A genome-wide genetic screen for host factors required for hepatitis C virus propagation

Qisheng Lia,1, Abraham L. Brassb,c,d,1, Aylwin Nge, Zongyi Hua, Ramnik J. Xavierc,e, T. Jake Lianga,2, and Stephen J. Elledged,2

aLiver Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892; bRagon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology, and Harvard Medical School, Charlestown, MA 02129; cGastrointestinal Unit, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114; dDepartment of Genetics, Harvard Medical School, Division of Genetics, Brigham and Women’s Hospital, Howard Hughes Medical Institute, Boston, MA 02115; eCenter for Computational and Integrative Biology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114

Contributed by Stephen J. Elledge, July 8, 2009 (sent for review May 28, 2009) Hepatitis C virus (HCV) infection is a major cause of end-stage liver (11, 12). Our previous functional genomic studies, and those of disease and a leading indication for liver transplantation. Current others, have revealed that viral dependencies on the host machinery therapy fails in many instances and is associated with significant are both intricate and extensive (13–15). Moreover, a great advan- side effects. HCV encodes only a few proteins and depends heavily tage may be gained in pursuing an unbiased genetic screening on host factors for propagation. Each of these host dependencies strategy in that unexpected connections and insights can be dis- is a potential therapeutic target. To find host factors required by covered. Given the complexity of the virus’ relationship with the HCV, we completed a genome-wide small interfering RNA (siRNA) host cell, and the relatively unexplored mechanisms of HCV screen using an infectious HCV cell culture system. We applied a replication, we hypothesized that additional host factors might be two-part screening protocol to allow identification of host factors found by completing an unbiased whole-genome siRNA screen involved in the complete viral lifecycle. The candidate genes found with the HCVcc system. included known or previously identified factors, and also implicate many additional host cell proteins in HCV infection. To create a Results more comprehensive view of HCV and host cell interactions, we The siRNA Screen. We used a two-part screen employing the infec- MICROBIOLOGY performed a bioinformatic meta-analysis that integrates our data tious JFH-1 genotype 2a virus and the Huh 7.5.1 human hepato- with those of previous functional and proteomic studies. The cellular cell line to find host proteins required by the HCVcc system. identification of host factors participating in the complete HCV Part one of the screen involved transfecting the Huh 7.5.1 cells with lifecycle will both advance our understanding of HCV pathogenesis siRNAs for 72 h, then challenging them with JFH-1 virus. After and illuminate therapeutic targets. 48 h, the cells were stained and imaged for expression of the HCV core protein as a marker for productive viral infection (Fig. 1A). To HCV ͉ RNA interference ͉ viral host factors ͉ functional genomics ͉ find host factors involved in later stages of viral infection, we viral lifecycle undertook part two of the screen, by exposing fresh Huh 7.5.1 cells to culture supernatant from part one for 48 h, then detecting core epatitus C virus (HCV) causes a chronic infection resulting in protein expression. This approach detects proteins needed for the Hprogressive liver damage. The virus enters hepatocytes via complete viral lifecycle, from viral-host receptor binding to the interactions between the viral envelope proteins, E1 and E2, and completion of a second round of viral infection. In addition, genes four known host receptors, CD81, Claudin-1, SBR-I, and occludin functioning in either anti-viral responses or safeguarding the cells (1). Subsequent to entry, host ribosomes bind to the internal against the stress of infection, may be detected in this screen. ribosomal entry site (IRES) of the HCV genome, and translate viral The screen was optimized using siRNAs against the host proteins, polyproteins on the rough endoplasmic reticulum (ER) (2). Host CD81 (part one) and ApoE (part two). ApoE, a host protein and viral proteases process the polyprotein into both structural involved in lipoprotein biosynthesis, has been shown to be required (core, and envelope proteins, E1 and E2) and nonstructural pro- for infectious particle formation (16). siRNAs against CD81 re- teins (p7, NS2–3, NS3, NS4A, NS4B, NS5A, and NS5B) (3). sulted in inhibition of part one infection (5–6-fold, Fig. 1A and Fig. Oligomerization of NS4B distorts the host ER into membranous S1A and B). siRNAs targeting ApoE, however, did not affect webs, which house HCV replication complexes (RCs), within which infection in part one, but did inhibit HCV propagation in part two the RNA-dependent RNA polymerase, NS5B, transcribes viral by 2–3 fold, in keeping with its previously assigned role in infectious genomic RNAs (2). Progeny viral genomes generated in the RCs particle formation (Fig. 1A and Fig. S1A and B). The levels of are translocated to lipid droplet-containing organelles and assem- infection were verified using quantitative RT-PCR and found to be ble into virions, which then traffic to the cell surface for release (4). proportional to the levels determined by imaging (Fig. S1C and D). However, although progress has been made in the areas of entry This platform was then used for a genetic screen with a whole- and replication, the later stages of the viral lifecycle—assembly, genome siRNA library (Dharmacon siGenome, 19,470 pools of budding, and maturation—remain less explored. four siRNAs per gene). Pools were classified as hits (decreased The in vitro study of HCV replication has benefited from the use infection) if the percentage of infected cells was Ͻ50% of the plate of the replicon system, consisting of subgenomic or whole genome viral RNAs stably expressed in permissive tissue culture cells. Using the replicon system, several recent functional screens, using either Author contributions: Q.L., A.L.B., T.J.L., and S.J.E. designed research; Q.L., A.L.B., and Z.H. performed research; A.N. and R.J.X. contributed new reagents/analytic tools; Q.L., A.L.B., partial or genome-wide siRNA libraries, have identified a number A.N., R.J.X., T.J.L., and S.J.E. analyzed data; and Q.L., A.L.B., A.N., R.J.X., T.J.L., and S.J.E. of host factors involved in HCV replication (5–7). However, the wrote the paper. replicon system is unable to address the role of host factors in the The authors declare no conflict of interest. full HCV lifecycle. The infection competent HCV cell culture 1Q.L. and A.L.B. contributed equally to this work. system (HCVcc) recapitulates the complete lifecycle, thereby per- 2To whom correspondence may be addressed: E-mail: [email protected] mitting a greater range of host-viral interactions to be studied or [email protected]. (8–10). Two efforts have used the HCVcc system using limited This article contains supporting information online at www.pnas.org/cgi/content/full/ candidate gene siRNA screens of either 65 or 140 curated targets 0907439106/DCSupplemental.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0907439106 PNAS Early Edition ͉ 1of6 Downloaded by guest on September 23, 2021 A Part One tested the four siRNAs from each pool separately, and found that 262 out of the 521 total pools selected (50%) were confirmed with NT CD81 ApoE at least two siRNAs, decreasing the likelihood of off-target effects [Dataset S1, Materials and Methods, the list of all genes scoring in the primary round is shown in Dataset S2 (17)]. Forty-four genes decreased viral propagation with multiple siRNAs predominantly in part two, implicating them in late-stage viral infection (Dataset S1). 100 19 120 siRNA Screen Bioinformatics and Meta-analysis. Among the host Part Two factors whose depletion decreased HCV infection in the primary NT CD81 ApoE screen, we recovered both positive controls, CD81 and ApoE, and over thirty proteins previously implicated by functional genomic studies to be needed for the replication of HCV or West Nile virus (WNV), a related flavivirus (Dataset S1 and Dataset S2). The screen recovered six of the 26 host proteins determined by Randall et al. to decrease JFH-1 levels by Ͼ3-fold after intensive siRNA 100 17 37 targeting (Dataset S2) (11). Among these shared candidates are the kinases MAPK1 and Raf1/MAPK3, the RNA-binding protein, ELAVL1 and the RNA-helicase, DDX3X. Proteomic studies, B including the HCV infection mapping project (I-MAP) and exper- Part One iments by Randall et al., have reported a direct physical interaction between Raf1/MAPK3, Stat3 and DDX3X and one or more of the HCV-encoded proteins (11, 20). This screen detected 15 of 96 factors recently reported by Tai et al. in a whole-genome siRNA screen using an HCV subgenomic replicon (Dataset S2) (5); the common genes include the kinases PI4KA and NUAK/SNARK, and the enzymes HAS1, CTSF, and IDS. NUAK/SNARK, an AMPK-related kinase functioning in the cellular stress response, was one of nine host factors found by Ng et al. to be necessary for HCV replicon function in a siRNA screen targeting approximately 4,000 genes (7). A critical role for PI4KA was also apparent in an HCVcc-based screen using a preselected library of genes implicated in endocytic trafficking (12). In that study, 7 of the 140 genes tested were needed by HCV, including the actin-regulating kinase, ROCK2, which we also recovered in our C Part Two screen (12). Consistent with Tai et al., we observed that multiple subunits (ARCN1, COPA, and COPB1) of the Golgi-associated retrograde transport complex, coatomer 1 (COP1), were needed for JFH-1 infection (Dataset S2) (5). The requirement for two COP1 subunits (COPA and COPB2) for HCV replication, was also observed in the trafficking-focused screen (12). Interestingly, our study detected 14 genes in common with those previously found in an RNAi screen for human proteins required for WNV infection, among them the enzymes BCKDHA, RPS6KL1, and USP11 (Dataset S2) (14). The WNV screen de- tected host factors involved in viral entry through replication, and thus would not find genes detected in part two of our study. However, the small degree of overlap (4.9%), between the host genes needed by these related viruses could arise in part from viral-specific host factor requirements, as well as differences in experimental methodologies. For example, none of the HCV Fig. 1. The siRNA screen. (A) siRNAs against CD81 or ApoE inhibit HCV siRNA screening efforts identified the ER-associated-degradation infection (green: HCV core). Percent infected (lower left corner) is relative to pathway (ERAD) as important, even though multiple components control throughout (Fig. S1A and B). NT, Nontargeting siRNA #2. Magnifica- were required for WNV replication (14), indicating differential ϫ tion, 20 .(B and C) The results of the HCVcc siRNA screen part one (B) and part dependencies. two (C) are shown with the siRNA SMARTpools ranked in order of average Z-score, from lowest (decreased infection) to highest (increased infection). Analysis for functionally enriched categories demonstrates a The position of known HCV-host factors and several newly identified genes significant number of HCV host factors reside in the nucleus (82, that scored in the screen are indicated. P ϭ 0.002). This includes ribonucleoprotein complex compo- nents (e.g., RBM22, SRRM2, UBA52), and transcription factors (e.g., BRF1, E2F2, ERCC5, FOXE3, MLXIPL, SMAD5, mean in either part one and two, or in part two alone, and did not SMAD6, TRRAP, WWTR1). Classification of the genes into decrease cell numbers to Ͻ50% of the plate mean in part one. Using higher level molecular function and biological process categories these criteria, we selected 407 pools for further validation (2% of showed significant enrichment for kinases (14 in total, P ϭ 0.01), the total genes screened, Fig. 1B). siRNA pools whose transfection factors involved in protein metabolism and modification (P ϭ increased core protein expression by Ͼ150% of the plate mean in 0.03), oncogenesis (P ϭ 0.006), nucleic acid binding (P ϭ 0.01) either part one or two, were also selected (increased infection) for and nucleic acid metabolism (P ϭ 0.03, Fig. S1E and F and validation (114 pools, 0.6%, Fig. 1C). In the validation round, we Dataset S3).

2of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0907439106 Li et al. Downloaded by guest on September 23, 2021 ADAMTS4 SRI FCGRT STMN2

TRRAP CALCOCO2 B2M NMT1 ELAVL1 EEF1A1 UBA52 UBQLN4 EFEMP1 BCAN PRKRIP1 C20orf20 FN1 KPNA1 GNB2 PTMA KHDRBS1 p7 LCK JUN CYBA IKBKE SRC STIM2 MAPK7 HCK TRIO XPNPEP1 MORF4L1 FES PIK3R1 EIF2AK2 PRPF40A PRKACA ZNF467 RBM4 ZNF148 RINT1 FMNL1 APLP2 SIAH1 ARFIP2 TAF1 NS3 STAT3 AKT1 TBK1 RAC1 ITGB1 TGFB1I1 FYN TICAM1 USHBP1 GNB2L1 YY1AP1 CDC6 SORBS3 WDR37 CDKN1A F CCNB2 MBP FLT4 AGT EWSR1 GRB2 ERCC5 C1QBP MAPK1 FRS3 TGFBR1 CORE BCAR1 E2F2 MYO3A CHUK CREBBP YY1 BCL6 NUAK2 TRAF2 YWHAZ CNOT3 MARK3 YWHAE CDCA3 WWTR1 IRF3 YWHAB CNOT2 SRRM2 CNOT1 VIM NPAT KCNK3 PPIB PROX1 TP73 EP300 PSME1 TFAP2A TP53 FADD TNFRSF1A TWIST1 FAS TBP TOP1 ZEB1 STAT1 RIPK2 HIPK3 BRF1 SMAD3 RAF1 MCM2 LIMS2 RXRA JAK1 JAK2 DDX3X MICROBIOLOGY KRT8 PML SMURF1 PPP2R1A PKM2 PLSCR1 KRT18 NR4A1 CDK6 SMURF2 DNAJA3 NS5A NS4A FHL2 PPIA NS5B NOC4L RAB14 GSK3B HSPA1A NUP62 LYN ARHGEF10L NS2 SMAD6 SORBS2 E1 E2 SMAD5 ITGA7 VAPA VAP B CDH1 NCL UBQLN1 DNAJB1 DLGAP4 CANX TUBB2C OSBP VAM P 1 HCV-encoded proteins HCV-interacting proteins (human) (HHCV) HCVcc siRNA screen hits HCV-interacting proteins that also scored in the HCVcc siRNA screen Interactions between HCV-encoded proteins and HCV-interacting proteins Interactions between HCVcc siRNA screen hits and HCV-interacting proteins

Fig. 2. Network showing connections between HCV proteins, HCV-interacting human proteins and candidate proteins from the HCVcc siRNA screen. Network extension analysis of HCV-host protein interactions derived from the infection mapping project (I-MAP, 20) revealed extensive first-order (direct) interactions between host factors implicated in the I-MAP and those found in the HCVcc siRNA screen (enrichment P ϭ 0.0001).

We encountered several pathways and macromolecular com- tected. Components of the nucleolus were well represented in plexes consistent with known phases of the viral lifecycle. HCV the nuclear-associated factors enriched for by the screen (TOP1, entry depends on four host cell receptors, and our screen recovered TCOF1, PNO1, NOP2, NOL6, NOP5/NOP58, NOC4L, two, CD81 and claudin 1. Our results confirm that cyclophilin A HEATR1, and IMP4). TCOF1 physically associates with nucle- (CypA) is the peptidyl-prolyl cis-trans isomerase required for HCV olar proteins, and is the gene responsible for Treacher Collins infection (18). Therefore, CypA is the likely target of the anti-HCV Syndrome, a severe congenital craniofacial developmental dis- cyclosporin analogue, Debio-025, thus demonstrating that RNAi ease (22). Mice deficient in TCOF1 display ribosome biogenesis can find high-yield leads for host-directed anti-viral therapies deficiencies suggesting that cells depleted in TCOF1 may be (HDAVs, 19). Depletion of three of five signal peptidase compo- unable to meet the increased demand for protein production nents (SPCS1, 2 and 3), which process the HCV envelope proteins required by HCV (22). Four of nine components of the Ccr4-Not E1 and E2 in the rER (3), inhibited infection in part two, validating complex (CNOT1, CNOT2, CNOT3, and CNOT6L) were re- their role in pathogenesis (P ϭ 6.14ϫ10Ϫ4)andthedesignofthis quired for HCV infection. The Ccr4-Not complex is a global portion of the screen. Glycosylation of the E1 and E2 viral envelope regulator of gene expression that functions in transcription and proteins is needed for HCV entry (21), suggesting an explanation polyadenylation (23). Mutation of each of the components of the for the role of OST48/DDOST, a component of the rER oligosac- orthologous yeast Ccr4-Not complex revealed that each subunit charyltransferase complex, which we detected as a late-acting HCV regulates the expression of a largely unique set of genes (24). host factor. Our efforts to find HIV-dependency factors (HDFs) Therefore, the components identified in this screen may control also found that OST48 was required for late-stage viral replication, distinct sets of HCV host factors. consistent with its playing a role in glycosylation of the viral envelope spike (13). Integration of HCV-Host Factor Interactions. To provide a more Previously unappreciated HCV dependencies were also de- comprehensive view of HCV-host interactions, we integrated the

Li et al. PNAS Early Edition ͉ 3of6 Downloaded by guest on September 23, 2021 Interactions between HCVcc siRNA screen hits and:

HCV CORE-interacting proteins HCV E1-interacting proteins HCV F-interacting proteins ARHGEF10L STAT3 ARHGEF10L SMURF2 SMURF2 SMURF2 EWSR1 MCM2 WWTR1 PPP2R1A NR4A1 TBP JUN NR4A1 CANX C1QBP TOP1 YWHAE MAPK1 RAB14 AGT KCNK3 TOP1 SMURF1 RXRA BRF1 SMAD3 TP53 KRT8 YWHAZ ZEB1 TFAP2A MAPK1 HSPA1A HCV E2-interacting proteins HCV p7-interacting proteins SMURF2 TNFRSF1A MARK3 FLT4 ARHGEF10L RIPK2 ZNF148 RAF1 YWHAB JAK1 UBQLN1 UBQLN4 CHUK KRT18 EP300 SRRM2 ITGB1 NR4A1 CANX PRPF40A TWIST1 STAT3 JAK2 E2F2 CDKN1A FMNL1 GNB2L1 ZNF467 MAPK1 YY1 GNB2L1 PROX1 PPIA BCAR1 CDC6 PML STAT1 STAT3 PRKRIP1 RAC1 RAC1 HIPK3 FAS IRF3 CREBBP FADD CCNB2 TP73 EWSR1 EIF2AK2 HCV NS3-interacting proteins RAC1 NPAT CHUK PRPF40A CNOT3 PLSCR1 HSPA1A RBM4 NOC4L ADAMTS4 FN1 USHBP1 VIM BCAN SMAD5 TRIO TICAM1 PSME1 PPP2R1A NOC4L ZNF467 ELAVL1 RAC1 ZEB1 FES IRF3 ARFIP2 MBP STAT3 TBK1 HCV NS4A/B-interacting proteins KPNA1 SMURF2 VIM HCV NS2-interacting proteins MAPK1 SMAD3 GNB2L1 BCL6 IKBKE ARHGEF10L PRPF40A SMAD6 PSME1 JUN KHDRBS1 SMURF1 ZNF148 FRS3 GNB2 NR4A1 UBQLN1 UBA52 STMN2 TP53 CHUK RAF1 TOP1 MAPK7 EEF1A1 PRKACA MAPK1 BRF1 HSPA1A TFAP2A EFEMP1 EWSR1 TAF1 YY1AP1 FCGRT C20orf20 XPNPEP1 DDX3X SRI HCV NS5B-interacting proteins PSME1 RAF1 TOP1 IRF3 HIPK3

SIAH1 TGFB1I1 B2M MORF4L1 RINT1 NUP62 CALCOCO2 TUBB2C PKM2 NCL PPIB

CDK6 OSBP VAM P 1 FLT4 SORBS3 MCM2 VAP B VAPA TFAP2A TBP LYN BRF1 NMT1 GRB2 TOP1 FYN MAPK1 LCK ZNF148 EFEMP1 TP53 SRC CDCA3 HSPA1A GNB2L1 RAF1 PTMA DNAJA3 TRAF2 JAK1 STAT3 CHUK HCV NS5A-interacting proteins AKT1 RIPK2 MYO3A EIF2AK2 TGFBR1 HCK APLP2 RAC1 SMAD6 PRKRIP1 PIK3R1 UBA52 NUAK2 SMURF2

HIPK3 CDH1 DLGAP4

LIMS2 GSK3B SORBS2

HCV-interacting proteins (human) (HHCV) HCVcc siRNA screen hits HCV-interacting proteins that also scored in the HCVcc siRNA screen Interactions between the HCVcc siRNA screen hits and HCV-interacting proteins

Fig. 3. Network clusters derived from Fig. 2 showing interactions between HCV-host factors from the siRNA screen and host proteins interacting with HCV core, E1, E2, F, p7 or NS components (from the infection mapping project (I-MAP, 20) thereby placing the siRNA screen hits in the network neighborhood of the respective HCV proteins. For clarity, interactions between HCV proteins and their human interacting partners are not displayed, but are shown in Fig.2.

functional results of our study with the proteomic and literature 0.0001, Fig. 2). This places the candidates detected using RNAi into mining data of the HCV infection mapping project (I-MAP), the interaction neighborhood of the HCV proteome (Fig. 3). undertaken by Chassey et al. (Fig. 2) (20). The I-MAP used parallel HCV and WNV belong to the Flaviviridae family, whose yeast two-hybrid screens employing HCV proteins as prey and two members include the human pathogens dengue virus and Jap- human cDNA libraries as bait. The resulting protein-protein inter- anese encephalitis virus (25). We explored the possibility of action (PPI) network was then extended using literature mining and uncovering common features presented by host-Flaviviridae a large human PPI database. In addition to providing functional interactions. Among the overlapping hits and first-order inter- evidence for the importance of nine proteins found by the I-MAP actions between host factors required for HCV, WNV and (CHUK, CTGF, DDX3X, PI4KA, RAF1, STAT3, FBLN5, dengue virus siRNA screens (14), a striking enrichment of RNF31, SMURF2), our network extension analysis revealed nu- pathway components and common network modules involved in merous first-order (direct) interactions between 63 HCV host TGF-␤ signaling (P ϭ 0.0002), ErbB signaling (P ϭ 0.002), factors found in this screen and those found in the I-MAP (P ϭ MAPK signaling (P ϭ 0.004), focal adhesion (P ϭ 0.006), and

4of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0907439106 Li et al. Downloaded by guest on September 23, 2021 wide HCV replicon screen detected common network modules A STIM2 Ubiquitin-mediated proteolysis (p=0.04) associated with TGF-␤ signaling, perhaps delineating the por- PRPF40A RAD51 tion of the viral lifecycle that relies on this pathway (P Ͻ 0.0005, MAPK signaling pathway UBA52 UBE2I IRF3 (p=0.004) RIPK2 Fig. 4 B and C) (5). TGFb-signaling pathway BRF1 PIAS2 SMAD6 CBLL1 TOP1 (p=0.0002) An estimated 25–30% of HIV-infected individuals are chroni- SMAD5 CDH1 STK3 CHUK SMURF2 cally coinfected with HCV (26). Defining host factors and pathways CSNK2A1 SMURF1 PAK1 RAB34 MYO3A upon which both HIV and HCV depend could suggest a common TGFBR1 KLHL1 GAB1 RAF1 MAPK1 RAB33B treatment strategy. Comparing the host factors recovered from the ErbB signaling pathway SHC1 RAC1 RABGEF1 (p=0.002) NUAK2 HCVcc and HIV siRNA screens reveals ten genes, DDX3X, PXN PLK4 CYBA FLT4 ATP6V0C Focal adhesion DNAJB1, ETF1, HEATR1, MAP4, NMT1, OST48, SPCS3, ZAP70 STAT3 MARK3 PITPNM2 PTK2B (p=0.006) SUV420H1, and Rap9p40/RABEPK that are needed for replica- TEAD1 VPS11 EWSR1 ZNF467 RBPMS GNB2L1 ZNF148 YAP1 tion of both viruses (13). Rab9p40 functions in the movement of late ERCC5 TEAD3 ARHGAP9 WDR37 WWTR1 SLC9A3R2 endosomes to the trans Golgi network (TGN), and along with its RBM9 BCKDHA NOL5A TNPO2 MT2A AP3S2 BCAP31 CITED2 GSC USP11 interaction partners, Rab9 and PIKfyve, is required for the particle RBM22 ZNF608 assembly of multiple viruses (HIV, Marburg, Ebola and measles TCOF1 ELAVL1 ADAMTS4 AP3B1VAM P 1 TFAP2A FOXA2 RPS6KL1 virueses), although the mechanism is not understood (27, 28). This HCVcc siRNA screen hits study, together with that of Tai et al., adds HCV to the list of WNV siRNA screen hits Overlapping siRNA hits from WNV & Dengue screens Rab9p40-dependent viruses. Overlapping siRNA hits from HCVcc & WNV screens Overlapping siRNA hits from HCVcc, WNV & Dengue screens Discussion 1st-order (direct) interactions between siRNA hits from HCVcc, WNV & Dengue screens Previous efforts have discovered important steps in HCV replica- tion, yet many events remain uncharacterized (2, 3, 25). Using B ZEB1 SRI RNAi and the HCVcc system, we have completed a genome-wide TGFb-signaling pathway PPP2R1A CISH (p=5x10-10) ITGA7 TRIM62 screen to find host factors required by HCV. The screen was FHL3 MARK3 HIPK3 validated by the ‘‘rediscovery’’ of Ͼ30 host factors previously SMAD2 NUAK1 SMURF2 SKIL MAPK1 implicated in HCV pathogenesis, including two host coreceptors, HSPA1A TGFBR1 NUAK2 SMAD4 SMAD6 CD81 and claudin 1. We then performed a bioinformatic meta- EWSR1 UBA52 CHUK MYO3A analysis to synthesize the information from our own work and MICROBIOLOGY ACVR1 RIPK2 SMAD5 earlier functional and proteomic screens for both HCV and related PRPF40A Flaviviridae host factors. This analysis revealed a strong statistical siRNA hit from HCVcc screen enrichment for several host cell pathways and complexes, as well as Overlapping siRNA hit from HCVcc and HCV replicon screens Bridging component in network neighborhood multiple direct interactions between the functionally defined data Interaction up to second order and one from a comprehensive proteomic study (20). Many inter- esting connections have emerged from the integration of these efforts that will serve as a resource for future investigations. TGFb signaling pathway C (p=0.0003) VEGF signaling pathway We also identified ten host genes that are needed by HCV and (p=0.003) TBK1 HIV. These genes may represent common dependencies that could RGPD5 GNB2L1 IRF3 SERPINB2 RAN CYBA RAF1 be exploited in instances of coinfection. The observation that STAT3 SMURF2 SMAD5 NOX1 RAC1 MAPK1 Rab9p40 is needed by the HCV replicon, demonstrates that it acts SMAD6 BCAT1 ZNF467 ZNF148 before viral particle formation, perhaps influencing the generation SMURF1 RIPK2 of the membranous web, given the juxtaposition of this structure UBA52 CHUK and the TGN. Rab9p40 contains a predicted ␤-propeller structure BCKDHA TOP1 DNAJB1 USP11 KCNK3 C20orf20 MAP3K14 homologous to known phosphatidyl inositol (PI)-binding domains YEATS4 TRRAP BCKDHB TBL3 NCOA6 COPB1 HSF1 HSPA1A MLXIPL (28), which might provide a functional link to PI (4)-phosphate

siRNA hit from HCVcc screen generated in the ER by PI4KA (29). siRNA hit from HCV replicon screen TGF-␤ levels are elevated in patients living with HCV, with even 1st-order (direct) Interaction between siRNA hits from HCVcc screen 1st-order (direct) Interaction between siRNA hits from HCVcc and HCV replicon screens higher levels being present in HIV-HCV coinfected individuals (30). Elevated TGF-␤ levels enhance HCV replication in vitro and correlate with accelerated liver fibrosis in vivo (31, 32). Gene Fig. 4. Common features of host-Flaviviridae interactions. (A) Common expression profiling of Huh 7.5.1 liver cells infected with the JFH-1 network modules for HCV-dependency factors identified in siRNA screens for ␤ Flaviviridae. The network was generated using first-order (direct) interactions virus discovered increased transcription of TGF- pathway genes, between siRNA hits from HCV, WNV, and dengue virus screens to identify including Smad7 and 9 (33). SMAD7 dampens the pathway’s common functional modules potentially used by Flaviviridae. Network clus- activity via a TGF-␤-induced negative feedback loop (33, 34). Our ters obtained were examined for enrichment (P Ͻ 0.05) in KEGG signaling data provide further evidence of the importance of this pathway for pathways. (B and C) Common network modules for HCV host factors identified HCV replication and lend functional relevance to the above- in the HCVcc and HCV replicon siRNA screens. (B) Network cluster generated mentioned gene expression profiling studies, in that both positive using second order interactions for overlapping siRNA hits reveals a significant (SMAD5) and negative regulators (SMAD6, SMURF1, and ␤ ϭ ϫ Ϫ10 enrichment for the TGF- -signaling pathway (P 5 10 ). (C) First order SMURF2) of the TGF-␤ pathway were detected. Together, these interactions identify connections between HCV replicon screen hits and a ␤ number of TGF-␤- and VEGF pathway-associated siRNA hits from the HCVcc results suggest that intact TGF- pathway homeostasis may benefit screen. Other binary or triple-member connections include subunits (BCKDHA the virus. and BCKDHB) of the branched chain keto acid dehydrogenase E1 enzyme Whereas RNAi technology represents a revolution in mamma- complex, and components (TRRAP and YEATS4) of the NuA4 histone acetyl- lian genetics, it also has caveats and must be interpreted with transferase complex. significant caution. Major concerns include false positives, arising from off-target effects (OTEs), where the promiscuous actions of the siRNA results in the observed phenotype being because of the ubiquitin-mediated proteolysis (P ϭ 0.04) was observed (Fig. depletion of a second unintended target, as well as false negatives, 4A). These may represent important shared functional modules stemming in part from inefficient targeting or nonspecific toxicity used by Flaviviridae. A comparison of this study to the genome- (17). We attempted to minimize false positives by testing our

Li et al. PNAS Early Edition ͉ 5of6 Downloaded by guest on September 23, 2021 primary candidates with multiple individual siRNAs (Dataset S2) may therefore provide targets upon which to focus the search for (17). We also integrated several HCV host factor data sets, and causal genetic variations that affect HCV infection. revealed statistically enriched complexes and pathways. To preserve new connections and minimize bias, we performed our bioinfor- Experimental Methods matics analysis after the selection of our validated gene set. Thus it See SI Text for additional experimental details. appears prudent to view the gene candidates confirmed with two or more siRNAs, or in two or more studies, either proteomically or siRNA Screen: Part 1. siRNAs were transfected into the Huh 7.5.1 cells at a 50 nM final concentration, using Oligofectamine (Invitrogen) in a 384-well format functionally, as being the highest confidence host genes involved in (Corning 3712). After 72 h, the cells were infected with the JFH-1 genotype 2a HCV infection. However, given the variation in screening meth- virus. After 48 h, the media was replica plated onto a plate with Huh 7.5.1 cells. odologies, and the current levels of false negatives, it is likely that The part 1 cells were then fixed and stained with anti-HCV core 6G7 monoclonal many genes uniquely indicated in these studies will ultimately be antibody provided by Drs. Harry Greenberg and Xiaosong He, (Stanford Univer- shown to partake in the viral lifecycle. sity) using an Alexa Fluor 488 antibody (A11001, Invitrogen). Cells were imaged The genes identified by these efforts represent a starting point for with an Image Express Micro (Molecular Devices) and analyzed with Metamorph Cell Scoring software (Molecular Devices Inc.). defining a complete set of HCV-host interactions. HCV host factors are potentially valuable targets for therapies to combat the more Part 2. Forty-eight hours after the transfer of the part one supernatant, the part recalcitrant HCV strains. An example of an HDAV against HCV two cells were stained for HCV core expression and imaged as in part 1. is Debio 025, which inhibits HCV infection by interfering with the host CypA protein (35). Our confirmation of HCV’s reliance on Cell Lines. Huh 7.5.1 cells were grown in DMEM 10% FBS. CypA illustrates that forward genetics can find host proteins that can be targeted to block infection. Virus. JFH-1 was propagated and infectivity was titrated as described (8, 37). In light of the heterogeneous course of chronic HCV infection, ACKNOWLEDGMENTS. We thank the ICCB-Longwood: C. Shamu, S. Chang, S. the variable response of patients to therapy, and the estimated 20% Rudnicki, S. Johnston, K. Rudnicki, D. Wrobel, and Z. Cooper. Funding support: of individuals that are exposed to HCV but clear the infection, a A.L.B. (Harvard Center for AIDS Research), A.N. (Crohns and Colitis Foundation of reasonable assumption is that polymorphisms in HCV host factors America), R.J.X. (Center for the Study of Inflammatory Bowel Disease Genetics may in part underlie these observations. There is mounting evi- and Genomics Core and National Institutes of Health Grant DK043351). The ICCB-Longwood is in part supported by a grant to T. Mitchison (National Cancer dence from genome wide association studies that rare polymor- Institute). This work was supported by the Intramural Research Program of the phisms may collectively account for a sizable percentage of the National Institute of Diabetes and Digestive and Kidney Diseases, National Insti- variation observed in heritable traits, including the course of tutes of Health, and the New England Regional Center of Excellence for Biode- fense and Emerging Infectious Diseases (National Institutes of Health Grant U54 chronic infections (36). Functional genomic and proteomic studies AI057159 to Dennis Kasper). S.J.E. is an Investigator with the Howard Hughes of HCV infection, and the meta-analyses of these combined efforts, Medical Institute.

1. Ploss A, et al. (2009) Human occludin is a hepatitis C virus entry factor required for 20. de Chassey B, et al. (2008) Hepatitis C virus infection protein network. Mol Syst Biol 4:230. infection of mouse cells. Nature 457:882–886. 21. Goffard A, Dubuisson J (2003) Glycosylation of hepatitis C virus envelope proteins. 2. Moradpour D, Penin F, Rice CM (2007) Replication of hepatitis C virus. Nat Rev Biochimie 85:295–301. Microbiol 5:453–463. 22. Sakai D, Trainor PA (2009) Treacher Collins syndrome: Unmasking the role of Tcof1/ 3. Lindenbach BD, Rice CM (2005) Unravelling hepatitis C virus replication from genome treacle. Int J Biochem Cell Biol 41:1229–1232. to function. Nature 436:933–938. 23. Collart MA, Timmers HT (2004) The eukaryotic Ccr4-not complex: A regulatory platform 4. Miyanari Y, et al. (2007) The lipid droplet is an important organelle for hepatitis C virus integrating mRNA metabolism with cellular signaling pathways? Prog Nucleic Acid Res production. Nat Cell Biol 9:1089–1097. Mol Biol 77:289–322. 5. Tai AW, et al. (2009) A functional genomic screen identifies cellular cofactors of 24. Azzouz N, et al. (2009) Specific roles for the Ccr4-Not complex subunits in expression hepatitis C virus replication. Cell Host Microbe 5:298–307. of the genome. RNA 15:377–383. 6. Supekova L, et al. (2008) Identification of human kinases involved in hepatitis C virus 25. Lindenbach BD, Rice CM (2003) Molecular biology of flaviviruses. Adv Virus Res replication by small interference RNA library screening. J Biol Chem 283:29–36. 59:23–61. 7. Ng TI, et al. (2007) Identification of host genes involved in hepatitis C virus replication by small interfering RNA technology. Hepatology 45:1413–1421. 26. Andersson K, Chung RT (2006) Hepatitis C Virus in the HIV-infected patient. Clin Liver 8. Wakita T, et al. (2005) Production of infectious hepatitis C virus in tissue culture from Dis 10:303–320. a cloned viral genome. Nat Med 11:791–796. 27. Murray JL, et al. (2005) Rab9 GTPase is required for replication of human immunode- 9. Zhong J, et al. (2005) Robust hepatitis C virus infection in vitro. Proc Natl Acad Sci USA ficiency virus type 1, filoviruses, and measles virus. J Virol 79:11742–11751. 102:9294–9299. 28. Shisheva A (2008) PIKfyve: Partners, significance, debates and paradoxes. Cell Biol Int 10. Lindenbach BD, et al. (2005) Complete replication of hepatitis C virus in cell culture. 32:591–604. Science 309:623–626. 29. Balla A, Balla T (2006) Phosphatidylinositol 4-kinases: Old enzymes with emerging 11. Randall G, et al. (2007) Cellular cofactors affecting hepatitis C virus infection and functions. Trends Cell Biol 16:351–361. replication. Proc Natl Acad Sci USA 104:12884–12889. 30. Blackard JT, et al. (2006) Intrahepatic cytokine expression is downregulated during 12. Berger KLC, et al. (2009) Roles for endocytic trafficking and phosphatidylinositol HCV/HIV co-infection. J Med Virol 78:202–207. 4-kinase III alpha in hepatitis C virus replication. Proc Natl Acad Sci USA 106:7577–7582. 31. Lin W, et al. (2008) HIV increases HCV replication in a TGF-beta1-dependent manner. 13. Brass AL, et al. (2008) Identification of host proteins required for HIV infection through Gastroenterology 134:803–811. a functional genomic screen. Science 319:921–926. 32. Wilson LE, et al. (2006) Progression of liver fibrosis among injection drug users with 14. Krishnan MN, et al. (2008) RNA interference screen for human genes associated with chronic hepatitis C. Hepatology 43:788–795. West Nile virus infection. Nature 455:242–245. 33. Walters KA, et al. (2009) Genomic analysis reveals a potential role for cell cycle perturba- 15. Konig R, et al. (2008) Global analysis of host-pathogen interactions that regulate tion in HCV-mediated apoptosis of cultured hepatocytes. PLoS Pathog 5:e1000269. early-stage HIV-1 replication. Cell 135:49–60. 34. Inoue Y, Imamura T (2008) Regulation of TGF-beta family signaling by E3 ubiquitin 16. Chang KS, Jiang J, Cai Z, Luo G (2007) Human apolipoprotein e is required for infectivity ligases. Cancer Sci 99:2107–2112. and production of hepatitis C virus in cell culture. J Virol 81:13783–13793. 35. Flisiak R, et al. (2009) The cyclophilin inhibitor Debio 025 combined with PEG 17. Echeverri CJ, et al. (2006) Minimizing the risk of reporting false positives in large-scale RNAi screens. Nat Methods 3:777–779. IFNalpha2a significantly reduces viral load in treatment-naïve hepatitis C patients. 18. Yang F, et al. (2008) Cyclophilin A is an essential cofactor for hepatitis C virus infection Hepatology 49:1460–1468. and the principal mediator of cyclosporine resistance in vitro. J Virol 82:5269–5278. 36. Goldstein DB (2009) Common genetic variation and human traits. N Engl J Med 19. Flisiak R, et al. (2008) The cyclophilin inhibitor Debio-025 shows potent anti-hepatitis 360:1696–1698. C effect in patients coinfected with hepatitis C and human immunodeficiency virus. 37. Kato T, et al. (2007) Production of infectious hepatitis C virus of various genotypes in Hepatology 47:817–826. cell cultures. J Virol 81:4405–4411.

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