle. Todetermine whether the autophagy machinery performs an an-tiviral or proviral function during HCV infection, we monitoredthe effect of shRNA-mediated knock-down of key autophagyproteins on the establishment of HCV infection. We targetedBeclin-1 and Atg4B, because Beclin-1, in complex with Vps34,the class III phosphatidylinositol-3-kinase [PI(3)K] promotesformation of autophagic vesicles (18), and Atg4B is a cysteine-protease essential for LC3 lipidation during autophagosomeformation and de-lipidation for recycling after autophagosomematuration (14, 15, 19–22). As shown in Fig. 2A, Beclin-1 andAtg4B expression was strongly inhibited in Huh-7 cells bylentivirus-based vector-mediated shRNA technology. As ex-pected, the induction of autophagy by thapsigargin (Tg) wasmarkedly decreased in these cells, as measured by the frequency

Author contributions: M.D., P.G., S.F.W., and F.V.C. designed research; M.D. performedresearch; M.D., P.G., S.F.W., and F.V.C. analyzed data; and M.D. and F.V.C. wrote the paper.

The authors declare no conflict of interest.

1To whom correspondence should be addressed. E-mail: fchisari@scripps.edu.

This article contains supporting information online at www.pnas.org/cgi/content/full/0907344106/DCSupplemental.

14046–14051 � PNAS � August 18, 2009 � vol. 106 � no. 33 www.pnas.org�cgi�doi�10.1073�pnas.0907344106

Dow

nloa

ded

by g

uest

on

June

3, 2

020

of cells containing punctate LC3-GFP (Fig. S1), confirming thereduction of autophagic vesicle formation that has been previ-ously described for other cell types (23, 24).

Next, Atg4B- and Beclin-1-deficient cells were infected withHCV at low multiplicity of infection (MOI) and HCV expansionwas measured over the next 6 days by determining extracellularinfectious viral titers. This analysis revealed that, HCV expan-sion was suppressed up to 100-fold in Atg4B-deficient cells andalmost completely abolished in Beclin-1-deficient cells as com-pared to control cells (Fig. 2B). The intracellular HCV RNAcontent paralleled extracellular HCV infectivity with reductionsof up to 10,000-fold in Atg4B- and Beclin-1-down-regulated cells(Fig. 2C). To confirm and extend these observations, we alsotested the effect of shRNA-mediated inhibition of Atg12, acritical component of a ubiquitin-like conjugation system thatenables elongation of the autophagosome (3). As expected,because LC3 lipidation depends on Atg5 and its conjugation withAtg12 (25, 26), both LC3-II species and the induced punctateGFP-LC3 distribution were reduced in Atg12 shRNA-treatedHuh-7 cells as compared to control cells (Fig. S2 A). Importantly,HCV expansion was greatly reduced in Atg12-deficient cells(Fig. S2B), reinforcing the requirement of autophagy machineryproteins for efficient HCV infection. Consistent with theseobservations, ectopic expression of a dominant negative Atg5mutant (Atg5K130R) that is defective for conjugation with Atg12and suppresses autophagic vesicle formation (25, 27) also inhib-ited HCV infection (Fig. S3).

Collectively, these results demonstrate that key proteins re-sponsible for different steps of autophagic vesicle formation arerequired for initiation of HCV infection in Huh-7 cells and thusrepresent proviral factors for HCV.

The Autophagy Machinery Is Not Required for HCV Particle Entry orSecretion. To identify the autophagy-dependent step(s) in theHCV life cycle, we performed single cycle infections in Beclin-1-,Atg4B-, and Atg12-deficient and control cells. Intracellular andextracellular (i.e., secreted) infectious particle production werereduced to a similar degree in Beclin-1-, Atg4B-, and Atg12-deficient cells, (Fig. 3A and Fig. S2C), indicating that, in contrastto the role of autophagy proteins in poliovirus particle release(8), the autophagy machinery is not required for HCV particlesecretion. Moreover, accumulation of intracellular HCV RNA

was also significantly reduced in autophagy protein-down-regulated cells (Fig. 3A and Fig. S2C), suggesting that theautophagy machinery is required for either virus entry, RNAtranslation or replication.

To examine the dependence of HCV entry on autophagy, westudied the impact of shRNA-mediated inhibition of autophagyprotein expression on HCV pseudoparticle (HCVpp) entry intoHuh7 cells (28). Autophagy protein-down-regulated and controlcells were infected with HCVpp that contain envelope glycop-roteins from the JFH-1 (genotype 2a) and H77 (genotype 1a)strains of HCV. As shown in Fig. 3B and Fig. S2D, HCVpp entrywas comparable in autophagy protein-deficient and control cells(Fig. 3B and Fig. S2D). Collectively, these results suggest that theautophagy machinery is required for HCV infection at a post-entry step, most likely HCV RNA translation and/or replication.

The Autophagy Machinery Is Required for HCV Replicon Replication.To determine whether the autophagy machinery modulatesHCV RNA translation and/or replication, we examined theability of autophagy protein-down-regulated cells to support

Fig. 1. Enhanced autophagic vesicle content in HCV-infected cells. (A)Analysis by immnunoblotting of endogenous LC3 lipidation in cell extracts ofHuh-7 cells infected for 24 h with HCV or treated with thapsigargin (Tg) (500nM) or with ALLN (10 �M) for 16 h, or untreated (mock), as indicated. �-actinexpression was examined as a protein loading control. The gels are represen-tative of three independent experiments. (B) Representative confocal images.LC3-GFP transfected Huh-7 cells were grown in normal medium (mock),infected for 24 h with HCV, or treated with ALLN or with Tg, as described in A.The nuclei were stained by Hoechst solution (blue). (C) Quantification of thefrequency of Huh-7 cells displaying a punctate distribution of LC3-GFP. Resultsrepresent the means of two independent experiments.

Fig. 2. Autophagy machinery is required for HCV spread. Huh-7 cells weretransduced with lentiviral vectors expressing shRNA against Beclin-1 (shRNAdenoted as sh49 and sh50) or Atg4B (sh800 and sh801). The selected shRNAsequence and multiplicity of infection (MOI) used to transduce Huh-7 cells didnot compromise the cell viability. At 8 days posttransduction, transduced andcontrol cells were harvested and seeded in equal numbers. Twenty andseventy-two hours later MTT was added and cellular proliferation was mon-itored. The results for the Beclin-1-(sh49) transduced cells were 99.3 � 27.3%of the nontransduced cells, set to 100. (A) The relative abundance of Beclin-1and Atg4B proteins in the Huh-7 cells was analyzed by immunoblotting.�-actin expression was used as a protein loading control. (B) Beclin-1- andAtg4B-deficient and nontransduced (control) Huh-7 cells were infected withHCV at an MOI of 0.01. The infectivity of their virus-containing cell superna-tants was determined at different times postinoculation, as indicated. Resultsdisplay average infectious titers, expressed as focus-forming units (ffu)/ml(mean � SD; n � 2). (C) The intracellular HCV RNA levels were determined byRT-qPCR 6 days postinoculation (MOI of 0.01) in Beclin-1- and Atg4B-deficientand control cells (mean � SD; n � 2). GE, genome equivalent.

Dreux et al. PNAS � August 18, 2009 � vol. 106 � no. 33 � 14047

MIC

ROBI

OLO

GY

Dow

nloa

ded

by g

uest

on

June

3, 2

020

replication of an HCV subgenomic replicon. Identical amountsof JFH-1 subgenomic replicon RNA containing the neomycinmarker gene (neo/SGR) were introduced into autophagy pro-tein-down-regulated and control cells. Replicon-replicating cellswere selected for neomycin resistance as described previously(29). As shown in Fig. 4A and Fig. S4A, down-regulation ofBeclin-1, Atg4B, and Atg12 strongly suppressed JFH-1 repliconreplication despite comparable RNA transfection efficiency(Fig. S5A) and cell viability as determined by trypan bluestaining. Similar results were obtained using a Con1 (genotype1b) subgenomic replicon (Fig. 4A, Fig. S4A, and Fig. S5B). Torule out the possibility of nonspecific effects on neomycinsensitivity by suppression of the autophagy machinery, weconfirmed these findings by using a subgenomic reporter repli-

con containing the renilla luciferase gene (Rluc/SGR). While thetransfection efficiency was comparable in all cell populations(Fig. S5C), the Rluc activity was reduced by up to 50-fold at alltime points posttransfection in Beclin-1, Atg4B, and Atg12-deficient cells (Fig. 4B and Fig. S4B). Collectively, these resultsindicated that autophagy machinery proteins are required forRNA translation and/or replication.

Next, we asked whether autophagosomes might facilitate thetethering of HCV replication complex proteins to cellular mem-branes, a process that is known to be required for HCVreplication (2). Consistent with a previous report using the H77

Fig. 3. The autophagy machinery regulates a postentry step, upstream or atthe level of HCV replication. Huh-7 cells were transduced or not (control) withlentiviral vectors expressing shRNA against Beclin-1, Atg4B, and GFP. (A)Extracellular and intracellular infectious virus and intracellular RNA produc-tion during 24 h in a single step infection at MOI of 10 (mean � SD; n � 3).(Upper) Accumulation of extracellular and intracellular HCV infectious parti-cles was determined 24 h after inoculation, and expressed as a percentage ofthat in control cells. (Lower) Intracellular HCV RNA content was determined at24 h post inoculation and expressed as a percentage of that in control cells. (B)Cell entry of HCVpp harboring E1E2 glycoproteins derived from HCV strainsH77 (1a) or JHF-1 (2a) and control particles harboring the VSV-G glycoprotein(VSV-Gpp) expressed as a percentage of that in control cells (mean � SD; n �4). To assess the specificity of E1E2 glycoprotein-mediated cell entry, anti-E2antibody (Ab) was added at 20 �g/mL. ND, not determined.

Fig. 4. Autophagy machinery regulates translation and/or delivery of in-coming viral RNA to the translation apparatus. Huh-7 cells were transducedwith lentivirus expressing shRNA against Beclin-1, Atg4B, and GFP. (A) Analysisof JFH1 and Con1 neo/SGR replication. Neomycin selected cells were fixed andstained with Crystal Violet. A replication defective GND mutant (GDD-to-GNDmutation in the NS5B protein) neo/SGR was used as a positive control for theneomycin selection. Results are representative of two independent experi-ments. (B) Analysis of Rluc/SGR translation/replication by monitoring Rlucactivity at different times posttransfection. To assess the specificity of Rlucactivity, control cells were treated with 5 �M of 2�-C-methyladenosine, a HCVpolymerase inhibitor (denoted inh). For each independent experiment, Rlucactivity was normalized to cell density and expressed as a percentage of thatdetermined in control cells at 72 h posttransfection (mean � SD; n � 4). (C)Translation of replication-deficient Rluc/SGR RNAs. Intracellular RNA levelsand Rluc activity of replication deficient Rluc/SGR were determined at 6 hposttransfection. Rluc activities are statistically reduced in autophagy proteindeficient cells compared to shRNA GFP expressing cells (P values �0.05 frompaired Student’s t test). In parallel transfections, Rluc activity expressed frompRL-TK plasmid (irrelevant-Rluc activity) was determined. For each indepen-dent experiment, Rluc activity was normalized to cell density and expressed asa percentage of that determined in control cells (mean � SD; n � 3).

14048 � www.pnas.org�cgi�doi�10.1073�pnas.0907344106 Dreux et al.

Dow

nloa

ded

by g

uest

on

June

3, 2

020

strain of HCV (30), we did not observe any colocalization ofHCV JFH1 proteins involved in viral replication (NS4A/4B andNS5A) with LC3-GFP in infected cells (Fig. S6), in the absenceor presence of the vesicle acidification inhibitor Bafilomycin A1(Fig. S7). These results suggest that the autophagy machinery ismost likely not required to maintain an active HCV replicationcomplex.

The Autophagy Machinery Is Required for Translation and/or Deliveryof Incoming Viral RNA to the Translation Apparatus. Interestingly,Rluc activity was compromised in autophagy protein-deficientcells �3-fold already at 5 h posttransfection, suggesting thatautophagy proteins might play a role in HCV RNA translationinitiation. To specifically monitor viral translation, we used areplication-defective Rluc/SGR construct that contains an inac-tivation mutation (GDD-to-GND) in the active site of the viralpolymerase (NS5B), and we analyzed Rluc activity expressedfrom the HCV IRES, as previously described (31). While thelevel of intracellular Rluc/SGR RNA was comparable in auto-phagy protein-deficient and control cells (Fig. 4C and Fig. S4C),HCV IRES-dependent translation measured by Rluc activitywas strongly reduced in Beclin-1, Atg4B, and Atg12-down-regulated cells as compared to control and GFP shRNA-expressing cells (Fig. 4C and Fig. S4C). Reduced Rluc expressionwas not due to a general effect on ectopic gene translationefficiency or protein stability, since Rluc activity derived fromcap-dependent translation of a RNA polymerase II transcribedRNA was not significantly different in autophagy protein-deficient and control cells (Fig. 4C and Fig. S4C). Collectively,these results suggested that the autophagy machinery is requiredfor HCV IRES-dependent translation of the incoming viralRNA, or for delivery of the incoming RNA to the translationapparatus, perhaps by targeting it to the appropriate cellularfactor(s) or location.

The Autophagy Machinery Is Required for the Initiation of HCVReplication But Not to Maintain It. Our results point to a crucial roleof the autophagy machinery for translation of incoming viralRNA and the establishment of replication either in HCV RNAtransfected cells (Fig. 4 and Fig. S4) or in de novo infected cells(Figs. 2 and 3 and Figs. S2 and S3). To determine whetherautophagy proteins are required only for initiation of translationand the establishment of HCV replication or if they are alsorequired to maintain ongoing HCV translation and replication,we analyzed the impact of autophagy protein down-regulation onHCV RNA and protein content in stable replicon cells. WhereasBeclin-1 and Atg4B protein content was greatly reduced inshRNA-transduced replicon cells (Fig. 5A), HCV RNA andprotein (illustrated by NS5A expression) levels were unchanged(Fig. 5A). Similar results were obtained in Huh-7 cells harboringH77 (genotype 1a) and Con1 (genotype 1b) subgenomic repli-cons or a JFH-1 full-length replicon (Fig. 5A), suggesting that theautophagy machinery is not required to maintain ongoing HCVreplication.

To rule out the possibility that stable replicon cells might beartefactually autophagy-independent because of their priorselection, Beclin-1, Atg4B, and Atg12 were down-regulated innonselected infected cells virtually all of which were HCV E2positive (Fig. S4D). While Beclin-1 and Atg4B expression (Fig.5B) and LC3 lipidation (Fig. S4E) were strongly reduced inthese cells, intracellular levels of infectious particles and HCVRNA were unchanged (Fig. 5B and Fig. S4E), confirming thatthe autophagy machinery is not required to maintain replica-tion of established infection. Furthermore, production ofinfectious virus particles (Fig. 5B and Fig. S4E) were notsignificantly reduced in down-regulated cells, confirming thatautophagy proteins aren’t required for HCV particle secretion(Fig. 3A).

Collectively, these results indicate that the autophagy machin-ery is required for translation of incoming RNA and, therefore,HCV replication in de novo infected cells, but not for translationof progeny HCV RNA once replication is established.

DiscussionIn this study, we analyzed the impact on HCV infection of severalproteins that regulate distinct molecular events leading to au-tophagy vesicle formation, including its initiation (e.g., Beclin-1),and maturation by the Atg12 (e.g., Atg12 and Atg5) and LC3(e.g., Atg4B) conjugation systems. We showed that dominantnegative inhibition and down-regulation of different regulatorsof the autophagy pathway strongly suppress productive HCVinfection. Our results are in agreement with a recent reportsuggesting that the down-regulation of LC3 and Atg7, a proteinthat mediates LC3 lipidation, decreases the HCV RNA contentin cells electroporated with JFH-1 HCV RNA (32). In addition,our results demonstrate that autophagy machinery proteinsmodulate a post entry-step of HCV infection and, through theanalysis of single step infection assays, reveal a functional

Fig. 5. Autophagy machinery does not regulate established HCV replication.(A) Replicon bearing-Huh-7 cells were transduced with lentiviral vectors ex-pressing shRNAs against Beclin-1, Atg4B, and GFP. Protein and RNA levelswere determined 10 days posttransduction. (Upper Left) NS5A protein levelsin replicon cells as analyzed by immunoblotting. (Upper Right) IntracellularHCV RNA levels in H77 (1a), con-1 (1b), JFH-1 (2a) SGR, or JFH-1 full-length (FLR)replicon-bearing Huh-7 cells monitored by RT-qPCR (mean � SD; n � 2). GE,genome equivalent. (Lower) Efficiency of down-regulation of Beclin-1, Atg4B,as analyzed by immunoblotting. Beclin-1 and Atg4B content were analyzed inestablished JFH-1 SGR replicon cells (denoted Replicon cells) and were com-pared to shRNA-treated cells before HCV infection (denoted DR before infec-tion). �-actin expression was used as a protein loading control. (B) Huh-7 cellsthat were virtually all infected by HCV were transduced with lentiviral vectorsexpressing shRNA against Beclin-1, Atg4B. (Left) Relative levels of Beclin-1 andAtg4B in the infected cells, as analyzed by immunoblotting. �-actin expressionwas used as a protein loading control. (Right) Extracellular and intracellularinfectivity and intracellular HCV RNA levels were determined 8 days posttrans-duction and are expressed as a percentage of those in control cells (mean � SD;n � 2).

Dreux et al. PNAS � August 18, 2009 � vol. 106 � no. 33 � 14049

MIC

ROBI

OLO

GY

Dow

nloa

ded

by g

uest

on

June

3, 2

020

interaction upstream or at of the level of RNA replication.Consistent with those results, we showed that the autophagymachinery is required for the translation and/or delivery ofincoming viral RNA to the translation apparatus and, therefore,for the establishment of productive infection. Importantly, ourresults demonstrate that autophagy proteins are required for theinitiation of HCV RNA translation/replication, but not oncethese processes are established, indicating that different hostfactors are required for translation of the incoming viral RNAversus the progeny HCV RNA once replication is established.

Our results indicate that the content of autophagy vesicles isincreased in HCV-infected cells, extending recent results ob-tained in cells infected with the H77 strain of HCV and in cellstransfected with JFH1 strain HCV RNA (30, 32). Sir et al. (32)proposed that the unfolded protein response (UPR) contributesto the induction of autophagy in HCV RNA-transfected cells.However, our results point to a role of autophagy proteins at avery early stage of the HCV life cycle, when HCV proteins areproduced from the incoming RNA, which should be independentof the UPR. Interestingly, for other pathogens, pattern recog-nition receptors (PRR) and the innate signaling pathway havebeen reported to trigger autophagy in infected cells (4). Notably,cytosolic RNA-sensing protein kinase PKR and eIF2� phos-phorylation regulate virus- and starvation-induced autophagy(7, 33). It is tempting to speculate that recognition of theincoming HCV RNA by RNA-sensing molecules induces auto-phagy and hence, favors its initial translation. Alternatively,constitutive basal autophagic vesicle formation might be re-quired for initial HCV RNA translation. In this regard, weobserved a reduction of LC3 lipidation in Atg12-deficient Huh-7cells even in the absence of an autophagy inducer (Fig. S2 A),that was associated with a reduction in susceptibility of thesecells to HCV infection. Therefore, it is likely that the establish-ment of HCV infection depends on the basal level of autophagyrather than its induction.

Formation of a membrane-associated replication complex,composed of viral proteins, replicating RNA and altered cellularmembranes, is a hallmark of all positive-strand RNA virusesinvestigated so far (34). HCV proteins form a multiproteincomplex in an intracellular membrane complex, thought to bederived from ER membranes, which is the site of RNA repli-cation (2, 35). Interestingly, the viral components of certainpositive-strand RNA viruses (e.g., poliovirus, mouse hepatitisvirus and dengue virus) that subvert the autophagy machineryfor their replication, co-localize with autophagy markers, sug-gesting that the translation/replication complexes of those vi-ruses might assemble on autophagic vesicles (8, 10–12). How-ever, HCV proteins do not colocalize with autophagy proteins ininfected cells suggesting that the HCV replication complex doesnot assemble on autophagic vesicles but rather, that HCVexploits the autophagy pathway by a currently undefined mech-anism that appears to be different from other autophagy-dependent viruses. It is conceivable that the autophagy pathwaysupports HCV replication by sequestering or destroying HCVreplication restriction factor(s). Alternatively, it is tempting tospeculate that the autophagy pathway might provide an initialmembranous support for translation of incoming RNA, beforeaccumulation of viral proteins and eventual virus-induced cel-lular modifications. The lack of an effect of autophagy proteindown-regulation on previously established HCV replicationsupports this hypothesis.

How the incoming HCV RNA is targeted to the appropriatecellular factors or location for initial translation remains un-known. It is believed that IRES-mediated HCV genome trans-lation, polyprotein processing and RNA replication occur withinER-derived membranes. Notably, an internal signal sequencelocated at the C terminus of the core protein targets the nascentpolypeptide to the ER membrane for translocation of E1 gly-

coprotein into the ER lumen (2). Interestingly, a model ofautophagosome maturation from specialized domains of the ERhas been proposed (36–38). Therefore, the possible continuumbetween ER and autophagic vesicles and/or autophagy-inducedlocal ER rearrangements, through a dynamic connection be-tween ER and autophagy vesicles (37), might be of importance,by providing a specialized platform for translation of the incom-ing HCV RNA.

Materials and MethodsPlasmid and Reagents. The antibodies used for immunoblotting and immu-nostaining were specific for E2 (C1); NS5A (MS5); Beclin-1 (Santa Cruz Biotech-nology); Atg4B (Abcam); LC3 (Novus Biologicals); actin (Sigma Aldrich). Acetyl-L-leucyl-L-leucyl-L-norleucinal (ALLN) and thapsigargin (Tg) were purchasedfrom Sigma Aldrich. 2�-C-methyladenosine was kindly provided by W. Zhong(Gilead Sciences). The SG and Full-length JFH-1-replicon have been previouslydescribed (39). The subgenomic (SG)-Con1 replicon (S1179I) was kindly pro-vided by C. Rice (Rockefeller University, New York, NY) (40). The SG-H77replicon (Htat2ANeo/QR/VI/KR//KR5A/SI) bearing Huh-7 cells were kindly pro-vided by S. Lemon (University of Texas Medical Branch, Galveston, TX) (41). TheJFH-1 Rluc/SGR or GND JFH-1 Rluc/SGR plasmids carry bicistronic RNA contain-ing the luciferase reporter gene in the first cistron and wild-type (wt) orreplication-deficient (encoding a GDD-to-GND mutation in NS5B) JFH-1 sub-genomic replicon in the second cistron, respectively.

Lentiviral Particle Production and Huh-7 Cell Transduction. Vesicular stomatitisvirus glycoprotein (VSV-G)-pseudotyped lentiviral particles were produced in293T cells by co-transfection of lentiviral vector encoding short hairpin RNAs(shRNAs) (Mission, Sigma Aldrich) with vectors encoding compatible packag-ing proteins and VSV-G, as described previously (42). Forty-eight hours post-transfection, cell supernatants were collected, filtrated, and used to transduceHuh-7 cells. Specific shRNA clones were selected according to the targetprotein down-regulation efficiency and the absence of cytotoxic effects. Forall of the experiments displayed, we used freshly shRNA transduced Huh-7 toavoid any compensatory cellular mechanism in autophagy deficient cells thatmight misleading the interpretation of the results. The sequence of the shRNAare described in SI Text.

Preparation of Viral Stock and Infection. JFH-1 virus was generated by trans-fection, and viral stocks were produced by infection of Huh-7 cells at amultiplicity of infection (MOI) of 0.01, as described previously (43). High-titerstocks of cell-culture adapted virus (D183) (29) were prepared by infection ofa Huh-7.5.1 cell subclone (clone 2) and used for single step infection experi-ments (MOI of 10). Cell extracts containing intracellular infectious HCV par-ticles were prepared by 6 freeze-thaw cycles of infected cells as described (39).Infectivity titers in cell culture supernatants and cell extracts were determinedby end-point dilution and E2-specific immunofluorescence as described (43).Intracellular HCV RNA levels were determined by HCV-specific reverse tran-scription-real-time quantitative PCR (RT-qPCR) and were normalized for glyc-eraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels as described(43).

Production of HCVpp and Cell Entry Assays. Viral pseudoparticles named HCVppand VSV-Gpp harbored the glycoproteins of HCV and VSV-G, respectively, andwere produced by transfection in 293T cells of vectors encoding viral glycop-roteins, packaging proteins, and GFP-transfer vector. For infection assays,target cells were seeded 24 h before inoculation at an MOI of 0.1. Thenmedium was removed and dilutions of viral supernatants were added to thecells and incubated for 4 h. Supernatants were then removed and the infectedcells kept in regular medium (DMEM, 10% FCS) for 72 h before analysis of thepercentage of GFP-positive cells by FACS analysis.

Reporter Replicon Replication Assays. In vitro transcribed RNAs were intro-duced into Huh-7 cells by electroporation as described in SI Text. RNA trans-fection efficiency and cell viability were assessed by measuring the intracel-lular neo/SGR RNA content 6 h posttransfection and by determining celldensities posttransfection in the absence of selection, respectively. For renillaluciferase assay (wt and GND mutant Rluc/SGR), cells were lysed with cellculture lysis buffer (Promega). Lysates were harvested and mixed with lucif-erase assay substrate (Promega), as suggested by the manufacturer. RLUvalues were normalized to the number of viable cells for each sample deter-mined by a trypan blue staining. Cell viability was confirmed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity as-says.

14050 � www.pnas.org�cgi�doi�10.1073�pnas.0907344106 Dreux et al.

Dow

nloa

ded

by g

uest

on

June

3, 2

020

ACKNOWLEDGMENTS. We thank Takaji Wakita (National Institute of InfectiousDiseases, Tokyo) for kindly providing the infectious JFH-1 molecular clone andrepliconconstructs,DennisBurton(TheScrippsResearch Institute,LaJolla,CA)forprovidingtheC1anti-E2antibody,MichaelHoughton(Chiron,Emeryville,CA) forproviding the MS5 anti-NS5A antibody, Michinori Kohara (Tokyo MetropolitanInstitute of Medical Science, Tokyo) for providing the anti-NS4A/NS4B antibody,Inder Verma (Salk Institute, La Jolla, CA) for providing lentiviral plasmids,François-Loïc Cosset (Ecole Normale Superieure, Lyon, France) for providing the

vectors necessary for HCVpp production, Charles M. Rice (Rockefeller University,New York) for providing Con1-SGR plasmid, and Stanley Lemon (University ofTexas Medical Branch, Galveston, TX) for providing SGR-H77 bearing Huh-7 cells;Bryan Boyd, Josan Chung, and Christina Whitten for excellent technical assis-tance; Yvon Jaillais for his confocal expertise; Greg Meiffren and Urtzi Garaigortafor critical reading of the manuscript; and our colleagues for encouragement andadvice. This work was supported by National Institutes of Health Grant AI079043.This is paper no. 20097IMS from the Scripps Research Institute.

1. Shepard CW, Finelli L, Alter MJ (2005) Global epidemiology of hepatitis C virusinfection. Lancet Infect Dis 5:558–567.

2. Moradpour D, Penin F, Rice CM (2007) Replication of hepatitis C virus. Nat RevMicrobiol 5:453–463.

3. Reggiori F (2006) 1. Membrane origin for autophagy. Curr Top Dev Biol 74:1–30.4. Orvedahl A, Levine B (2009) Eating the enemy within: Autophagy in infectious diseases.

Cell Death Differ 16:57–69.5. Orvedahl A, et al. (2007) HSV-1 ICP34.5 confers neurovirulence by targeting the Beclin

1 autophagy protein. Cell Host Microbe 1:23–35.6. Ogawa M, et al. (2005) Escape of intracellular Shigella from autophagy. Science

307:727–731.7. Talloczy Z, Virgin HWt, Levine B (2006) PKR-dependent autophagic degradation of

herpes simplex virus type 1. Autophagy 2:24–29.8. Jackson WT, et al. (2005) Subversion of cellular autophagosomal machinery by RNA

viruses. PLoS Biol 3:e156.9. Wong J, et al. (2008) Autophagosome supports coxsackievirus B3 replication in host

cells. J Virol 82:9143–9153.10. Khakpoor A, Panyasrivanit M, Wikan N, Smith DR (2009) A role for autophagolyso-

somes in DEN-3 production in HepG2 cells. J Gen Virol.11. Lee YR, et al. (2008) Autophagic machinery activated by dengue virus enhances virus

replication. Virology 374:240–248.12. Prentice E, Jerome WG, Yoshimori T, Mizushima N, Denison MR (2004) Coronavirus

replication complex formation utilizes components of cellular autophagy. J Biol Chem279:10136–10141.

13. Klionsky DJ, et al. (2008) Guidelines for the use and interpretation of assays formonitoring autophagy in higher eukaryotes. Autophagy 4:151–175.

14. Kabeya Y, et al. (2000) LC3, a mammalian homologue of yeast Apg8p, is localized inautophagosome membranes after processing. EMBO J 19:5720–5728.

15. Kabeya Y, et al. (2004) LC3, GABARAP and GATE16 localize to autophagosomalmembrane depending on form-II formation. J Cell Sci 117:2805–2812.

16. Ohsaki Y, Cheng J, Fujita A, Tokumoto T, Fujimoto T (2006) Cytoplasmic lipid dropletsare sites of convergence of proteasomal and autophagic degradation of apolipopro-tein B. Mol Biol Cell 17:2674–2683.

17. Ogata M, et al. (2006) Autophagy is activated for cell survival after endoplasmicreticulum stress. Mol Cell Biol 26:9220–9231.

18. Cao Y, Klionsky DJ (2007) Physiological functions of Atg6/Beclin 1: A unique autoph-agy-related protein. Cell Res 17:839–849.

19. Fujita N, et al. (2008) An Atg4B mutant hampers the lipidation of LC3 paralogues andcauses defects in autophagosome closure. Mol Biol Cell 19:4651–4659.

20. Tanida I, Ueno T, Kominami E (2004) Human light chain 3/MAP1LC3B is cleaved at itscarboxyl-terminal Met121 to expose Gly120 for lipidation and targeting to autopha-gosomal membranes. J Biol Chem 279:47704–47710.

21. Tanida I, et al. (2004) HsAtg4B/HsApg4B/autophagin-1 cleaves the carboxyl termini ofthree human Atg8 homologues and delipidates microtubule-associated protein lightchain 3- and GABAA receptor-associated protein-phospholipid conjugates. J Biol Chem279:36268–36276.

22. Hemelaar J, Lelyveld VS, Kessler BM, Ploegh HL (2003) A single protease, Apg4B, isspecific for the autophagy-related ubiquitin-like proteins GATE-16, MAP1-LC3,GABARAP, and Apg8L. J Biol Chem 278:51841–51850.

23. Apel A, Herr I, Schwarz H, Rodemann HP, Mayer A (2008) Blocked autophagy sensitizesresistant carcinoma cells to radiation therapy. Cancer Res 68:1485–1494.

24. Jagannath C, et al. (2009) Autophagy enhances the efficacy of BCG vaccine by increas-ing peptide presentation in mouse dendritic cells. Nat Med 15:267–276.

25. Mizushima N, et al. (2001) Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells. J Cell Biol 152:657–668.

26. Fujita N, et al. (2008) The Atg16L complex specifies the site of LC3 lipidation formembrane biogenesis in autophagy. Mol Biol Cell 19:2092–2100.

27. Hamacher-Brady A, Brady NR, Gottlieb RA (2006) Enhancing macroautophagy protectsagainst ischemia/reperfusion injury in cardiac myocytes. J Biol Chem 281:29776–29787.

28. Bartosch B, Dubuisson J, Cosset FL (2003a) Infectious hepatitis C virus pseudo-particlescontaining functional E1–E2 envelope protein complexes. J Exp Med 197:633–642.

29. Zhong J, et al. (2006) Persistent hepatitis C virus infection in vitro: Coevolution of virusand host. J Virol 80:11082–11093.

30. Ait-Goughoulte M, et al. (2008) Hepatitis C virus genotype 1a growth and induction ofautophagy. J Virol 82:2241–2249.

31. Schaller T, et al. (2007) Analysis of hepatitis C virus superinfection exclusion by usingnovel fluorochrome gene-tagged viral genomes. J Virol 814591–4603.

32. Sir D, et al. (2008) Induction of incomplete autophagic response by hepatitis C virus viathe unfolded protein response. Hepatology 48:1054–1061.

33. Talloczy Z, et al. (2002) Regulation of starvation- and virus-induced autophagy by theeIF2alpha kinase signaling pathway. Proc Natl Acad Sci USA 99:190–195.

34. Miller S, Krijnse-Locker J (2008) Modification of intracellular membrane structures forvirus replication. Nat Rev Microbiol 6:363–374.

35. Gosert R, et al. (2003) Identification of the hepatitis C virus RNA replication complex inHuh-7 cells harboring subgenomic replicons. J Virol 77:5487–5492.

36. Simonsen A, Stenmark H (2008) Self-eating from an ER-associated cup. J Cell Biol182:621–622.

37. Axe EL, et al. (2008) Autophagosome formation from membrane compartmentsenriched in phosphatidylinositol 3-phosphate and dynamically connected to the en-doplasmic reticulum. J Cell Biol 182:685–701.

38. Bernales S, McDonald KL, Walter P (2006) Autophagy counterbalances endoplasmicreticulum expansion during the unfolded protein response. PLoS Biol 4:e423.

39. Gastaminza P, et al. (2008) Cellular determinants of hepatitis C virus assembly, matu-ration, degradation, and secretion. J Virol 82:2120–2129.

40. Blight KJ, Kolykhalov AA, Rice CM (2000) Efficient initiation of HCV RNA replication incell culture. Science 290:1972–1974.

41. Yi M, Lemon SM (2004) Adaptive mutations producing efficient replication of geno-type 1a hepatitis C virus RNA in normal Huh7 cells. J Virol 78:7904–7915.

42. Dreux M, et al. (2007) The exchangeable apolipoprotein ApoC-I promotes membranefusion of hepatitis C virus. J Biol Chem 282:32357–32369.

43. Zhong J, et al. (2005) Robust hepatitis C virus infection in vitro. Proc Natl Acad Sci USA102:9294–9299.

Dreux et al. PNAS � August 18, 2009 � vol. 106 � no. 33 � 14051

MIC

ROBI

OLO

GY

Dow

nloa

ded

by g

uest

on

June

3, 2

020