Supporting Information

Berger et al. 10.1073/pnas.0902693106 SI Methods GAC CGG-MGBNFQ. PCR conditions were 70 °C for 30 min, Generation of Cellular and Viral Constructs. Cellular genes were 95 °C for 6 min, and (95 °C for 15 sec, 55 °C for 30 sec, 72 °C for PCR amplified from Huh-7.5 cDNA with primers to allow 15 sec) ϫ 50 cycles. insertion of flanking restriction sites using high-fidelity Phusion DNA polymerase (Finnzymes). PCR amplicons were subcloned Cell Viability Assay. Cellular ATP levels were measured using the into pmGFP-C1 (a monomeric GFP version of pEGFP-C1; BD CellTiter-Glo Luminescent Cell Viability assay (Promega). Lu- ␮ Biosciences) or a modified pTrip-CMV-GFP (an HIV-based minescent reagent (100 l) was added directly to cells after vector) (1, 2) re-designed with PmeI in the multicloning site. treatment with siRNAs for 5 days in a 96-well format and RAB5A (GenBank Acc. #NM_004162) and RAB7L1 (GenBank incubated at room temperature for 10 min. Then luminescence ␮ Acc. #NM_003929) were cloned as C-terminal fusions in of 80 l was measured for 0.25 seconds per manufacturer’s pmGFP-C1 using the following (restriction sites italicized): instructions using a 96-well luminometer (Centro LB 960, RAB5A (forward 5Ј-GCA AGC TTc aac cat ggc tag tcg agg cgc Berthold Technologies). aa, reverse 5Ј-ACG GTA CCt tag tta cta caa cac tga ttc ct) and RAB7L1 (forward 5Ј-GCA AGC TTc gac cAT Ggg cag ccg cga Generation of HCV Pseudoparticles. HIV-HCV pseudotyped par- cca cc, reverse 5Ј-ACG GTA CCc tag cag cag gac cag ctg gag ga) ticles (HCVpp) were generated by co-transfecting 100-mm dishes of subconfluent 293T cells with 3 ␮g of plasmid expressing (Integrated DNA Technologies, IDT). Full-length PIK4CA se- ␮ quence (GenBank Acc. #NM_058004) was cloned and sequence HCV genotype 1a envelope proteins E1 and E2 and with 3 g of pNL4.3.Luc.RϪEϪ encoding the HIV retroviral backbone and verified as an N-terminal fusion in pTrip-CMV-GFP using the a firefly luciferase reporter (4–6), using Fugene6 (Roche). At following (restriction sites italicized): (forward 5Ј-GTT TAA 48 h posttransfection, supernatant was centrifuged at 1500 ϫ g ACA TGT GTC CAG TGG ATT TCC ATG GG, reverse 5Ј- and collected. Hepes buffer (Invitrogen) was added (20 mM final GTT TAA ACA TGT GTC CAG TGG ATT TCC ATG GG) concentration), and aliquots were stored at Ϫ80 °C. (IDT). We constructed pSG-JFH1-Rluc, which contains an HCV Sample Processing and Imaging for Immunofluorescence Assays. Cells internal ribosome entry site (IRES)-driven Renilla luciferase were fixed in cold methanol for 15 min, washed in 1X PBS, and followed by an encephalomyocarditis virus (EMCV) IRES that blocked in 0.1% saponin-50% goat serum (Chemicon). Samples drives expression of HCV nonstructural proteins NS3–5B fol- Ј were incubated overnight with primary antibodies as follows: lowed by the HCV 3 nontranslated region (NTR). The neomy- 1:200 anti-dsRNA (mAB J2, English and Scientific Consulting cin resistance cassette of the previously described pSGR-JFH1- Bt.) and 1:20,000 anti-NS5A (9E10, gift of C. M. Rice, The Neo (3) was replaced with Renilla luciferase in a 3-piece ligation. Rockefeller University, New York) in 10% goat serum. Alexa Segment no. 1 was PCR amplified from upstream of an AgeI site Fluor 594-conjugated secondary antibody (1:1000) (Molecular in pSG-JFH1-Neo to the partial core sequence followed by a Probes-Invitrogen) in 10% goat serum was added for 1 h. After BglII site using the following (restriction site italicized): forward 3 PBS washes and a rinse in H O, coverslips were mounted in Ј Ј 2 5 -CAT GAA TCA CTC CCC TGT GA; reverse 5 -TAG ATC Mowiol (Calbiochem). Samples were imaged using an Olympus TTG GGC GAC GGT TGG TG. Segment no. 2 consisted of DSU spinning disc confocal microscope with a 100ϫ oil objec- Ј Ј Renilla luciferase, cloned to incorporate a 5 BglII and 3 PmeI tive. Digital images were taken using Slidebook 4.1 software and site with the following (restriction sites italicized): forward processed using ImageJ (National Institutes of Health) and 5Ј-AAG ATC TAT GAC TTC GAA AGT TTA TGA TC; Adobe Photoshop. reverse 5Ј-TGT TTA AAC TTA TTG TTC ATT TTT GAG AAC TC. The pSG-JFH1-Neo was digested with AgeI and PmeI, Sample Processing for Electron Microscopy. Cells were fixed with ligated with segments 1 and 2, and sequence verified. A repli- 2% glutaraldehyde and 4% paraformaldehyde in 0.1 M sodium cation-defective pSGR-JFH1-RLuc-GND construct was gener- cacodylate buffer for 2 h, washed with sodium cacodylate buffer, ated by mutating the GDD active site in the viral RNA poly- then incubated with 1% osmium tetroxide in 0.1 M sodium merase NS5B to GND by introduction of Asn at the second cacodylate buffer for 1 h. Cells were washed with sodium amino acid and sequence verified. cacodylate buffer, rinsed in maleate buffer (pH 5.1), then stained with 1% uranyl acetate in maleate buffer for 1 h. Samples were Real-Time PCR Conditions. RT-PCR conditions for measuring processed through a series of dehydration steps ranging from cellular gene expression from 2 ␮l of extracted RNA (from 25%–100% ethanol with a final 100% propylene oxide incuba- Ϸ130-␮l samples) were 48 °C for 30 min, 95 °C for 10 min, and tion. Samples were infiltrated with 2:1 propylene oxide:Spurr’s (95 °C for 15 sec, 60 °C for 1 min) ϫ 50 cycles. Genotype 1b HCV resin (Sigma) for 30 min, then with 1:1 propylene oxide:Spurr’s replicon RNA was detected from 2 ␮l of extracted RNA using resin for 30 min, and finally with 100% Spurr’s resin for 1 h. Resin the following primers and probe: forward 5Ј-CCG GGA GAG was polymerized with embedded cells at 60 °C for 1–2 days. CCA TAG TGG TCT; reverse 5Ј-CCA AAT CTC CAG GCA Ninety-nm sections were cut with a Reichert-Jung Ultracut E TTG AGC; probe 5Ј- 6FAM-CAC CGG AAT TGC CAG GAC microtome and stained with uranyl acetate and lead citrate.

1. Zennou V, et al. (2000) HIV-1 genome nuclear import is mediated by a central DNA flap. 4. Zhang J, et al. (2004) CD81 is required for hepatitis C virus glycoprotein-mediated viral Cell 101:173–185. infection. J Virol 78:1448–1455. 2. Philippe S, et al. (2006) Lentiviral vectors with a defective integrase allow efficient and 5. Hsu M, et al. (2003) Hepatitis C virus glycoproteins mediate pH-dependent cell entry of sustained transgene expression in vitro and in vivo. Proc Natl Acad Sci USA 103:17684– pseudotyped retroviral particles. Proc Natl Acad Sci USA 100:7271–7276. 17689. 6. Connor RI, Chen BK, Choe S, Landau NR (1995) Vpr is required for efficient replication 3. Kato T, et al. (2003) Efficient replication of the genotype 2a hepatitis C virus sub- of human immunodeficiency virus type-1 in mononuclear phagocytes. Virology genomic replicon. Gastroenterology 125:1808–1817. 206:935–944.

Berger et al. www.pnas.org/cgi/content/short/0902693106 1of7 Fig. S1. Cell viability following treatment with indicated siRNAs. Cell viability was determined 5 days after electroporation of siRNAs and quantiﬁed by a luminescence-based cell viability assay (Promega) that measures intracellular ATP levels. SEM is shown. siRNAs that signiﬁcantly reduced viability (**, P Ͻ 0.001) as compared with IRR were discarded.

Berger et al. www.pnas.org/cgi/content/short/0902693106 2of7 Fig. S2. PI4K-III␣ isoform 2 is required for HCV replication. PI4K-III␣ isoform 2 (230 kDa) was speciﬁcally silenced using siRNAs (PIK4CA-230 #1 and #2) and compared with a PIK4CA siRNA pool that targets both isoforms. The effects of these siRNAs on subgenomic replicon replication were quantiﬁed by luciferase activity. Values are relative to irrelevant siRNA (IRR)-treated cells. SEM (n ϭ 4) is shown. **, P Ͻ 0.001, and *,P Ͻ 0.05, as compared with IRR.

Berger et al. www.pnas.org/cgi/content/short/0902693106 3of7 Table S1. Genes and siRNAs tested in RNA interference screen siGENOME UPGRADE siGENOME SMARTpool (primary screen) Gene Genbank accession no. pooled siRNAs Catalog no. Individual siRNAs

CDC42 NM࿝001791 GGAGAACCAUAUACUCUUG D-005057–01* GGAGAACCAUAUACUCUUG GAUUACGACCGCUGAGUUA D-005057–02 GAUUACGACCGCUGAGUUA GAUGACCCCUCUACUAUUG D-005057–03 GAUGACCCCUCUACUAUUG CGGAAUAUGUACCGACUGU D-005057–04 CGGAAUAUGUACCGACUGU ROCK2 NM࿝004850 GAGGAAAGCUGAUCAUGAA D-004610–02 GUAGAAACCUUCCCAAUUC GUAGAAACCUUCCCAAUUC D-004610–03 GCAACUGGCUCGUUCAAUU GCAACUGGCUCGUUCAAUU D-004610–05 GCAAAUCUGUUAAUACUCG GCAAAUCUGUUAAUACUCG D-004610–18† GCAGCAAUGGUAAGCGUAA RAB7L1 NM࿝003929 CAGAUUGACCGGUUCAGUA GAGAACGGUUUCACAGGUU D-010556–03 GAGAACGGUUUCACAGGUU GGGACUACAUCAAUCUACA D-010556–05 GGGACUACAUCAAUCUACA CAACAAGUGUGAUCUGUCC D-010556–06 CAACAAGUGUGAUCUGUCC EEA1 NM࿝003566 AGACAGAGCUUGAGAAUAA D-004012–01 AGACAGAGCUUGAGAAUAA GAGAAGAUCUUUAUGCAAA D-004012–02 GAGAAGAUCUUUAUGCAAA GAAGAGAAAUCAGCAGAUA D-004012–03 GAAGAGAAAUCAGCAGAUA GAACAAGACUAUACUAAGU RAB5A NM࿝004162 GCAAGCAAGUCCUAACAUU GGAAGAGGAGUAGACCUUA D-004009–02 GGAAGAGGAGUAGACCUUA AGGAAUCAGUGUUGUAGUA D-004009–03 AGGAAUCAGUGUUGUAGUA GAAGAGGAGUAGACCUUAC D-004009–04 GAAGAGGAGUAGACCUUAC PIK3C2G NM࿝004570 GUAAAGCCUUGAAUGAUGA D-006773–01 GUAAAGCCUUGAAUGAUGA GGACCGAGCUCCUUUCAUU D-006773–02 GGACCGAGCUCCUUUCAUU GCACAAAUGUCAGCCAUAA D-006773–03 GCACAAAUGUCAGCCAUAA GAAAGAAAUUGGCAGACUA PIK4CA NM࿝002650 GAGCAUCUCUCCCUACCUA D-006776–01 GAGCAUCUCUCCCUACCUA GUGAAGCGAUGUGGAGUUA D-006776–02 GUGAAGCGAUGUGGAGUUA CCACAGGCCUCUCCUACUU D-006776–03 CCACAGGCCUCUCCUACUU GCAGAAAUUUGGCCUGUUU D-006776–09 GCAGAAAUUUGGCCUGUUU PIK4CA NM࿝058004 230 #1 GGAUAAAGCUAUUCAGAAA 230 #2 CGGAUAAAGCCAAGACCAA CD81 NM࿝004356 M-017257–02* ACACA NM࿝198834 M-004551–02 ACTR2 NM࿝005722 M-012076–00 ACTR3 NM࿝005721 M-012077–01 ADAM10 NM࿝001110 M-004503–01 AMPH NM࿝001635 M-011569–00 AP1B1 NM࿝001127 M-011200–00 AP1M1 NM࿝032493 M-013196–00 AP1M2 NM࿝005498 M-012056–00 AP2A1 NM࿝014203 M-012492–00 AP2A2 NM࿝012305 M-012812–00 AP2B1 NM࿝001282 M-003627–01 AP2M1 NM࿝004068 M-008170–00 ARF1 NM࿝001658 M-011580–00 ARF3 NM࿝001659 M-011581–00 ARF5 NM࿝001662 M-011584–00 ARF6 NM࿝001663 M-004008–00 ARFIP2 NM࿝012402 M-012820–00 ARPC1B NM࿝005720 M-012082–01 ARPC2 NM࿝005731 M-012081–00 ARPC3 NM࿝005719 M-005284–00 ARPC4 NM࿝005718 M-008571–00 ARPC5 NM࿝005717 M-012080–00 ARRB1 NM࿝004041 M-011971–00 ARRB2 NM࿝004313 M-007292–00 ATG5 NM࿝004849 M-004374–03 ATG12 NM࿝004707 M-010212–02 ATM NM࿝138293 M-003201–02 ATP6V0A1 NM࿝005177 M-017618–00 BIN1 NM࿝004305 M-008246–00 CAMK1 NM࿝003656 M-004940–00 CAV1 NM࿝001753 M-003467–01 CAV2 NM࿝001233 M-010958–00

Berger et al. www.pnas.org/cgi/content/short/0902693106 4of7 siGENOME UPGRADE siGENOME SMARTpool (primary screen) Gene Genbank accession no. pooled siRNAs Catalog no. Individual siRNAs

CAV3 NM࿝001234 M-011229–00 CBL NM࿝005188 M-003003–01 CBLB NM࿝170662 M-003004–01 CBLC NM࿝012116 M-006962–00 CFL1 NM࿝005507 M-012707–00 CIB1 NM࿝006384 M-012261–00 CIB2 NM࿝006383 M-012230–00 CIB3 NM࿝054113 M-012901–00 CLTA NM࿝001833 M-004002–00 CLTB NM࿝001834 M-004003–00 CLTC NM࿝004859 M-004001–00 CLTCL1 NM࿝001835 M-011611–00 COPA NM࿝004371 M-011835–00 COPB2 NM࿝004766 M-019847–01 CTBP1 NM࿝001328 M-008609–01 DAB2 NM࿝001343 M-008522–00 DDEF2 NM࿝003887 M-011544–00 DIAPH1 NM࿝005219 M-010347–01 DNM1 NM࿝004408 M-003940–00 DNM2 NM࿝004945 M-004007–01 DNM3 NM࿝015569 M-013931–00 EFS NM࿝005864 M-012094–00 ELKS NM࿝015064 M-010942–00 ENTH NM࿝014666 M-021406–00 EPN1 NM࿝013333 M-004724–00 EPN2 NM࿝148921 M-004725–00 EPN3 NM࿝017957 M-021006–00 EPS15 NM࿝001981 M-004005–00 EPS15L1 NM࿝021235 M-004006–00 FASN NM࿝004104 M-003954–04 FYN NM࿝002037 M-003140–03 GAF1 NM࿝015470 M-004298–00 GIT1 NM࿝014030 M-004298–00 GNB2L1 NM࿝006098 M-006876–00 GORASP1 NM࿝031899 M-013510–00 GRB2 NM࿝002086 M-019220–00 HGS NM࿝004712 M-016835–00 HIP1 NM࿝005338 M-005001–01 HIP1R NM࿝003959 M-027079–00 IHPK3 NM࿝054111 M-006739–00 ITSN1 NM࿝003024 M-008365–00 ITSN2 NM࿝006277 M-009841–00 LIMK1 NM࿝002314 M-007730–01 MAP4K2 NM࿝004579 M-003587–01 MAPK8IP1 NM࿝005456 M-003595–00 MAPK8IP2 NM࿝012324 M-012462–00 MAPK8IP3 NM࿝015133 M-003596–01 NEDD4 NM࿝006154 M-007178–01 NEDD4L NM࿝015277 M-007187–01 NSF NM࿝006178 M-009401–00 PACSIN1 NM࿝020804 M-007735–00 PACSIN3 NM࿝016223 M-015343–00 PAK1 NM࿝002576 M-003521–03 PI4KII NM࿝018425 M-006770–01 PI4K2B NM࿝018323 M-006769–01 PICALM NM࿝007166 M-004004–02 PIK3CG NM࿝002649 M-005274–02 PIK4CB NM࿝002651 M-006777–02 PIP5K1A NM࿝003557 M-004780–02 PITPNM1 NM࿝004910 M-019888–00 PRKCM NM࿝002742 M-005028–00 PSCD3 NM࿝004227 M-019268–00 RAB11A NM࿝004663 M-004726–02 RAB11B NM࿝004218 M-004727–01

Berger et al. www.pnas.org/cgi/content/short/0902693106 5of7 siGENOME UPGRADE siGENOME SMARTpool (primary screen) Gene Genbank accession no. pooled siRNAs Catalog no. Individual siRNAs

RAB3A NM࿝002866 M-009668–01 RAB3B NM࿝002867 M-008825–00 RAB3C NM࿝138453 M-008520–00 RAB3D NM࿝004283 M-010822–01 RAB4A NM࿝004578 M-008539–01 RAB4B NM࿝016154 M-008780–01 RAB5B NM࿝002868 M-004010–01 RAB5C NM࿝004583 M-004011–01 RAB6A NM࿝002869 M-008975–01 RAB6B NM࿝016577 M-008548–00 RAB7B NM࿝177403 M-018225–00 RAB8A NM࿝005370 M-003905–00 RAB8B NM࿝016530 M-008744–00 RAC1 NM࿝018890 M-003560–02 RHOA NM࿝001664 M-003860–00 ROCK1 NM࿝005406 M-003536–01 SARA1 NM࿝020150 M-016756–00 SH3GLB1 NM࿝016009 M-017086–00 SH3GLB2 NM࿝020145 M-015810–00 SNAP91 NM࿝014841 M-032296–00 SREBF1 NM࿝004176 M-006891–00 STAU NM࿝004602 M-011894–00 SYNJ1 NM࿝003895 M-019486–01 SYNJ2 NM࿝003898 M-012624–00 SYT1 NM࿝005639 M-020044–00 SYT2 NM࿝177402 M-018809–00 TNIK XM࿝039796 M-004542–02 VAMP1 NM࿝014231 M-012497–00 VAMP2 NM࿝014232 M-012498–00 VAPA NM࿝003574 M-021382–00 VAPB NM࿝004738 M-017795–00 VAV2 NM࿝003371 M-005199–00 VIL2 NM࿝003379 M-017370–01 WAS NM࿝000377 M-028294–01 WASF1 NM࿝003931 M-011557–00 WASF2 NM࿝006990 M-012141–00 WASF3 NM࿝006646 M-012301–01

*Dharmacon product number. †siRNA not part of original SMARTpool for primary screen.

Berger et al. www.pnas.org/cgi/content/short/0902693106 6of7 Table S2. Real-time RT-PCR assays Target PCR primer-probe sets*

CDC42 Hs00741586࿝mH ROCK2 Hs00178154࿝m1 RAB7L1 Hs00187510࿝m1 EEA1 Hs00185960࿝m1 RAB5A Hs00702360࿝s1 PIK3C2G Hs00362148࿝m1 PIK4CA Hs01090927࿝m1 GAPDH 4326317E 18S 4319413E

*Applied Biosystems product no.

Berger et al. www.pnas.org/cgi/content/short/0902693106 7of7