Cellular cofactors affecting hepatitis C virus infectionand replicationGlenn Randalla,b, Maryline Panisa, Jacob D. Cooperb, Timothy L. Tellinghuisena,c, Karen E. Sukhodoletsd,Sebastien Pfeffere,f, Markus Landthalere, Pablo Landgrafe, Sherry Kana, Brett D. Lindenbacha, Minchen Chieng,David B. Weirh, James J. Russog, Jingyue Jug,h, Michael J. Brownsteini, Robert Sheridanj, Chris Sanderj,Mihaela Zavolank, Thomas Tuschle, and Charles M. Ricea,l

aLaboratory of Virology and Infectious Disease, Center for the Study of Hepatitis C, and eHoward Hughes Medical Institute, Laboratory of RNA MolecularBiology, The Rockefeller University, New York, NY 10021; bDepartment of Microbiology, University of Chicago, Chicago, IL 60637; gColumbia GenomeCenter, New York, NY 10032; hDepartment of Chemical Engineering, Columbia University, New York, NY 10027; iJ. Craig Venter Institute, Rockville,MD 20850; jComputational Biology Center, Memorial Sloan–Kettering Cancer Center, New York, NY 10021; and kBiozentrum, Universitat Basel,CH-4056 Basel, Switzerland; and dDepartment of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110

Contributed by Charles M. Rice, May 25, 2007 (sent for review February 28, 2007)

Recently identified hepatitis C virus (HCV) isolates that areinfectious in cell culture provide a genetic system to evaluate thesignificance of virus– host interactions for HCV replication. Wehave completed a systematic RNAi screen wherein siRNAs weredesigned that target 62 host genes encoding proteins thatphysically interact with HCV RNA or proteins or belong tocellular pathways thought to modulate HCV infection. Thisincludes 10 host proteins that we identify in this study to bindHCV NS5A. siRNAs that target 26 of these host genes alterinfectious HCV production >3-fold. Included in this set of 26were siRNAs that target Dicer, a principal component of the RNAisilencing pathway. Contrary to the hypothesis that RNAi is anantiviral pathway in mammals, as has been reported for sub-genomic HCV replicons, siRNAs that target Dicer inhibited HCVreplication. Furthermore, siRNAs that target several other com-ponents of the RNAi pathway also inhibit HCV replication.MicroRNA profiling of human liver, human hepatoma Huh-7.5cells, and Huh-7.5 cells that harbor replicating HCV demon-strated that miR-122 is the predominant microRNA in eachenvironment. miR-122 has been previously implicated in posi-tively regulating the replication of HCV genotype 1 replicons.We find that 2�-O-methyl antisense oligonucleotide depletion ofmiR-122 also inhibits HCV genotype 2a replication and infectiousvirus production. Our data define 26 host genes that modulateHCV infection and indicate that the requirement for functionalRNAi for HCV replication is dominant over any antiviral activitythis pathway may exert against HCV.

antivirals � miR-122 � RNAi � HCVcc-siRNA

Hepatitis C virus (HCV) has a notable ability to establishpersistent infections in �70% of cases, resulting in 130

million chronically infected people throughout the world (1).This prevalence has spurred considerable interest in the study ofHCV–host interactions, on both cellular and molecular levels.The inability to grow HCV in cell culture led some groups tofocus on the identification of cellular proteins that interact withindividual HCV proteins or RNA elements, resulting in theaccumulation of a large number of putative HCV–host interac-tions. Unfortunately, the significance of most of these withrespect to the HCV life cycle is currently unknown (reviewed inref. 2). Over the past 6 years, cell culture systems have beendeveloped that enable the characterization of HCV replicationand entry (3–6). This effort recently culminated in the devel-opment of cell culture systems that reproduce the entire viral lifecycle (7–9). A number of virus–host interactions have beencharacterized by using these experimental systems. For example,CD81 has been demonstrated to play a role in HCV entry(10–12).

Sequence-specific gene silencing of RNAi is ideal for assessingthe genetic phenotypes associated with virus–host interactions.We have previously shown that siRNAs are highly effective atsilencing either host or viral RNAs in cells that contain repli-cating HCV, demonstrating the potential of RNAi as a tool tostudy HCV–host interactions (12–16). The goal of this study isto define host cofactors involved in HCV replication. We firstidentified 10 host proteins that interact with NS5A by using thetwo-hybrid system and copurification approaches. These werecombined with other published HCV–host interactions to pro-vide a sample set in which we could evaluate both the signifi-cance of these proteins for HCV replication and the utility ofsiRNA screens for identifying host genes and pathways thatmodulate HCV replication. siRNAs targeting 26 of the 62 hostgenes tested, including the RNAi ribonuclease Dicer, modulatethe production of infectious HCV by at least 3-fold. Interferencewith multiple components of the RNAi pathway involved inmiRNA biogenesis or the liver-specific miRNA miR-122 re-sulted in an inhibition of HCV replication. Thus, the RNAipathway and miR-122 in particular are required for optimalHCV replication and infectious virus production, consistent withrecent HCV replicon data from Jopling et al. (17).

ResultsIdentification of HCV NS5A-Interacting Proteins. This study presentsRNAi analysis of the significance of 62 host genes in HCVreplication and infectious virus production. The majority ofthese genes have been published to interact with HCV RNA orproteins [supporting information (SI) Table 2]. Additionally, weidentified a number of host interactions with HCV NS5A thatwere subsequently included in this analysis. Two approacheswere used to identify NS5A-interacting proteins. The first ap-proach involved the yeast two-hybrid system; details are pro-vided in Materials and Methods (18). A LexA DNA-bindingdomain-NS5A (1a H77) fusion protein was used to a HeLa

Author contributions: G.R., S.P., T.L.T., K.E.S., T.T., and C.M.R. designed experiments; G.R.,M.P., J.D.C., T.L.T., K.E.S., S.P., P.L., S.K., D.B.W., and M.C. performed experiments; M.L.,B.D.L., and M.J.B. contributed new reagents; G.R., J.D.C., D.B.W., J.J.R., J.J., R.S., C.S., M.Z.,and T.T. analyzed data; and G.R., J.D.C., T.L.T., P.L., and C.M.R. wrote the paper.

Conflict of interest statement: C.M.R. has equity in Apath, LLC, which has a commerciallicense for the Huh-7.5 cell line and certain HCVcc derivatives.

Abbreviations: HCV, hepatitis C virus; UPR, unfolded protein response.

cPresent address: Scripps Research Institute, Jupiter, FL 33411.

fPresent address: Centre National de la Recherche Scientifique– Unite Propre de Recherche2357, Institut de Biologie Moleculaire des Plantes, 67084 Strasbourg Cedex, France.

lTo whom correspondence should be addressed. E-mail: ricec@rockefeller.edu.

This article contains supporting information online at www.pnas.org/cgi/content/full/0704894104/DC1.

© 2007 by The National Academy of Sciences of the USA

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cDNA library whose translatable products are fused with anacidic transcriptional activation domain. Nine cDNAs encodingthree unique proteins that interacted with NS5A were identifiedfrom a library of 108 cDNA clones. They are (i) the nucleartransport protein IPO4, (ii) the receptor recycling proteinVPS35, and (iii) hsp90-associated protein 1, HSPA1A. Theseinteractions were specific, inasmuch as the identified clonesinteracted with HCV NS5A, but not with the irrelevant bicoidprotein or the closely related bovine viral diarrhea virus NS5A.

Our second approach involved the identification of cellularkinases that bind to NS5A in a complex that phosphorylatesNS5A in vitro. The Kinetworks KPKS screen (Kinexus; Kinet-works, Vancouver, BC, Canada) is a method that can detect andquantify 75 protein kinases in a complex mixture of proteinsbased on recognition by specific antibodies. Purified prepara-tions of GST and GST-NS5A were prepared and subjected tothis analysis. Twelve candidate protein serine/threonine kinasesthat specifically associated with GST-NS5A were identified.Seven protein kinases match with the characterized profile of theNS5A kinase, including kinase inhibitor profiles, pH optimum,and divalent metal ion preferences (19). These kinases wereidentified as CDK6, ERK6/MAPK12, RAF1, AKT1, PDPK1,GSK3�, and GSK3�. The summary of NS5A–protein interac-tion data is shown in SI Table 2. Interestingly, the putative yeast

homologues of four of these genes, AKT1, PDPK1, GSK3�, andGSK3�, have been reported to phosphorylate NS5A in vitro (20).

A Systematic RNAi Screen Identified Host Genes That Modulate HCVReplication. We next tested the significance of 62 host genes forHCV replication. These host genes encode proteins that phys-ically interact with HCV RNA or proteins, including the NS5A-interacting proteins identified above, or alternatively, that be-long to signaling pathways thought to modulate RNA virusreplication. A list of host genes, sites of HCV interaction, andassociated references is available in SI Table 3. Huh-7.5 cellswere transfected with at least two different siRNAs per gene(described in SI Table 4) and then infected with HCV over asliding window, beginning at 24, 48, or 72 h after transfection.After 48 h of infection, cellular supernatants were collected fortitration of infectious virus, whereas intracellular HCV RNAswere quantified by quantitative real-time RT-PCR. Cell viabilityassays measuring intracellular ATP levels were taken fromparallel samples at the time of harvesting (SI Fig. 4). Changes inHCV RNA and virus levels were then calculated for each siRNA.

The fold change in HCV RNA or virus levels after silencingof the target gene, relative to the median value of all genes tested,is shown in Table 1. Twenty-six of the 62 genes that were targetedmodulate HCV infectious virus production �3-fold. Real-time

Table 1. Changes in HCV replication after siRNA targeting of host gene RNAs

siRNA* Virus† HCV RNA siRNA Virus HCV RNA

HCV � �230 � �10000 MAPK12 �2.0 � 0.5��� �1.3 � 0.8DDX3X �42 � 19�� �1600 � 800 NCL �1.8 � 0.4�� 4.5 � 1.6EIF2S3 �30 � 3.7��� �55 � 34 CDC2 �1.8 � 0.5��� 1.8 � 0.3STAT3 �13 � 4.4��� �8.3 � 2.4 EIF2AK3 �1.8 � 0.4�� �2.0 � 0.1CD81 �11 � 0.6�� �6.1 � 2.6 IPO4 �1.7�� 1.9 � 0.5ELAVL1 �9.1 � 2.8��� �3.3 � 0.4 HNRPL �1.5 � 1.8�� �1.6 � 0.4VAP-ABC‡ �8.7 � 1.7��� �5.9 � 0.3 XBP1 �1.4 � 1.8��� �1.5 � 0.7DICER1 �7.5 � 2.5��� �3.1 � 0.7 CSNK2A1 �1.3 � 0.1��� 1.4 � 0.2HSPBP1 �6.4 � 0.7�� �5.7 � 2.3 TP53 �1.3�� 1.7 � 0.6GRB2 �6.3 � 0.9��� �1.3 � 0.3 EIF2B3 �1.2 � 0.1��� �3.5 � 0.6HM13 �6.2 � 1.8�� �10 � 4.0 CALR �1.2 � 1.8�� 2.4 � 0.1RAF1 �5.6 � 1.8�� �3.6 � 3.1 SCD �1.2 � 2.3��� �1.1 � 0.5EIF2AK2 �5.5 � 0.7��� �1.9 � 0.7 VPS4A �1.1 � 1.3��� �1.4 � 1.1PSMA7 �5.4 � 1.7�� �1.3 � 0.8 AKT1 1.0 � 0.1��� 1.7 � 0.5SRCAP �4.9 � 1.4��� �1.4 � 0.5 SSB 1.0 � 0.3��� �2.0PTBP1 �4.7 � 1.6��� �2.1 � 1.0 CDK2 1.1 � 0.5��� 1.3 � 0.6GAPDH �4.6 � 0.5��� �2.2 � 0.3 ISG15 1.1 � 0.2�� �1.2 � 0.5EIF4E �4.4 � 0.8��� 3.6 � 1.3 SCARB1 1.1�� 2.7 � 1.5VPS35 �4.4 � 1.1��� �2.3 � 0.4 USP18 1.1 � 0.2�� 3.5 � 2.4RANBP5 �4.4 � 1.1��� �4.1 � 0.8 PRMT1 1.2 � 0.6��� 1.9 � 0.1HNRPC �3.9 � 0.9��� �3.0 � 0.2 PCBP1 1.2 � 0.6��� �2.3 � 0.1ACTN1 �3.9 � 0.7�� �4.0 � 1.6 RPL3 1.3 � 0.0��� 2.3 � 0.8RELA �3.4 � 0.8�� �3.5 � 0.7 PRMT5 1.3 � 0.0��� 1.8 � 0.5MAPK1 �3.2��� 2.0 � 0.8 LSM1 1.3 � 0.6��� �7.5 � 1.9PCBP2§ �3.1���, 3.4�� �1.8, 1.8 ISGF3G 1.5�� 2.7 � 3.0RPL22 �2.9 � 0.3�� �6.4 � 0.3 PRKACA 1.5��� 2.9 � 0.1PDPK1 �2.8 � 0.6��� �4.2 � 2.2 DNAJC14 1.5 � 1.1��� 1.6 � 0.2AHSA1 �2.5 � 0.5��� �3.2 � 1.0 HSPCA 1.7 � 0.4�� �1.6 � 0.4CDK6 �2.4 � 1.0�� 2.1 � 1.2 ADI1 2.0 � 1.1��� 1.2 � 1.0SEC11L1 �2.4 � 0.4��� �2.5 � 0.7 SNX1 2.4 � 1.3�� 2.9 � 0.1CANX �2.2 � 0.7�� �1.4 � 0.1 ATF6 6.8�� 3.8 � 0.4

Dots represent number of days between siRNA treatment and initial infection that produced the most extremeviral phenotype.*Names refer to the gene name of the siRNA target (HUGO nomenclature).†Values represent fold change in HCV levels plus SEM in specific siRNA-treated cells compared with controls.Values are based on the geometric mean of two replicate experiments. A negative value reflects a decrease inrelative HCV levels.

‡VAP-ABC siRNAs target VAPA, VAPB, and VAPC.§PCBP2 siRNAs produce an early increase in HCV levels, followed by a decrease.

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RT-PCR assays (described in SI Table 5) were developed forthese genes to measure the expression levels of each siRNA’starget RNA. The percent inhibition of target gene expression 2days after siRNA transfection is shown in SI Fig. 5. For eachsiRNA, the target gene expression decreased at least 60%, withan average inhibition of �85%, (see SI Fig. 5). Thus, each siRNAinhibited the RNA accumulation of its intended target gene.

A few trends are apparent from the data. First, changes inHCV infectious virus production generally parallel the changesin HCV RNA levels for most siRNAs. This correlation isexpected for defects in a stage of the virus life before thepackaging and release of infectious virus. Second, there is nocorrelation between the effect of siRNAs on HCV replicationand cell viability, suggesting that most phenotypes do not resultfrom changes in cellular physiology. In total, 26 genes modulateHCV replication by the following criteria: (i) Introduction ofsiRNAs targeting the genes alter infectious HCV production by�3-fold and (ii) a decrease in target gene RNA accumulationafter siRNA treatment. These genes and their regions of HCVinteraction are shown in SI Fig. 6.

RNAi Is Required for Efficient HCV Infection. We extended thesestudies for one of the 26 ‘‘hits’’ from the initial screen, the RNAienzyme Dicer. Dicer was included in this screen, because anumber of groups postulated that RNAi might be an innateantiviral defense in mammals (21). We initially hypothesized thatif RNAi were antiviral against HCV, then silencing Dicer wouldincrease HCV replication. However, we observed the oppositephenotype: silencing Dicer inhibited HCV replication �7-fold.Conflicting data have been published on this topic using HCVgenotype 1 replicons. The microRNA miR-122 was reported tobe required for HCV replication (17); however, Dicer siRNAs,which would interfere with miR-122 biogenesis, were subse-quently reported to enhance HCV subgenomic replication (22).We decided to reexamine the role of RNAi in HCV replicationby using the infectious HCV genotype 2a isolate.

We investigated the role of RNAi in the HCV life cycle bysilencing multiple components of the RNAi pathway and testingthe effects on HCV replication and infectious virus productionas above. Additionally, different siRNAs that target distinctDicer sequences were used to minimize the possibility of non-specific off-target effects that can be associated with RNAiexperiments. All siRNAs tested reduced target RNA accumu-lation by at least 80% with no appreciable effects on cell viability(Fig. 1 C and D). We found that siRNAs targeting genes involvedin miRNA biogenesis (DICER1, Drosha/RNASEN, andDGCR8) and the RISC effector complex (EIF2C1–4) inhibitedHCV replication (Fig. 1 A and B).

We next examined the miRNA environment associated withHCV infection to identify miRNAs that are expressed within theenvironment of HCV replication and to test whether HCVreplication interfered with miRNA biogenesis. Total RNA fromhuman liver, Huh-7.5 cells, and Huh-7.5 cells containing repli-cating genomic HCV-Con1 was electrophoretically separatedand �21-nt RNAs were gel-purified, cloned, sequenced, andannotated. The relative abundances of cloned miRNAs areplotted in Fig. 2. Seventy-one distinct miRNAs, including fourpreviously unidentified miRNAs (miR-100604, -100819,-100854, and -100871; SI Table 6) were isolated in liver cells.Strikingly, miR-122 was the most frequently cloned miRNA,representing 72% of total miRNAs. It was also the most highlyexpressed miRNA in Huh-7.5 cells and Huh-7.5 � HCV cells,representing 23% and 15% of the cloned miRNAs, respectively.

Some differences between Huh-7.5 cells that harbor replicatingHCV and the parental Huh-7.5 cell line with respect to theexpression of low-abundance miRNAs include higher expression ofthe miR-322, 197, 532–5p, and 374 miRNA families and the absenceof miR-146a, 30a, 23a, and 191 families in cells with replicating

HCV. The miRNA expression patterns match with previous North-ern blot analysis, showing that the levels of miR-122, -21, and -130are unaffected by HCV replication (13). Thus, HCV replicationdoes not grossly affect miRNA biogenesis.

A recent study implicated miR-122 in the modulation of HCVreplication by using genotype 1 replicons (17). The miR-122-binding site is conserved between genotype 1 and 2a, suggestingthat miR-122 might have a similar function in the production ofinfectious genotype 2a HCV (Fig. 3A). We tested this possibilityby transfecting 2�-O-methyl oligos targeting either a randomsequence or the miR-122 sequence into Huh-7.5 cells andmeasuring the effects on HCV replication. miR-122 depletionconsistently inhibited HCV replication and infectious virusproduction over a number of time points (Fig. 3 B and C). Thesedata demonstrate that the requirement for miR-122 in HCVreplication is conserved between genotype 1b replicons andinfectious genotype 2a HCV. Thus, the requirement of the RNAipathway for infectious HCV production likely reflects a need formiR-122 expression.

DiscussionHost genes modulate viral infection and are an underappreciatedtarget for antiviral therapy. We identified 26 human genes thatmodulate HCV replication and implicated the RNAi pathway itselfas a key regulator of HCV replication. Significant host proteins thatinteract with the structural genes include CD81, the dead boxhelicase DDX3X, the cellular proteases signal peptidase(SEC11L1), and signal peptide peptidase (HM13). CD81 is atetraspanin that promotes HCV entry via its interaction with HCVE2. DDX3X binds to the core protein; its role in the HCV life cycleis currently unknown (23). DED1, a yeast homolog of DDX3X, isalso required for the replication of brome mosaic virus, anotherpositive-strand RNA virus (24). The functional conservation ofDDX3X among diverse viruses and the degree of HCV inhibitionafter the silencing of DDX3X suggest that DDX3X plays a signif-

Fig. 1. Silencing RNAi machinery inhibits HCV replication. siRNAs targetinggenes intheRNAipathway,HCV,oranirrelevantsequence(IRR)were individuallyintroduced into Huh-7.5 cells, which were subsequently infected with HCV. (A)Relative HCV RNA levels were quantified by real-time RT-PCR for HCV and 18SRNA. Values are averages of two sets of triplicates � SEM. (B) Infectious HCV incellular supernatants were quantified by limiting dilution and expressed asID50/ml. (C) Percent inhibition of target RNA levels after siRNA treatment com-paredwith irrelevanttreatedsamples.Valuesarethe levelsof targetRNA/GAPDHRNA. (D) Cell viability (ATP levels) after siRNA treatment at the time of virusharvesting. ATP levels are normalized to the median of treated samples.

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icant role in HCV replication. Signal peptidase proteolyses theHCV structural genes, whereas signal peptide peptidase furtherproteolyses the core protein into the mature core (25).

Cellular proteins that interact with HCV nonstructural genesinclude AFT6, a component of the unfolded protein response(UPR), which is induced by NS4B (26). The UPR is a cellularpathway that is activated by various stresses to the endoplasmicreticulum, including HCV replication. Interestingly, the silencingof ATF6 results in an increase in HCV replication and virusproduction and is also associated with increased cell viability.This suggests that the UPR becomes induced in our experiments,and that the ATF6 pathway limits viral replication. NS5A-interacting proteins that modulate HCV replication includeVAP-A/B and VPS35, which function in vesicular trafficking(27–30). VAPA can bind to NS5B as well as hypophosphorylatedNS5A and has been proposed to serve as a scaffold for thereplication complex within lipid microdomains (31–34). Thecytoskeletal component ACTN1 also binds to NS5B and may beinvolved in replicase localization. PDPK1, RAF1, and EIF2AK2are kinases that bind to NS5A. It is unclear whether these kinasesphosphorylate NS5A or alternatively impact HCV replicationindirectly through their various (i.e., Ras/MAPK) signalingpathways. Similarly, GRB2 binds to NS5A and is a key compo-nent of RAS signaling. The roles of RANBP5, SRCAP, andAHSA1 in HCV replication remain unclear.

Some proteins identified in our screen bind to HCV RNAsequences that are associated with translation and/or RNAsynthesis. EIF2S3 binds to the 5� nontranslated region (5� NTR)of HCV and has been reported to modulate HCV translation(35). PTBP1 binds to both 5� and 3� NTRs, suggesting a possibleinteraction between the 5� and 3� termini of HCV RNA (35–37).RPL22 and GAPDH interact specifically with the 3� NTR (38).PTBP1, ELAVL1, and HNRNPC bind to the 3� termini of both(�) and (�) strands, suggesting they may be components of theRNA synthesis initiation complexes (39, 40).

Cellular stress response pathways also modulate HCV repli-cation, including the UPR, dsRNA activation of IFN signaling(PKR/EIF2AK2), oxidative stress (NF-�B/RELA, STAT3), andheat shock (Hsp70/HSPA1A). Numerous groups have observedthe activation of these pathways during HCV replication, so it islikely that these pathways are responding, at least in part, toHCV infection (41).

RNAi silencing of VAPA, VAPB, PTBP1, SSB (La), EIF2B3,ELAVL1, PSMA7, and RAF1 inhibit replication of HCV ge-notype 1 replicons (32, 42–44). Our data with infectious geno-type 2a HCV correlated with that published for each of thesegenes. SSB (La) and EIF2B3 siRNAs displayed a small degreeof inhibition of HCV RNA accumulation, whereas the remaininggenes are listed as ‘‘hits’’ in this screen. These are likely to playimportant roles in HCV replication, given that similar results

Fig. 2. Relative miRNA expression profile of liver, Huh-7.5 cell line, andHuh-7.5 with replicating genomic HCV-Con1. miRNAs of human, mouse, andrat were aligned in sequence alignments, and sequence groups were built(specified in SI Table 7). The number of miRNAs in one sequence group isindicated in brackets. The clone count of sequence groups relative to allmiRNA clones obtained from liver and Huh7 cell lines is depicted in thespecified color code. Tissues were hierarchically clustered based on the miRNAprofiles. miRNA expression patterns in the hepatocellular carcinoma cell linescluster together and separately from liver, as indicated by lines at the top.

Fig. 3. Interfering with miR-122 inhibits HCV replication. (A) Alignment ofmiR-122-binding sites in HCV 5� NTR of genotype 1b Con1 and genotype 2aJFH-1. (B and C) 2�-O-methylated RNAs targeting a random sequence (Rand) ormiR-122 (miR-122) were introduced into Huh7.5 cells, infected with HCV, andharvested at the indicated time points, and HCV RNA (B) and infectious virus(C) was quantified. Values are averages of triplicates � SEM. P value � 0.001.

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were found by different groups using different siRNA sequences,silencing approaches, HCV genotypes, and replication systems.

In this paper, the RNAi pathway was demonstrated to berequired for optimal HCV replication. The function of miR-122in HCV replication, however, remains unclear. Genetic evidenceexists for an HCV-specific function of miR-122. HCV RNAs thatcarry a mutation within the 5� NTR of the miR-122-binding sitedo not replicate but can be rescued by expression of a miR-122variant containing a complementing mutation (17). miR-122 didnot appear to greatly influence HCV translation in the repliconsystem, suggesting that miR-122 may regulate viral RNA syn-thesis or the trafficking of viral RNAs to subcellular compart-ments. Alternatively, HCV may interfere with the capacity ofmiR-122 to silence its endogenous substrates. In support of thishypothesis, HCV replication is associated with an increase inexpression of cholesterol biosynthesis genes that are regulated bymiR-122 (45–48).

The conserved expression of distinct miRNAs was identifiedby miRNA profiling of liver and Huh-7.5 cells, the most abun-dant of these being miR-122. miR-122 is expressed at high levelsin Huh-7.5 cells but not in other transformed liver cells, corre-lating with susceptibility of the cell type to HCV replication (17,49). However, HCV can be adapted to replicate in a number ofcell types that do not express detectable miR-122, suggesting thatmiR-122 may not be required for HCV replication in differentcellular environments (50). Although we cannot rule out a rolefor other identified miRNAs in HCV replication, the sheerabundance of miR-122 in liver (72% of total miRNAs) suggeststhat this interaction predominates.

Mounting evidence suggests that RNAi is not a robust anti-viral pathway in mammals (51). HCV core protein overexpressedoutside of its genomic context was reported to inhibit Dicer.However, we previously showed that replicating HCV did notinhibit RNAi, either with siRNAs or shRNAs targeting viral orcellular RNAs, or by altering the expression of endogenousmiRNAs (13, 22). If RNAi were indeed an antiviral pathwayagainst HCV, one would expect that HCV replication wouldactivate the RNAi machinery and small viral siRNAs would begenerated. To test this hypothesis, we identified �2,000 smallRNAs from Huh-7.5 cells containing actively replicating HCV;however, no HCV siRNAs were identified (13). Thus, althoughreplicating HCV was shown to be susceptible to transfectedHCV-specific siRNAs and not inhibitory to RNAi, replicatingHCV did not appear to be susceptible to endogenous RNAi.Similar studies with other viruses revealed a surprising depen-dence on the RNAi machinery: HCMV, HHV8, MHV68, SV40,and HIV encode viral miRNAs (13, 52–57).

Previous studies of subgenomic HCV replicons yielded con-flicting data; miR-122 was required for viral replication, yetDicer was reported to inhibit the replication of transfected HCVRNAs (17, 22). This suggested that the RNAi machinery mayhave two interactions with HCV, a microRNA interaction and anantiviral siRNA interaction. These data indicate that the re-quirement of functional RNAi for replication is dominant overany antiviral activity this pathway may exert against HCV. Theseresults also implicate the RNAi machinery as a potential anti-viral target to limit HCV replication.

Materials and MethodsCells and Virus. Huh-7.5 cells are a subline derived from Huh-7hepatoma cells that are highly permissive for the initiation ofHCV replication (58). Huh-7.5 cells containing the HCV-Con1replicon [Con1/Fl-neo(S2204I)] were used for miRNA profiling(59). Cells were maintained in DMEM supplemented withnonessential amino acids and 10% FBS, whereas replicon cellmedia also contained 0.75 mg/ml G418. HCV FL-J6/JFH is afull-length genotype 2a sequence that produces the full replica-tion cycle in cell culture (9). It is a chimera containing the JFH-1

5� nontranslated region (60), the J6 core through NS2 genes (61),and the JFH-1 NS3 through 3� nontranslated region.

RNAi Assay. The primary screen included 116 siRNAs that weredesigned to target 58 genes (two siRNAs per gene), whereas fourother host genes were targeted by gene-specific siRNA smartpools (Dharmacon, Lafayette, CO; SI Table 4). RNAi assayswere performed as described (14). Briefly, 2.5 � 106 Huh7 cellsin 0.4 ml of PBS pH 7.4 were electroporated with 1 nanomole ofsiRNA for five pulses of 870 V for 99 �s with 1-s intervals on aBTX (Holliston, MA) 820 electroporator. Cells were plated andinfected at 24, 48, or 72 h after electroporation with a multiplicityof 0.5 infectious HCV particles per cell for 6 h, rinsed with media,then maintained for 2 days at 37°C. Rand and miR-122 2�O-methyl RNAs were described previously (17).

HCV Quantification. Virus titers were determined by limitingdilution analysis as described (9). Viral RNA was quantified byreal-time RT-PCR analysis as described (62). Cellular RNAswere quantified by using ABI TaqMan assays, as recommended(Applied Biosystems, Foster City, CA). For SYBR green assaysof cellular genes, 0.75 �g of DNaseI-treated total RNA wasreverse-transcribed with SuperScript II (Invitrogen, Carlsbad,CA) and oligo(dT) for 1 h at 42°C, then heat-inactivated at 80°Cfor 20 min. One-twentieth of the cDNA mix was mixed with anequal volume of 2� SYBR Green Master Mix (Applied Biosys-tems) and the appropriate primers. PCR conditions were: 50°C,2 min; 95°C, 10 min (95°C, 15 sec; 55°C, 1 min) � 40 cycles (63).Results were analyzed with SDS 1.7 software (Applied Biosys-tems). Relative HCV RNA levels are the quantity of HCVnormalized to 18S RNA levels. Relative cellular gene RNAlevels are the quantity of the specific gene RNA normalized toGAPDH RNA levels.

NS5A Two-Hybrid Analysis. The LexA DNA-binding domain (pEG-202) was fused to the NS5A gene from the genotype 1a H77strain. It was used to screen a HeLa cDNA library that is fusedwith a B42 acidic transcriptional activation domain (pJG4/5).Plasmids were transformed into the Saccharomyces cerevisiaestrain EGY48, and interactors were assayed for growth inminimal dropout media lacking uracil, histidine, tryptophan, andLacZ expression. Nine cDNAs encoding three unique proteinsthat specifically interact with NS5A were identified from alibrary of 108 HeLa cDNA clones.

NS5A Kinase Complex Isolation. The mammalian expression andisolation of GST and GST-NS5A was performed as described,with minor modifications (19). Briefly, BHK21 cells in 150-mmdish were infected with 10 pfu per cell of vTF7–3 virus for 1 hin PBS plus 1% FBS, washed, then transfected with 170 �l oflipofectamine (Invitrogen), and 30 �g of plasmid DNA in 5 mlof Opti-MEM. After 24 h, cells were lysed in 5 ml of NETNbuffer (50 mM Tris�HCl, pH 7.5/120 mM NaCl/1 mM EDTA/0.5% Nonidet P-40) supplemented with 5 mM DTT/1 �g/mlaprotinin/1 �g/ml leupeptin/20 �g/ml PMSF. Lysates were clar-ified at 16,000 � g for 10 min at 4°C and incubated with 500 �lof a 1:1 slurry of glutathione agarose in lysis buffer for 30 min.Agarose beads were briefly centrifuged at 3,000 � g, washed,then resuspended in a final volume of 250 �l of kinase buffer (50mM Tris�HCl, pH 7.5/5 mM MnCl2/5 mM DTT). NS5A-associated kinase activity was assayed as described (19). Agarosebeads with associated proteins were then resuspended in SDS/PAGE sample buffer, heated to 95°C for 5 min, and centrifuged,then supernatants were removed and sent to Kinexus for theKPKS kinase screen.

12888 � www.pnas.org�cgi�doi�10.1073�pnas.0704894104 Randall et al.

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Total RNA Isolation, Small RNA Library Preparation, Sequencing, andSequence Annotation. Small RNA cloning was performed asdescribed (64) with the following modifications. Samples werecloned by using preadenylated 3� adapter oligonucleotides andT4 RNA ligase Rnl2 as described (13) (indicated by A, SI Table7). Radiolabeled 19- and 24-nt oligonucleotide size markerscontaining a PmeI site were added to total RNA before gelelectrophoresis. 3�-Adapter ligation products were gel-purifiedand ligated to a 5�-oligonucleotide by using T4 RNA ligase 1(NEB) plus ATP. After reverse transcription and a first PCRamplification step, the PCR product was digested with PmeI toeliminate the size markers. After a second PCR step, whichintroduced nonpalindromic restriction sites, the PCR productwas digested with BanI, concatemerized by using T4-DNA ligase,and ligated into pCR2.1 (Invitrogen). Colonies were screened byPCR for the presence and size of inserts and sequenced.

Annotation used GenBank (www.ncbi.nih.gov/GenBank/index.html), a human tRNA database (http://lowelab.ucsc.edu/GtRNAdb), sn/snoRNA databases (www-snorna.biotoul.fr/index.php, http://noncode.bioinfo.org.cn, and www.imb.uq.edu.au),

a miRNA database (http://microrna.sanger.ac.uk/sequences/index.shtml), the repeat element annotation of version 17 of thehuman genome assembly, version 6 of the mouse genome assembly,and version 3 of the rat genome assembly from http://genome.cse.ucsc.edu.

Two-hybrid system reagents were provided by Roger Brent. We thankKathleen Hefferon for critical reading and editing of the manuscript.This work was funded by the Public Health Service (National Institutesof Health Grants CA57973, CA85883, and AI40034), the Ellison MedicalFoundation, and the Greenberg Medical Research Institute (C.M.R).C.M.R. is an Ellison Medical Foundation Senior Scholar in GlobalInfectious Diseases. G.R. is supported by the American Cancer Society(Grant PF-02-016-01-MBC), the National Institute of Diabetes andDigestive and Kidney Diseases/Digestive Disease Research Core Center(Grant P30 DK42086), and Susan and David Sherman. T.L.T. wassupported by the Charles Revson Foundation for Biomedical Researchand the National Institutes of Health/National Institute of Allergy andInfectious Diseases Ruth L. Kirschstein National Research Serviceaward (5F32 AI51820-03). B.D.L. is supported by the National CancerInstitute Howard Temin Award (CA107092).

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