Cell Reports, Volume 16

Supplemental Information

Quantitative Lipid Droplet Proteome Analysis

Identifies Annexin A3 as a Cofactor

for HCV Particle Production

Kathrin Rösch, Marcel Kwiatkowski, Sarah Hofmann, Anja Schöbel, CordulaGrüttner, Marcus Wurlitzer, Hartmut Schlüter, and Eva Herker

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HCV PPI(Ramage)

HCVlipid dropletproteome

307 9 256

HCV RNAi(Li)

HCVlipid dropletproteome

308 8 88

HCV RNAi(Tai)

HCVlipid dropletproteome

309 7 46

HCV RNAi(Poenisch)

p-value = 0.013

p-value = 1.9 x 10-7 p-value = 1.1 x 10-4

p-value = 1.8 x 10-5

ARL8BVDAC1FAM213ASSBP1SAR1AVAPBHSD17B4

ZW10SSBCCT6APSMA5PSME2APOB

DDX3XDDOSTOSBPHNRNPKGNB2L1PPP2R1APSME1PPIAMCM2

ARL1CDC42COPACOPG1COPB1RAB7ACOPB2RAN

EPHX1VDAC3CLTCHNRNPKCOPAPRDX6RAN

F

Figure S1

1

C A T G G C A T C T T A T C T G G T TA AC A T G G C A T C T A T C T G G G T A A G

Splice site

CRISPR/Cas9

ATG GCA TCT TAT CTG GGT TGG ACA CCG AGG AAC AGT AAG AGA TTA TCC AGA CTT TAG M A S Y L G W T P R N S K R L S R L *

Allele 1: T insertion

sgRNA:G G G A U A U C A U G G C A U C U A U C

ATG GCA TCT GGG TTG GAC ACC GAG GAA CAG TAA M A S G L D T E E Q *

Allele 2: Δ4 deletion

C A T G G C A T C T T A T C T G G T A AG

Allele 1: T insertion

Allele 2: Δ4 deletionC A T G G C A T C T ATG G T A AG G A A

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Figure S2

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AffinityPurification

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105

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IgG(rb)

CD81(ms)

E2(ms)

ANXA3scbt (rb)

ANXA3sa (rb)

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ctio

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9 10 11 12 13 14 15 16 17 252418 19 20 21 22 23Input

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 252418 19 20 21 22 23

B

Figure S4

4

Supplemental Figure Legends

Figure S1: Analysis and validation of the lipid droplet proteome analysis, related to Figure 1. (A) Plot of the lipid droplet–associated proteins ranked according to their mean enrichment ratio (log2 HCV-infected/control) and centered around the median (Mean ± SD, n = 4, *p < 0.05, **p < 0.01, ***p < 0.001). Lines indicate 1.5-fold cut-off values in fold enrichment/depletion used for further analysis. (B) Scatterplot and regression analysis of all proteins identified in 4 independent experiments, in #1–#3 HCV-infected cells were cultured in light AA and control cells in heavy AA; in #4 cells were labeled reverse. Shown is the enrichment score at lipid droplets as log2 HCV-infected over control normalized to the median. (C) Pearson correlation coefficient r between the different experiments. (D) Volcano plot of p-values plotted against the mean enrichment ratios indicates in green the proteins selected for further analysis. (E) We performed PubMed searches to assess the fraction of the identified proteins that have been previously linked to either “lipid droplet” or “HCV” search terms. 35% of the proteins we detected in lipid droplet fractions have been previously annotated with the term “lipid droplet” while 37% were annotated together with term “HCV” (F) Venn diagrams indicating the overlap between the lipid droplet proteins identified in our screen with previously identified HCV protein-protein interaction partners (Ramage et al., 2015) or host dependency or restriction factors identified in RNAi screens (Li et al., 2009; Poenisch et al., 2015; Tai et al., 2009). P-values were calculated using GeneOverlap package in R.

Figure S2: CRISPR/Cas9-mediated knock out of ANXA3, related to Figure 2. Sequencing result of ANXA3 knockout and parental Huh7.5 cells. The binding site of the sgRNA is indicated. One allele harbors a T insertion while the second allele carries a 4 nucleotide deletion mutation. Both mutations leave the splicing site intact and, if translated, would lead to the production of an 18 or 10 amino acid peptide respectively.

Figure S3: ANXA3 is not associated with purified HCV and does not influence cPLA2 activity, related to Figure 6. (A) cPLA2 activity was assessed in HCV-infected ANXA3-knockdown and control cells by a commercial assay. Shown is the cPLA2 activity in mU/ml cell lysate. As positive control we used bee venom PLA2 provided with the kit (Mean ± SD, n = 3). (B) To analyze if ANXA3 is bound to HCV particles, Huh7.5 cells were infected with virus encoding FLAG-tagged E2 (Jc1FLAG-E2 NS5AB-EGFP). Cell supernatant was harvested, concentrated by polyethylene glycol precipitation and ultracentrifugation on a sucrose cushion, followed by affinity purification of FLAG-tagged virions with anti-FLAG agarose. (C) Purified HCVcc particles were analyzed by western blotting using core, E2, FLAG, ApoE, and ANXA3 antibodies. Of note, purified particles were re-loaded (right panel) to probe with the ANXA3 antibodies on a fresh membrane. While the viral proteins core and E2 as well as the host protein ApoE are integral components of the purified virions we did not detect ANXA3. (D) To further prove that ANXA3 is not part of virions we analyzed if ANXA3 antibodies can neutralize HCV. Therefore Jc1p7-GLuc-2A-NS2 virions were incubated with the indicated antibodies prior infection of naïve Huh7.5 cells. 2 days post infection HCV replication was analyzed by measuring the activity of secreted gaussia luciferase in the cell culture supernatant (Mean ± SD, measured in duplicate). While the positive controls anti-CD-81 and anti-E2 blocked HCV infection two different antibodies targeting ANXA3 had no effect.

Figure S4: ANXA3 is not required for baseline lipoprotein secretion in hepatoma cells, related to Figure 6. (A) Lipoprotein secretion was assessed by western blotting with ApoE and ApoB antibodies of culture supernatant of control or Con1 SGR–transfected cells. Quantification of ApoB and ApoE secretion by densitometric analysis of western blots (Mean ± SEM, n = 3–9). ApoE secretion levels are also shown in Figure 6. (B) Membrane floatation assay of HCV-infected ANXA3-knockdown and control cells to analyze the distribution of ApoE. The density profiles of each lane of ANXA3-knockdown and control cells was plotted using ImageJ and overlaid for easier comparison. Shown is one representative assay.

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Supplemental Tables

Tables S1–S4: Tables listing the peptides identified in each independent experiment, related to Figure 1.

Table S5: Proteins identified in LD fractions, related to Figure 1. Sheet “LD proteins” shows the Uniprot ID and the Gene Name of the proteins identified in all four experiments with the log2 enrichment HCV-infected/control, as well as the mean, the p-value and the PubMed ID search results for the gene names plus “lipid droplet (s) OR lipid bodies” and “HCV OR Hepatitis C”. Sheets “PubMed (LD)” and “PubMed (HCV) list the individual PubMed entries indicating the protein search term, the PMID, 1st author, title, and year of publication.

Table S6: Gene ontology enrichment analysis of the identified lipid droplet proteins, related to Figure 1. Gene ontology (GO) enrichment analysis of all proteins identified, enriched, or depleted of annotations for molecular function (MF), cellular compartment (CC), and biological process (BP), and KEGG and Reactome (REAC) biological pathways. Shown are annotations significantly enriched but not hierarchically filtered. Analysis was performed using the gProfileR package in R.

Table S7: List of proteins that were identified in our lipid droplet proteome and other genome/proteome-wide HCV screens, related to Figure 1. Shown is the Uniprot ID and the Gene Name of all proteins that were found in other screens as well as the log2 enrichment at lipid droplets.

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Supplemental Experimental Procedures

Plasmids pBR322 Jc1NS5AB-XFP constructs encoding HCV Jc1 and fluorescent proteins, selection markers, or the firefly luciferase between a duplicated NS5A-NS5B cleavage site were described previously (Webster et al., 2013a). In the pBR322 Jc1ΔE1E2NS5AB-FLuc replicon a portion of the E1/E2 genes from amino acids 313–567 in the Jc1 polyprotein is deleted (Webster et al., 2013b). pBR322 Jc1FLAG-E2 was described previously (Eggert et al., 2014) and pBR322 Jc1FLAG-E2 NS5AB-EGFP expressing FLAG-tagged E2 for the affinity purification of the virions and pBR322 Jc1FLAG-E2

NS5AB-EGFP-BSD for the co-immunoprecipitation and microscopy experiments were constructed by overlap extension PCR as described. pBR322 Jc1FLAG-E2 NS5A-HA was constructed from pBR322 Jc1FLAG-E2 and pBR322 Jc1ΔE1E2NS5A-

HA described previously (Vogt et al., 2013) and carries 3xHA and 6xHis tags in domain 3 of NS5A (at amino acid position 381). Wild type JFH1 (Wakita et al., 2005) was subcloned into pBR322 and Jc1 was described (Pietschmann et al., 2006). The HCV Jc1p7-GLuc-2A-NS3 construct was cloned by overlap extension PCR as described (Marukian et al., 2008). The Con1 subgenomic replicon has been described (Choi et al., 2004). To construct the ANXA3-HA expression plasmid, ANXA3 was PCR-amplified from plasmid SC116952 (Origene) using the primers listed below and cloned into the pEBB expression plasmid (Tanaka et al., 1995). Lentiviral shRNA constructs were cloned into the pSicoR-MS1 and pSicoR-MS2 vectors described previously using the primers listed below; control luciferase and scramble shRNAs have been described previously (Herker et al., 2010; Wissing et al., 2011). Additionally, a modified pSicoR-MS1-Puro vector was generated by replacing mCherry with a puromycin resistance gene using the primers listed below. shANXA3 and shNT were cloned into pSicoR-MS1-Puro using the primers listed below. Lentiviral core expression construct was described previously (Herker et al., 2010). FLAG-tagged HCV constructs were described previously (Ramage et al., 2015) and FLAG-core and NS5A-FLAG were subcloned into lentiviral LeGO-iCer2 vectors for stable expression (Weber et al., 2008). As a control, lentiviral vector LeGO-Cer2 was used (Weber et al., 2008). To modify the multiple cloning site of LeGO-iCer2 vector, additional restriction sites (XbaI and XhoI) were inserted using the primers listed below. To create an E2 FLAG-tagged E1/E2 expression construct containing the core signal peptide, PCR-amplified sequence from amino acids 171-750 of Jc1FLAG-E2 NS5AB-

EGFP was used and cloned into the modified LeGO-iCer2 vector. To knock out ANXA3 using the CRIPR/Cas9 system we selected the target sequence using the on-line tool on http://crispr.genome-engineering.org and cloned the DNA primer encoding the sgRNA sequence into pSpCas9 (BB)-2A-Puro (PX459).

Primers for cloning ANXA3-HA ANXA fw TCGGATCCGCCACCATGGCATCTATCTGGGTTG

ANXA-HA rev CTGCGGCCGCTCAAGCGTAATCTGGTACGTCGTATGGGTAACCACCGTCATCTCCACCACAGAT

Primers for cloning lentiviral shRNA constructs

ANXA3 fw TGGAGCTTTCCTCAATTAAATTCAAGAGATTTAATTGAGGAAAGCTCCTTTTTTC

ANXA3 rev TCGAGAAAAAAGGAGCTTTCCTCAATTAAATCTCTTGAATTTAATTGAGGAAAGCTCCA

ANXA2 fw TGCAAGTCCCTGTACTATTATTCAAGAGATAATAGTACAGGGACTTGCTTTTTTC

ANXA2 rev TCGAGAAAAAAGCAAGTCCCTGTACTATTATCTCTTGAATAATAGTACAGGGACTTGCA

ANXA4 fw TGAAACATTCTGAAGATGTATTCAAGAGATACATCTTCAGAATGTTTCTTTTTTC

ANXA4 rev TCGAGAAAAAAGAAACATTCTGAAGATGTATCTCTTGAATACATCTTCAGAATGTTTCA

ANXA5 fw TGAAAGGTGTTTGACAAGTATTCAAGAGATACTTGTCAAACACCTTTCTTTTTTC

ANXA5 rev TCGAGAAAAAAGAAAGGTGTTTGACAAGTATCTCTTGAATACTTGTCAAACACCTTTCA

ARF4 fw TGCAGACATATCTTCATTAATTCAAGAGATTAATGAAGATATGTCTGCTTTTTTC

7

ARF4 rev TCGAGAAAAAAGTACTCCTATGCAATATTATCTCTTGAATAATATTGCATAGGAGTACA

VIL1 fw TGGAACACCTGTCCATTGAATTCAAGAGATTCAATGGACAGGTGTTCCTTTTTTC

VIL1 rev TCGAGAAAAAAGGAACACCTGTCCATTGAATCTCTTGAATTCAATGGACAGGTGTTCCA

PABPC1 fw TGAACGGCTGTTTCCTCTTATTCAAGAGATAAGAGGAAACAGCCGTTCTTTTTTC

PABPC1 rev TCGAGAAAAAAGAACGGCTGTTTCCTCTTATCTCTTGAATAAGAGGAAACAGCCGTTCA

LMAN2 fw TGTGACCGTTTGCTAAATAATTCAAGAGATTATTTAGCAAACGGTCACTTTTTTC

LMAN2 rev TCGAGAAAAAAGTGACCGTTTGCTAAATAATCTCTTGAATTATTTAGCAAACGGTCACA

HMGCS1 fw TGTACTAATCTCCAATTAAATTCAAGAGATTTAATTGGAGATTAGTACTTTTTTC

HMGCS1 rev TCGAGAAAAAAGTACTAATCTCCAATTAAATCTCTTGAATTTAATTGGAGATTAGTACA

RAB32 fw TGTGCTGGGGTGGTAATAAATTCAAGAGATTTATTACCACCCCAGCACTTTTTTC

RAB32 rev TCGAGAAAAAAGTGCTGGGGTGGTAATAAATCTCTTGAATTTATTACCACCCCAGCACA

DPP4 fw TGCAGGCCCATGTAGTCAAATTCAAGAGATTTGACTACATGGGCCTGCTTTTTTC

DPP4 rev TCGAGAAAAAAGCAGGCCCATGTAGTCAAATCTCTTGAATTTGACTACATGGGCCTGCA

DDX1 fw TGAAAGTCTGTAGTCTTAAATTCAAGAGATTTAAGACTACAGACTTTCTTTTTTC

DDX1 rev TCGAGAAAAAAGAAAGTCTGTAGTCTTAAATCTCTTGAATTTAAGACTACAGACTTTCA

NT fw TGCGCGATAGCGCTAATAATTTTCAAGAGAAATTATTAGCGCTATCGCGCTTTTTTC

NT rev TCGAGAAAAAAGCGCGATAGCGCTAATAATTTCTCTTGAAAATTATTAGCGCTATCGCGCA

Primers for cloning the sgRNA sequence into the Cas9 expression plasmid ANXA3 sgRNA fw CACCGGGGATATCATGGCATCTATC ANXA3 sgRNA rev AAACGATAGATGCCATGATATCCCC Primers for cloning additional restriction sites and sp/E1/FLAG-E2 into LEGO iCer2 MCS LeGO MCS fw AATTCGACCTCGAGTCTAGAGC LeGO-MCS rev GGCCGCTCTAGACTCGAGGTCG sp/E1/FLAG-E2 fw TAACTCGAGAATGGGTTGCTCCTTTTCTATCTTCTTGCT sp/E1/FLAG-E2 rev GCTTAGCGGCCGCTCATGCTTCGGCCTGGCCCAA Primers for cloning pSicoR-MS1-Puro pSicoR-MS1-Puro fw ATGCTAGCGCTACCGGTCGCCACCATGACCGAGTACAAGCCCACGGTG pSicoR-MS1-Puro rev GAGAATTCTTAGGCACCGGGCTTG

Cell lines and culture conditions HEK293T cells were obtained from the American Type Culture Collection, Huh7.5 cells were from Charles M. Rice. Cells were grown under standard cell culture conditions in high glucose DMEM supplemented with 10% FBS (Biochrom Superior) and 1% GlutaMax (Gibco). Cells were transfected with FuGENE 6 (Promega) or calcium phosphate.

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Antibodies and reagents The following antibodies were obtained commercially: anti-ANXA3 (clone T-18, Santa Cruz), anti-ANXA3 (HPA013431, Sigma), anti-core (clone C7-50, Santa Cruz), anti-NS5A (genotype 2a, clone 2F6/G11, IBT), anti-NS5A (genotype 1b, clone 388, Santa Cruz), anti-FLAG (F7425, F1804, Sigma), anti-HA (clone Y-11, Santa Cruz), anti-ADRP (ab52355, Abcam), anti-α-Tubulin (clone B-5-1-2, Sigma), anti-β-Actin (clone AC-15, Sigma), anti-ApoE (ab52607, Abcam), anti-ApoB (ab31992, Abcam), anti-E2 (clone AP-33, Genentech), anti-CD81 (clone M38, Biozol), anti-IgG2a (mouse, clone eBM2a, eBioscience), anti-IgG (rabbit, 2378704, Millipore), anti-GM130 (sc-16268, Santa Cruz), anti-Calnexin (sc-6465, Santa Cruz), anti-Calreticulin (ADI-SPA-600-F, Enzo Life Science), Alexa 488-, Alexa 594-, and Alexa 647-conjugated secondary antibodies (all donkey, IgG (H+L), Life Technologies), HRP-labeled secondary antibodies (Jackson Laboratories). Enzymes for molecular cloning were purchased from New England Biolabs, cell culture reagents from Gibco/Life Technologies, and fine chemicals, if not noted otherwise, from AppliChem or Sigma. Lipid droplets were stained with BODIPY (Life Technologies) or LD540 (Spandl et al., 2009).

In vitro transcription of HCV RNA and production HCV virus stocks HCV viral stocks were prepared as described (Herker et al., 2010). Briefly, plasmids encoding the different HCV

strains were linearized with SspI, purified by phenol-chloroform extraction and in vitro transcribed using the MegaScript T7 kit (Ambion). For RNA transfection, Huh7.5 cells were trypsinized, washed once in Opti-MEM (Invitrogen), and resuspended in cytomix buffer (120 mM KCl, 5 mM MgCl2, 0.15 mM CaCl2, 2 mM EGTA, 1.9 mM ATP, 4.7 mM GSH, 25 mM HEPES, 10 mM potassium phosphate buffer, pH 7.6) at 107 cells per ml. 400 µl of the cell suspension were mixed with 10 µg HCV RNA and pulsed at 260 V and 950 µF using the Gene Pulser II (Biorad). Culture supernatant of Huh7.5 cells transfected with HCV RNA was harvested, filtered, and concentrated by polyethylene glycol 8000 precipitation. For infection experiments, naïve Huh7.5 cells were incubated with virus preparations for 4 h at 37 °C.

Determination of viral titers (TCID50) Viral titers were calculated by determining the tissue culture infectious dose TCID50 essentially as described (Lindenbach and Rice, 2005). Huh7.5 cells stably expressing the HCV reporter RFP-NLS-IPS (Jones et al., 2010) were seeded in 96-well plates and infected with serial dilutions of culture supernatants or concentrated viral stocks. 3–4 days later, cells were fixed with 4% paraformaldehyde for 1 h and infection was monitored by translocation of RFP-NLS into the nucleus. To analyze intracellular infectivity, HCV RNA–transfected cells were trypsinized and 1x105 cells were resuspended in 1 ml DMEM and lysed by multiple cycles of freeze and thaw. Cell debris was removed by centrifugation for 5 min at 1000 rpm and TCID50 was determined as described above.

HCV infection and viral spreading To measure HCV spreading 2 x 105 naïve or lentiviral-transduced Huh7.5 cells were seeded in 6-well plates, incubated for 24 h at 37 °C, and infected with HCV Jc1 reporter strains. 24 h post infection cells were split 1:3 and seeded into new 6-well plates. At day 2, 4, and 6 post infection cells were harvested and fixed with 4% paraformaldehyde. Viral spreading was measured by detecting the fluorescent marker proteins by flow cytometry using the LSR Fortessa (BD Bioscience) flow cytometer and analyzed using FlowJo software (Treestar).

Lentivirus production and transduction Lentiviral particles were produced as previously described (Naldini et al., 1996). Briefly, 293T cells were co-transfected with the pSicoR-MS1 shRNA constructs, a packaging construct (pCMVΔR8.91), and a construct expressing the glycoprotein of vesicular stomatitis virus (VSV-G) (pMD.G). Pseudotyped lentiviral particles were concentrated using ultracentrifugation. Lentiviral transductions were carried out in the presence of 4 µg/ml polybrene (Sigma) for 4 h at 37 °C.

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Knockout of ANXA3 using the CRISPR/Cas9 system We created clonal ANXA3 knockout cell lines using the CRISPR/Cas9 system as described (Ran et al., 2013). Briefly, the pSpCas9 (BB)-2A-Puro (PX459) expressing the sgRNA targeting the ANXA3 locus was transfected into Huh7.5 cells using FuGENE 6. Clonal cell lines were analyzed by amplification and sequencing of the genomic locus using the primers ANXA3 gDNA fw 5’-AGCAAATGGGATACCCAATGT-3’ and ANXA3 gDNA rev 5’-CCTTTCCGCCTTGGTGTCAT-3’ and by quantitative RT-PCR and western blotting.

Subgenomic HCV replicon assays For subgenomic replicon assays, cells were electroporated with in vitro transcribed replicon RNA as described before. 24 h post transfection cells were seeded in 10 cm cell culture dishes and selected with 1 mg/ml G418 (Sigma). After 3 weeks of selection, cells were fixed with 4% paraformaldehyde for 20 min and stained with crystal violet.

Luciferase HCV replicon assays To quantify firefly luciferase activity we used the luciferase assay system according to the manufacturer’s instructions (Promega). Briefly, Huh7.5 cells were electroporated with in vitro transcribed delta E1/E2 luciferase replicon RNA as described before. Cells were resuspended in 13 ml DMEM and seeded in a 12-well plate with 1 ml/well. 4 h, 24 h, 48 h, and 72 h after transfection cells were washed with PBS and lysed with 150 µl 1x cell culture lysis buffer and stored at -20 °C until all samples were harvested. Afterwards samples were transferred into a 96-well plate and centrifuged for 5 min at 1000 rpm to remove cell debris. Supernatant was transferred into a new 96-well plate. 20 µl of sample was mixed with 100 µl of luciferase assay reagent and measured using an Infinite M200 plate reader (Tecan). In addition, the protein concentration was determined using the DC Protein Assay (Biorad).

Lipid droplet isolation and western blotting Lipid droplets were isolated as described (Miyanari et al., 2007). Briefly, cells were scraped in PBS, lysed in hypotonic buffer (50 mM HEPES, 1 mM EDTA and 2 mM MgCl2, pH 7.4) supplemented with protease inhibitors with 30 strokes in a tight-fitting Dounce homogenizer. After spinning for 5 min at 1500 rpm, post-nuclear fractions were mixed with equal volumes of 1.05 M sucrose in isotonic buffer (50 mM HEPES, 100 mM KCl, 2 mM MgCl2) and placed at the bottom of SW60 Ti (Beckman) centrifuge tubes, overlaid with isotonic buffer containing 1 mM Phenylmethanesulfonyl fluoride (AppliChem) and centrifuged for 2 h at 100,000 g, 4 °C. Proteins from the floating lipid droplet fraction were harvested using a bended blunt cannula. Lipid droplets were washed in isotonic buffer and centrifuged for 10 min at 21,000 g, 4 °C. The underlying buffer was removed and protein levels were determined by DC Protein Assay (Biorad). For western blot analysis, cells were lysed in RIPA buffer (1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS in PBS supplemented with protease inhibitor cocktail (Sigma)) for 30 min on ice, followed by SDS-PAGE. For chemiluminescent detection, we used ECL and ECL Hyperfilm (Amersham).

SILAC-labeling, HCV-infection, and lipid droplet isolation for mass spectrometry For isotope metabolic protein labeling we used the SILAC Protein Quantitation kit according to the manufacturer’s instructions (Thermo). Briefly, Huh7.5 cells were cultured in heavy media supplemented with 13C6 L-lysine-2HCl and 13C6 L-arginine-HCl (100 µg / ml DMEM) or in light media containing L-lysine-2HCl and L-arginine-HCl (100 µg / ml DMEM) for at least six passages. Incorporation efficiency was determined by mass spectrometry, using cell lysate separated by SDS-PAGE. Gels were stained with 4x Roti load loading dye (Carl Roth) and analyzed by mass spectrometry. After the incorporation was confirmed, light or heavy Huh7.5 cells were infected with Jc1NS5AB-mKO2-BSD or Jc1NS5AB-

EGFP reporter strains and cultured for two to three weeks. Infection rates were measured by flow cytometry as described above. In one half of the experiments we selected HCV-infected cells with 10 µg/ml blasticidin for two weeks. One day prior the lipid droplets isolation, equal cell numbers of HCV-infected and control cells were seeded in 150 cm plates. For lipid droplet isolation equal numbers of HCV-infected and control cells were mixed and lysed in sucrose LD-isolation buffer (0.25 M sucrose, 1 mM EDTA, 1 mM DTT, supplemented with protease inhibitor

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cocktail) using a Dounce homogenizer. After centrifugation for 5 min at 1000 g, post-nuclear fractions were placed at the bottom of SW60 Ti (Beckman) centrifuge tubes and overlaid with LD isolation buffer (50 mM potassium phosphate buffer pH 7.4, 100 mM KCl, 1 mM EDTA, 1 mM Phenylmethanesulfonyl fluoride). After centrifugation for 2 h at 100.000 g, 4°C, floating lipid droplet fraction were harvested using a bended blunt cannula and again placed in centrifuge tubes, overlaid with LD isolation buffer and centrifuged for 2 h at 100.000 g, 4°C. We performed two sequential sucrose density centrifugations of post-nuclear supernatant followed by three washing steps in LD isolation buffer to increase the purity. After removal of the washing buffer, lipid droplet fractions were mixed with 4x Roti load loading dye and incubated on ice for 1h. Afterwards, the lipid droplet–associated proteins were separated by SDS-PAGE. Gels were stained with RotiBlue (Carl Roth) and analyzed by mass spectrometry.

Mass spectrometry Tryptic in-gel digestion was performed as described (Shevchenko et al., 2006). After digestion the samples were evaporated and dissolved in 0.1% formic acid (FA, dissolved in HPLC-H2O) for LC-MS/MS-analysis. LC-ESI-MS/MS analyses were performed on a quadrupole-time-of-flight mass spectrometer (Q-TOF Premier, Micromass/Waters) or on a linear trap quadrupole (LTQ) orbitrap mass spectrometer (Orbitrap Fusion, Thermo Scientific). Both instruments were coupled with an ESI-source to a nano-UPLC system (nanoAcquity, Waters; Dionex UltiMate 3000 RSLCnano, Thermo Scientific). LC-ESI-MS/MS analysis on Q-TOF mass spectrometer was performed as described (Kwiatkowski et al., 2015). For LC-ESI-MS/MS analysis on orbitrap mass spectrometer samples were loaded (5 µl/min) on a trapping column (Acclaim PepMap µ-precolumn, C18, 300 µm x 5 mm, 5 µm, 100 Ǻ, Thermo Scientific; buffer A: 0.1% FA in HPLC-H2O; buffer B: 0.1% FA in acetonitrile (ACN)) with 2% buffer B. After sample loading the trapping column was washed for 5 min with 2% buffer B (5 µl/min). The peptides were eluted with a flow-rate of 200 nl/min onto the separation column (nanoAcquity UPLC column, BEH 130 C18, Waters; 75 µm × 250 mm, 1.7 µm, 100 Ǻ, custom made, Waters; 200 nl/min, gradient: 2−30% B in 30 min). The spray was generated from a fused-silica emitter (I.D. 10 µm, New Objective) at a capillary voltage of 1700 V. MS/MS measurements were carried out in data dependent acquisition mode (DDA) using top speed mode and a HCD collision energy of 28%. Every second a MS scan was performed over an m/z range from 400–1300, with a resolution of 120000 FWHM at m/z 200 (maximum injection time = 50 ms, AGC target = 2e5). MS/MS spectra were recorded in the ion trap (rapid mode, maximum injection time = 200 ms, AGC target = 1e4).

Data analysis Data from LC-ESI-Q-TOF-MS/MS analysis were analyzed using the open-source software framework OpenMS (Sturm et al., 2008) and the OpenMS Proteomic Pipeline (TOPPAS) (Kohlbacher et al., 2007). For peptide and protein identification LC-MS/MS raw data was processed as described previously (Kwiatkowski et al., 2015) with the exception that the MS/MS data were searched against a human decoy-database (Swiss-Prot, www.uniprot.org, downloaded November 10, 2014, 20,161). For SILAC quantification the raw data files were converted to *.mzXML in profile mode using massWolf file converter. Further data processing was carried out with TOPPAS. The *.mzXML files were converted to *.mzML. For subsequent data processing the mzML files were filtered (only MS1 level, rt-range [s]: 1800-5000) and smoothed (savitsky golay algorithm, frame length: 13, polynomial order: 4). SILAC pairs were detected and quantified using SILACAnalyzer (Nilse et al., 2010) with the following parameters: one missed cleavage, retention time threshold 10 s, intensity cutoff 20 counts, intensity correlation 0.7 and a model deviation of 1.8. A peptide required at least three isotopic peaks and maximal seven isotopic peaks to be taken into account by the SILACAnalyzer. Detected SILAC pairs were exported as *.consensusXML and matched with peptide identifications (*idXML) using IDMapper (retention time tolerance: 10 s, m/z-tolerance: 1 Da). Results were exported as *.csv and further statistical processing was carried out using mathematica. Only unique peptides were taken into account for SILAC quantification. The LC-MS/MS data from orbitrap analysis were processed with MaxQuant (version 1.5.2.8) (Cox and Mann, 2008). Peptide and protein identification was carried out with Andromeda against a human (Swiss-Prot, www.uniprot.org, downloaded November 10, 2014, 20,161) and a contaminant database (cRAP-database, http://www.thegpm.org/crap, 298 entries). The search was performed with a tolerance of 6 ppm on MS- and 0.6 Da on MS/MS-level. Carbamidomethylation was considered as a static modification on cysteine residues. Oxidation on methionine as well as 13C6-label on lysine and arginine were considered as variable modifications. The search was performed with a false discovery rate (FDR) of 0.01 on both peptide and protein level. SILAC quantification was

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carried out with MaxQuant (Cox et al., 2009), wherein only unique peptides were taken into account for SILAC quantification. To correct for different cell numbers or numbers of lipid droplets we centered the detection ratios of light over heavy peptides (L/H) or vice versa in swapped labeling conditions (H/L) by dividing through the median of the identified proteins as described (Ting et al., 2009). Of note, under the cell culture conditions we used, we don’t detect significant lipid droplet accumulation upon infection with Jc1-based HCV constructs. To rule out any bias we additionally normalized our MS data to PLIN2 levels (analysis not shown) and found similar results as when we normalize to the median. Normalizing quantitative MS data to the median is however preferable (Ting et al., 2009).

Membrane floatation assay Membrane floatation assays of subgenomic replicon cells were essentially performed as described (Vogt et al., 2013). Briefly, 6 x 106 of Huh7.5 or Huh7.5 Con1 replicon cells were scraped in PBS, resuspended in 750 µl 0.25 M sucrose/PBS supplemented with protease inhibitors (Sigma) and lysed on ice with 50 strokes in a tight-fitting Dounce homogenizer. After spinning for 10 min at 1000 g, the supernatant was transferred into a new tube and protein levels were determined using a DC Protein Assay (Biorad). 0.75 mg of protein was adjusted in volume to 500 µl with 1.05 M sucrose and mixed with 500 µl 60% iodixanol (Sigma). A discontinuous iodixanol gradient (20%, 10%) was layered on top of the lysate fraction and centrifuged at 200,000 g for 16 h at 4 °C using a SW60 Ti rotor. From top to bottom eight 500 µl fractions were harvested. 40 µl of each fraction were analyzed by SDS-PAGE and western blotting. To analyze intracellular ApoE distribution, membrane floatation assays of HCV-infected cells were performed in larger scales. Briefly, 2 x 106 of lentiviral transduced Huh7.5 cells were infected with Jc1FLAG-E2 NS5AB-EGFP-BSD and cultured in media containing 10 µg/ml basticidin for 8 days. Cells were scraped in PBS, resuspended in 1.5 ml 0.25 M sucrose/PBS containing protease inhibitors (Sigma) and lysed on ice with 200 passages in a tight-fitting Dounce homogenizer. Lysates were cleared by centrifugation at 1000 g for 10 min at 4 °C. Supernatants were transferred into a new tube and protein levels were determined using a DC Protein Assay (Biorad). 3 mg of protein was adjusted to a final volume of 2 ml with sucrose/PBS. Samples were mixed 1:1 with 2 ml 60% iodixanol (Sigma) and transferred into a centrifugation tube and overlaid with 4 ml 20% iodixanol and 4 ml 10% iodixanol. Gradients were centrifuged at 200,000 g for 16 h at 4 °C using a SW40 rotor. 25 fractions were collected from top to bottom and analyzed by SDS page and western blotting.

Iodixanol gradient centrifugation To determine HCV particle density we performed iodixanol (Sigma) gradient centrifugation as described (Catanese et al., 2013). Briefly, iodixanol gradients were prepared with 6.5 ml of 10% (wt/vol) (8.3 ml of 60% iodixanol, 1 ml 0.1 M EDTA, 1.6 ml 0.15 M Tris/HCl pH 8.0, 39.6 ml 0.25 M sucrose) and 6.5 ml of 40% (33.3 ml of 60% iodixanol, 1 ml 0.1 M EDTA, 1.6 ml 0.15 M Tris/HCl pH 8.0, 14,1 ml 0.25 M sucrose) iodixanol dilutions using a two chamber gradient maker. Supernatant of Jc1NS5AB-EGFP transfected cells was centrifuged at 1000 g for 5 min to remove cell debris. 1 ml of supernatant was loaded on top of a gradient and centrifuged at 34,500 rpm for 16 h at 4 °C. From top to bottom twenty 500 µl fractions were harvested. The first eleven fractions were used to isolate viral RNA, determine the infectivity (TCID50), and the density using a refractometer (DR 201-95, Krüss).

Proteinase K digestion protection assay Proteinase K digestion protection assay was performed as previously described (Gentzsch et al., 2013). Briefly, Huh7.5 cells were transfected with in vitro transcribed HCV Jc1 RNA and seeded in 6-well plates. 48 h post transfection cells were resuspended in 170 µl proteinase K buffer (50 mM Tris/HCl, pH 8, 10 mM CaCl2, 1 mM DTT) and lysed by five repeats of freeze and thaw on dry ice. Afterwards the lysate was divided into three 50 µl portions. One portion was treated with 100 µg/ml proteinase K (Macherey Nagel) on ice for 30 min, the second portion was treated with 1% Triton X-100 for 5 min prior to proteinase K digestion, while 50 µl was left untreated. Proteinase K digestion was blocked by 5 mM phenylmethanesulfonyl fluoride. Protease protection of core was analyzed by western blotting. Signal intensities reflecting the core levels were quantified using image J.

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2D blue native SDS-PAGE For 2D blue native SDS-PAGE Huh7.5 cells were transfected with in vitro transcribed Jc1FLAG-E2 NS5AB-EGFP-BSD

RNA. 6 days post transfection, 1 x 106 cells were washed with PBS and lysed in 80 µl lysis buffer (0.75 M aminocaproic acid, 50 mM Tris-Bis, pH 7.0) supplemented with 15 µl 10% n-dodecyl-β-D-maltopyranoside for 30 min on ice. Lysates were centrifuged at 14.000 rpm for 10 min at 4 °C. Supernatants were transferred into a new tube and 1 µg/mL pepstatin, 2 µg/ml antipain, 20 µg/ml leupeptin were added. Samples were mixed with 10 µl 5% Coomassie Brilliant Blue G (Sigma) in 0.5 M aminocaproic acid and equal volumes of 2x non-reducing sample buffer (62.5 mM Tris/HCl, pH 6.8, 25% glycerol, 0.1% bromophenol blue). 40 µl of sample was loaded onto a 4–20% polyacrylamide gradient gel (Biorad) and electrophoresis was performed at a constant voltage of 150 V for 2.5 h at 4 °C. For the second dimension, sample lanes from blue native page were cut out and incubated in 2 x SDS sample buffer (150 mM Tris-HCl, pH 6.8, 1.2% SDS, 30% glycerol, 0.002% bromophenol blue, 15% ß-mercaptoethanol) for 30 min. After equilibration, blue native page gel stripes were placed horizontally onto a 15% polyacrylamide SDS gel (Biorad) and overlaid with SDS sample buffer. Electrophoresis was performed for 40 min at 200 V. Core complexes were analyzed by western blotting.

Immunofluorescence staining and confocal microscopy Cells grown on coverslips were fixed in 4% paraformaldehyde for 1 h at room temperature, washed with PBS, and permeabilized in 0.1% Triton X-100 for 5 min. After incubation in blocking solution (5% BSA, 1% fish skin gelatin, 50 mM Tris in PBS) for 1 h, cells were incubated with primary antibodies in blocking solution for 1 h at RT or o/n at 4 °C, washed and incubated with secondary antibodies for 1 h. For lipid droplet staining, fixed cells were stained for 20 min with BODIPY (Invitrogen) or LD540 (Spandl et al., 2009), diluted in PBS solution. Coverslips were embedded in Mowiol (Calbiochem) mounting medium (Longin et al., 1993). CLSM was performed on a Nikon C2+ confocal laser scanning microscope. The Nikon C2+ microscope was equipped with four lasers: a 405 nm diode laser (100 mW, Coherent Inc.), a 488 nm DPSS Laser (10 mW, Melles Griot GmbH), a 543 nm HeNe laser (5 mW, Melles Griot GmbH) and a 642 nm diode laser (45 mW, Melles Griot GmbH). We used a 60x violet corrected oil objective with a NA of 1.4 for imaging (Plan Apo VC 60x H, Nikon). For colocalization analysis we used coloc2 in Fiji (Schindelin et al., 2012). We analyzed individual cells of several independent experiments and calculated the degree of colocalization using the Manders' colocalization coefficients (MCC) and Pearson’s correlation coefficient (PCC).

MTP activity measurement Total cellular MTP activity was quantified using an MTP activity assay kit (Roar Biomedical Inc.) according to manufacturer’s instructions. Briefly, the cells were washed with PBS and scraped in homogenization buffer (150 mM NaCl, 10 mM Tris/HCl pH 7.4, 1 mM EDTA and 10 µl/ml protease inhibitor cocktail (Sigma)). Cells were lysed by sonication and protein levels were determined using a DC Protein Assay (Biorad). 75 µg of protein was incubated with 1 µl donor and 1 µl acceptor particles in 200 µl total volume homogenization buffer for 3–6 h at 37 °C. MTP activity was determined using an Infinite M200 plate reader (Tecan).

Measurement of ApoE/ApoB secretion 1 x 105 lentiviral-transduced Huh7.5 cells were seeded in a 12-well plate. To exclude cross-reaction with serum proteins, cells were washed once with Opti-MEM (Invitrogen) and incubated with Opti-MEM for 18 h at 37 °C. 30 µl of the cell culture supernatant was analyzed by SDS-PAGE and western blotting with ApoE and ApoB antibodies. Signal intensities reflecting the ApoE and ApoB levels were quantified using image J.

HCV neutralization assay Antibody-mediated neutralization of HCV infectivity was determined by using an HCVcc reporter virus expressing secreted gaussia luciferase (Jc1p7-GLuc-2A-NS2) and a renilla luciferase assay system according to the manufacturer’s instructions (Promega). HCV particles were incubated with various antibody concentrations (0.5–50 µg/ml) for 1h at 37 °C and then used to infect naïve Huh7.5 cells. 4h hours later, cells were washed twice with DMEM and cultured with 100 µl media for 48h at 37 °C. Afterwards 50 µl of supernatant was inactivated with 50 µl of 2x renilla

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luciferase lysis buffer for 1h at RT. HCV infection was determined by measuring luciferase activity as described before.

RNA isolation and quantitative RT-qPCR Total cellular RNA was isolated using RNA Stat reagent (TelTest) and treated with the TURBO DNA-free DNAse (Ambion). Viral RNA from the culture supernatant was isolated with the Nucleo-Spin RNA Virus kit (Macherey Nagel). RNA levels were adjusted to carrier RNA input that was added in excess prior to RNA isolation. cDNA was synthesized using Superscript III reverse transcriptase (Invitrogen) with random hexamer primers, followed by RNase H (New England Biolabs) digestion. For quantitative PCR, we used the primers listed below from the PrimerBank (Wang et al., 2012) and SYBR green mastermix (Fermentas) on a 7900HT Fast Real-time PCR System (Applied Biosystems).

Primers used for qPCR

ANXA3 fw GAGAGGTCAAATGCACAGCG ANXA3 rev CTGGTGGAGTCACTAGGGC ANXA2 fw TCTACTGTTCACGAAATCCTGTG ANXA2 rev AGTATAGGCTTTGACAGACCCAT ANXA4 fw GGAGGTACTGTCAAAGCTGCT ANXA4 rev GGCAAGGACGCTAATAATGGC ANXA5 fw GTTTGGCAGGGATCTTCTGGA ANXA5 rev TCATAAAGCCGAGAGGGTTTCA ARF4 fw CCCTCTTCTCCCGACTATTTGG ARF4 rev GCACAAGTGGCTTGAACATACC

VIL1 fw GGCAAGAGGAACGTGGTAGC VIL1 rev CGGTCCATTCCACTGGATGA PABPC1 fw CAGGCTCACCTCACTAACCAG PABPC1 rev GGTAGGGGTTGATTACAGGGT LMAN2 fw GTACGTCTGACCCCTGACGA LMAN2 rev CTGTGCCGTGGACTTTGAAG

HMGSC1 fw CTCTTGGGATGGACGGTATGC HMGSC1 rev GCTCCAACTCCACCTGTAGG RAB32 fw CTGCCCGGTTCCTAGTGGA RAB32 rev GGGATTTGTTCTCTGCTCTCAAG DPP4 fw TACAAAAGTGACATGCCTCAGTT DPP4 rev TGTGTAGAGTATAGAGGGGCAGA DDX1 fw TCTCCGAGATGGGTGTAATGC DDX1 rev ACCTCCTCCTAAGATCAATGGG

β-actin fw CATGTACGTTGCTATCCAGGC β-actin rev CTCCTTAATGTCACGCACGAT GAPDH fw AAGGTGAAGGTCGGAGTCAAC GAPDH rev GGGGTCATTGATGGCAACAATA 18S rRNA fw GTAACCCGTTGAACCCCATT 18S rRNA rev CCATCCAATCGGTAGTAGCG

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JFH1 fw CGGGAGAGCCATAGTGG JFH1 rev AGTACCACAAGGCCTTTCG

Quantification of HCV core and ApoE protein by ELISA The amount of HCV core or ApoE protein was quantified using the Hepatitis C Virus Core Antigen ELISA kit (BioCat) or the Human ApoE ELISA Pro kit (Mabtech), respectively, according to manufacturer’s instructions.

Co-immunoprecipitation For E2 immunoprecipitation shRNA-expressing Huh7.5 cells were transfected with HCV Jc1FLAG-E2 NS5AB-EGFP-BSD

RNA. Equal transfection rates were confirmed by flow cytometry of EGFP. Cells were washed with PBS and lysed with 1 ml NP-40 lysis buffer (50 mM Tris/HCl pH 7.4, 150 mM NaCl, 1% NP-40, supplemented with protease inhibitor cocktail (Sigma)) for 1 h on ice. Afterwards cell debris was removed by centrifugation for 10 min at 11.000 rpm, 4 °C. For capturing FLAG-tagged E2, 1 mg protein was incubated with 30 µl of FLAG M2 affinity gel (Sigma) for 3 h at 4°C. Beads were then washed five times with ice-cold NP-40 lysis buffer and analyzed by SDS-PAGE and western blotting. For NS5A immunoprecipitation shRNA-expressing Huh7.5 cells were transfected with HCV Jc1FLAG-E2 NS5A-HA

RNA. After washing with PBS cells were lysed with 1 ml low stringency 0.1 % NP-40 lysis buffer (50 mM Tris/HCl pH 7.4, 150 mM NaCl, 0.1% NP-40, supplemented with protease inhibitor cocktail (Sigma)) for 1 h on ice. Cell debris was removed by centrifugation for 10 min at 11.000 rpm, 4 °C. For capturing HA-tagged NS5A, 1 mg protein was incubated with 30 µl of HA affinity gel (Sigma) for 3 h at 4°C. Beads were then washed five times with ice-cold 0.1 % NP-40 lysis buffer and analyzed by SDS-PAGE and western blotting.

Affinity purification of HCV particles HCV particles were isolated as described previously (Merz et al., 2011). Briefly, supernatant of Huh7.5 cells infected with HCV Jc1FLAG-E2 NS5AB-EGFP was harvested and concentrated by polyethylene glycol 8000 precipitation. Afterwards approx. 30 ml of the concentrate was loaded on top of an iodixanol gradient (60%, 10%, PBS) and centrifuged at 96,281 g for 20 h at 4 °C using an SW28 rotor. Following centrifugation, the 27 ml from top of the gradient were discarded and the remainder of the gradient was harvested in 1 ml fractions. To purify HCV particles, virus preparations obtained from fraction 7 and 8 were incubated with 0.5 ml of FLAG M2 affinity gel (Sigma) according to manufacture’s instructions. Samples were analyzed by SDS-PAGE and western blotting.

Quantification of cPLA2 activity The cPLA2 activity was quantified using the cPLA2 Assay kit (Cayman) according to manufacturer’s instructions.

Bioinformatics and statistical analysis For bioinformatics and statistical analysis we used R (R Core Team, 2015), RStudio (RStudio Team, 2015), and GraphPadPrism (GraphPad Software, Inc). Data was analyzed and plotted using gdata (Warnes et al., 2015b), lattice (Sarkar, 2008), gplots (Warnes et al., 2015a), and ggplot2 (Wickham, 2009) packages. Statistical analysis of overlaps with other HCV screens was performed using GeneOverlap (Shen, 2013). For automated PubMed searches we used RISMed (Kovalchik, 2015), MyGene for ID conversion (Adam Mark et al., 2014), and cisPath for protein network analysis and visualization (Wang, 2015). We used the gProfileR package for gene ontology annotation analysis with a p-value cutoff of 0.05 and “strong” hierarchical filtering (Reimand et al., 2015). Statistical analysis was performed using unpaired two-tailed student's t-test and in case of normalized data one sample t-test.

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Supplemental References

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