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Originally published In Press as doi:10.1074/mcp.M600121-MCP200 on August 29, 2006.
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Molecular & Cellular Proteomics 5:2319-2325, 2006.
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

Research

Modification of Host Lipid Raft Proteome upon Hepatitis C Virus Replication*,S

Petra Mannová, Ruihua Fang, Hong Wang, Bin Deng, Martin W. McIntosh, Samir M. Hanash and Laura Beretta{ddagger}

From the Public Health Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Hepatitis C virus (HCV) replication complex resides in detergent-insoluble subcellular domains or lipid rafts. We used two proteomics approaches to characterize the protein content of lipid rafts isolated from Huh7 cells and its modification upon HCV replication. Using two-dimensional electrophoresis and mass spectrometry, we identified ~100 protein spots in the isolated lipid rafts; among them, 39 were reproducibly modified in HCV replicon cell lines as compared with control cell lines. We also used stable isotope labeling by amino acids in cell culture (SILAC) combined with one-dimensional electrophoresis separation and mass spectrometry. Using this approach, we identified 1036 individual proteins based on peptides selected with at least 95% confidence; among them, 413 proteins were identified with at least two peptides. Quantification analysis identified 150 proteins modified by at least 2.5-fold (110 up-regulated and 40 down-regulated) in HCV-replicating cells compared with controls. Protein identifications and quantifications obtained by both proteomics approaches were largely concordant. Modulated proteins included a majority of proteins involved in vesicular and protein trafficking and in cell signaling. Remarkably for a large number of proteins, their up-regulation in lipid rafts of HCV replicon cells was due to their relocalization. By using small interfering RNAs directed to the modulated small GTPases Cdc42 and RhoA, we observed an increase in HCV replication, whereas reduction of syntaxin 7 expression resulted in decreased replication of HCV. Our findings indicate that protein subcellular relocalization occurs in HCV-containing cells that can directly affect HCV replication.

Hepatitis C virus (HCV)1 is a small, enveloped, plus strand RNA virus in the Flaviviridae family. Chronic infection occurs in the majority of HCV-infected individuals and is associated with a high subsequent risk for liver cirrhosis and hepatocellular carcinoma. Active replication complexes of HCV are localized in detergent-insoluble membrane domains or lipid rafts (1, 2). Lipid rafts are involved in cell signaling and intracellular trafficking (3, 4). Some viruses utilize these subcellular membrane domains for cell entry, virion assembly, or budding (5–7) or perturb cell signaling pathways by interfering with proteins in lipid rafts (8, 9). We recently reported the up-regulation of N-Ras in detergent-insoluble membrane domains and subsequent activation of the phosphatidylinositol 3-kinase-mTOR pathway in Huh7 cell lines expressing a full-length HCV replicon, leading to protection against apoptosis and reduced HCV replication (10).

To further identify protein modifications in lipid rafts resulting from HCV replication, we utilized the standard proteomics approach of two-dimensional (2-D) PAGE followed by mass spectrometry analysis of individual protein spots. We also took advantage of recent progress in mass spectrometry sensitivity (using linear ion trap quadrupole (LTQ)-FT-MS) and labeling techniques for quantitative proteomics (using stable isotope labeling by amino acids in cell culture or SILAC (11)). To this end, two cell populations are grown in media containing either a naturally occurring amino acid or a stable isotope-labeled analog. Because the stable isotope analogs are heavier then their naturally occurring counterparts, protein quantification occurs directly at the level of the peptide mass spectrum using the difference in signal intensity between the peptides derived from isotope-labeled or normal amino acid proteins. In the present study we investigated protein modifications occurring in the detergent-insoluble subcellular fraction upon HCV replication in Huh7 cells followed by small interfering RNA (siRNA) inhibition of selected modified proteins for their functional assessment on HCV replication.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Isolation of Detergent-insoluble Fractions—
Human hepatoma Huh7 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (Cellgro/Mediatech) and 1% penicillin/streptomycin (Invitrogen). Stable Huh7 clones SFL-2 and SFL-3 expressing the selectable full-length HCV-1b genome and T1 clone expressing the neomycin resistance gene were described previously (10). These cells were grown in complete DMEM with 400 µg/ml G418 (Invitrogen).

Detergent-insoluble fractions were isolated as described previously (10). Briefly postnuclear supernatants were treated with 1% Nonidet P-40, mixed with 72% sucrose, overlaid with 55 and 10% sucrose, and centrifuged at 35,000 rpm in a Beckman SW41 Ti rotor for 18 h at 4 °C. Proteins from detergent-insoluble fractions were precipitated with acetone, 10% trichloroacetic acid, 0.07% ß-mercaptoethanol for 2 h at –20 °C. Following centrifugation at 2500 x g for 10 min, pellets were washed with acetone, 0.07% ß-mercaptoethanol.

2-D PAGE and Q-TOF-MS Analysis—
2-D PAGE was performed as described previously (10). Briefly proteins were solubilized in buffer composed of 6 M urea, 2 M thiourea, 2% Nonidet P-40, 2% ß-mercaptoethanol, and 1% ASB-14. Protein samples (120 µg) were supplemented with 2% ampholytes (pH 4–8; Gallard/Schlessinger) and applied onto isoelectric focusing tube gels (0.2-mm diameter). Isoelectric focusing was performed using pH 4–8 carrier ampholytes at 700 V for 16 h followed by 1000 V for an additional 2 h. Gels were equilibrated in 125 mM Tris (pH 6.8), 10% glycerol, 2% SDS, 1% dithiothreitol, and bromphenol blue and loaded onto the gradient (11–14%) polyacrylamide second dimension gel. After separation, proteins were visualized by a photochemical silver-based staining (12). The gels were digitized with a Kodak charge-coupled device camera, and spots were quantified using BioImage 2-D Analyzer software (BioImage, Ann Arbor, MI). The protein spots of interest were excised and destained in 15 mM potassium ferricyanide and 50 mM sodium thiosulfate for 10 min followed by three washes with water and dehydration in 100% acetonitrile for 5 min. The proteins were digested in gel with trypsin (Promega) in 200 mM sodium bicarbonate at 37 °C overnight. Peptides were extracted twice with 10% acetonitrile, 10% formic acid and analyzed by nanoflow capillary LC-ESI/Q-TOF MS/MS in the Q-TOF micro (Waters, Manchester, UK). MS/MS spectra were acquired in the automated MS to MS/MS switching data-dependent acquisition mode. Doubly and triply charged ions were selected and fragmented with argon as the collision gas. The acquired spectra were automatically processed (background subtract: polynomial order, 1; below curve, 40%; smooth: smooth window, ±3; number of smooths, 2; Savitzky Golay was selected; centroid top, 80%) and searched against the non-redundant UniProt/Swiss-Prot protein sequence database release 49.7 combined with sequences of individual HCV proteins (the European HCV Database) using ProteinLynx Global Server 1.1 (Micromass), permitting oxidation (Met), carboxymethyl (Cys), ±100-ppm peptide MS tolerance, and ±0.1-Da MS/MS tolerance. Up to one missed tryptic cleavage was considered. Typically ProteinLynx scores higher than 100 or their corresponding MASCOT scores higher than 47 (p < 0.05) were considered significant.

SILAC Experiment—
SFL-3 and Huh7 cells were cultured in lysine-deficient DMEM (Caisson Laboratories) supplemented with 10% dialyzed fetal bovine serum (Invitrogen), 1% penicillin-streptomycin, and either 12C6-labeled L-lysine for Huh7 cells or 13C6-labeled stable isotope L-lysine for SFL-3 cells at a final concentration of 100 mg/liter (Invitrogen). Each cell line was grown for two passages encompassing at least six population doublings. Detergent-insoluble fractions were prepared as described above, and proteins were solubilized in buffer composed of 6 M urea, 2 M thiourea, 1% octyl glucoside, 2% ß-mercaptoethanol, and 1% ASB-14. Equal amounts of proteins were mixed (120 µg total), resolved by 12% SDS-PAGE, and stained with colloidal Coomassie Blue (Pierce). Fourteen sections of the gel lane were cut. These sections were in-gel digested with trypsin as described above. Peptides from individual fractions were analyzed by LC-LTQ-FT-MS/MS (Thermo Electron Corp., Waltham, MA). MS/MS spectra were analyzed by the search/identification program Comet (13) against a combined database of HCV proteins (the European HCV Database) and human International Protein Index FASTA database version 3.06 (released May 10, 2005, contained 49,923 entries) and three trypsin protein sequences (two bovine and one porcine). For the database search, the parent mass (monoisotopic mass) tolerance was set to ±2.1 daltons, and fragment mass tolerance was set to ±1.0 dalton. Dynamic modifications were set on Met (15.9994) and Lys (6.0201) residues. Peptide and protein identifications were than analyzed by PeptideProphet (14) and ProteinProphet (15) programs, respectively, to estimate statistical confidence of the identifications. Peptides labeled with [13C6]lysine were identified by including a modification on lysine with a mass shift of 6.0468, which counts for six carbon atoms in each lysine, in the search parameter. Only peptides with a PeptideProphet score ≥0.95 were retained. MS spectra were analyzed using the program msInspect. The features identified in the MS spectra were then compared with those peptides identified in MS/MS spectra with a PeptideProphet score ≥0.95 using a practical extraction and report language (PERL) program developed in-house. This step involves comparing the theoretical monoisotopic mass of the peptide with the monoisotopic mass of its parent ion feature using a mass tolerance of ±5 ppm. The peak area of the feature was taken as the abundance of its corresponding peptide. The ratios of the labeled peptides (i.e. those peptides with all of their lysines shifted by 6.0468) over their corresponding normal peptides were then calculated.

Western Blot—
Proteins were resolved by 12% SDS-PAGE and electrotransferred onto a nitrocellulose membrane (Amersham Biosciences). Membranes were incubated with the following antibodies: anti-RhoA, anti-Bax, anti-Cdc42 (Santa Cruz Biotechnology), anti-Rab9A (Affinity BioReagents), anti-syntaxin 7 (Synaptic Systems), anti-SR-BI (Abcam), and anti-Sar1b (Upstate Biotechnology, Lake Placid, NY). Horseradish peroxidase-conjugated immunoglobulins (DakoCytomation) were used as secondary antibodies, and proteins were visualized with ECL chemiluminescence reagent (Amersham Biosciences). Quantification of immunodetected protein bands was performed using ImageJ software (Research Services Branch/National Institute of Mental Health, National Institutes of Health).

siRNA Assays—
SFL-2 and SFL-3 cells were transfected with 50 or 100 nM siRNA specific to RhoA, Bax, Cdc42 (Santa Cruz Biotechnology), syntaxin 7, or Rab9A or with non-targeting siRNA (Dharmacon). Forty to 70 h post-transfection, protein extracts were analyzed by Western blot, and total RNA was purified with TRIzol (Invitrogen) and used for real time PCR analysis.

Real Time PCR—
One microgram of DNase 1-treated total RNA was reverse transcribed using the Moloney murine leukemia virus reverse transcriptase (Invitrogen), the antisense HCV primer, and the reverse actin primer for 50 min at 42 °C. cDNA mixtures (1:10) were mixed with 2x iQ SYBR Green Supermix (Bio-Rad) and primers specific for the conservative 5'-noncoding region in the HCV genome: 5'-CTCGCAAGCCACCCTATCAGGCAGTA-3' (antisense) and 5'-CGGGAGAGCCATAGTGGTCTGCG-3' (sense). Reactions were performed in the Bio-Rad iCycler MyiQ real time PCR detection system and analyzed by MyiQ software (Bio-Rad). Actin quantifications, using primers 5'-TGGACTTCGAGCAAGAGATGG-3' and 5'-GGAAGGAAGGCTGGAAGAGTG-3', were performed as internal controls.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Comparative Proteomics Analysis of Detergent-insoluble Fractions by 2-D PAGE—
Detergent-insoluble subcellular fractions were isolated by flotation assay in sucrose gradient from stable Huh7 cell lines SFL-2 and SFL-3, which express the full-length HCV-1b genome, and from two control cell lines, Huh7 and T1, as described previously (10). Proteins were precipitated and separated by 2-D PAGE. The integrated intensities in silver-stained gels for 31 protein spots were increased at least 2-fold in both the SFL-2 and SFL-3 replicon cell lines compared with the control cell lines in two independent experiments. Likewise eight proteins were down-regulated in the same experiments (Fig. 1).


Figure 1
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  		FIG. 1. Representative silver-stained 2-D gel of detergent-insoluble subcellular fraction isolated from HCV replicon cell lines. Protein spots reproducibly modified in the detergent-insoluble fraction of HCV containing cell lines are numbered (the down-regulated spots are indicated in italics).

 
These protein spots were analyzed using Q-TOF-MS. Protein identification was obtained for 31 protein spots that yielded 27 distinct proteins (Table I and Supplemental Table SI). Two protein identifications (Rab14 and RhoA) were obtained for spot number 4. Proteins VIP36 and serum paraoxonase/arylesterase 2 occurred as four and three isoforms, respectively, that differed based on charge and/or mass. All isoforms of these two proteins were concomitantly up-regulated. Up-regulated proteins can be categorized into four major groups: proteins involved in vesicle and protein trafficking (members of the Rab GTPases family and other GTPases, syntaxin 7, and VIP36), proteins involved in cell signaling and apoptosis (N-Ras, RhoA, prohibitin, and Bax), proteins involved in response to unfolded proteins and protein processing (transitional endoplasmic reticulum ATPase p97 and protein-disulfide isomerase), and proteins involved in cell metabolism. Down-regulated proteins were predominantly members of the tropomyosin family.


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  		TABLE I Selected up-regulated proteins in lipid raft fraction of HCV replicon cell lines

 
We also selected for identification 13 protein spots that were unchanged in the HCV replicon cell lines compared with the control cell lines. Corresponding proteins were identified as membrane-associated progesterone receptor component 1, cytochrome b5, glycoprotein 25L2, heat shock cognate 71-kDa protein, ATP synthase {delta} chain, ATP synthase D chain, 60 S acidic ribosomal protein P2, cytochrome c oxidase polypeptide Va, VIP36, Rab11A, and ATP synthase ß chain (Supplemental Table SI).

Comparative Proteomics Analysis of Detergent-insoluble Fractions following Stable Isotope Labeling in Cell Culture—
SFL-3 HCV replicon cells and Huh7 cells were cultured in the presence of stable isotope-labeled or normal L-lysine, respectively. The detergent-insoluble fractions were isolated separately for each cell line, and equal amounts of protein were subsequently mixed. Following separation by SDS-PAGE, the gel lane was cut into 14 sections ranging from 15 to 150 kDa (Fig. 2). Proteins in each gel section were digested and submitted to mass spectrometry analysis using LTQ-FT. Peptides were identified based on a PeptideProphet score ≥0.95. These peptides corresponded to a total of 1036 individual proteins, including 413 cellular proteins identified with at least two unique peptides and three HCV proteins (NS3, NS5A, and NS4B) (Supplemental Table SII). Statistical confidence of protein identifications was calculated by ProteinProphet (Supplemental Table SII).


Figure 2
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  		FIG. 2. Coomassie Blue-stained SDS-PAGE gel of proteins isolated from the detergent-insoluble subcellular fraction of HCV cell line and control cells. The gel lane was cut into 14 sections as indicated.

 
For quantification, we calculated the SILAC ratio by comparing the signal intensities of lysine-containing unique peptides derived from stable isotope-labeled lysine ("heavy," SFL-3) and normal lysine-labeled ("light," Huh7) proteins. We also detected unique peptides only found either as stable isotope-labeled or as normal lysine-labeled. Those peptides are therefore referred to as heavy or light only. Quantification data were obtained for 268 proteins, 110 of which were up-regulated (SILAC ratio ≥2.5-fold or peptides present as heavy only) and 40 proteins that were down-regulated (SILAC ratio ≤0.3-fold or peptides present as light only) in detergent-insoluble fractions isolated from HCV replicon cells compared with controls (Table I and Supplemental Table SII).

Up-regulated proteins include proteins involved in vesicular transport and protein trafficking (27 proteins, predominantly Rab GTPases). Other up-regulated proteins included proteins involved in cell signaling and apoptosis (17 proteins, e.g. N-Ras, Cdc42, RhoA, and scavenger receptor BI) and in protein processing and response to unfolded proteins (eight proteins). The latter group includes transitional endoplasmic reticulum ATPase p97, NPL4, and Derlin-1. Several proteins involved in protein biosynthesis (seven proteins), such as subunit 12 of eukaryotic translation initiation factor 3 and heterogeneous nuclear ribonucleoprotein K (hnRNP K) were also up-regulated. Finally we detected up-regulation of 28 proteins involved in metabolism. Three HCV proteins were also identified. Peptides from NS3 and NS4B were present only as stable isotope-labeled (heavy only) as expected; NS5A peptide did not contain any lysine. Down-regulated proteins included seven proteins associated with vesicular trafficking (members of the Rab family associated with early endosomes and phosphatidylinositol 4-kinase) and members of the keratin family of the intermediate filaments.

Integration of Data from Both Proteomics Strategies—
We compared protein identifications and quantifications obtained using both proteomics approaches. Quantification by SILAC was obtained for 15 of the 27 distinct proteins identified by 2-D PAGE. Similar quantification data were obtained from both approaches for the large majority of these 15 proteins including Sar1b, Rab18, N-Ras, Rab9A, RhoA, catechol O-methyltransferase, transitional endoplasmic reticulum ATPase p97 subunit, Rab7, syntaxin 7, and serum paraoxonase/arylesterase 2 (Table I). VIP36 was detected as multiple protein spots by 2-D PAGE and in more then one gel section by the SILAC approach, suggesting the presence of post-translationally modified isoforms. In both data sets, some of these isoforms were up-regulated, whereas others were not modulated. The large majority of the unchanged proteins identified by the 2-D gel approach were also confirmed by SILAC. Therefore, protein identifications and quantifications obtained by the two proteomics approaches used in this study are highly concordant. The combination of SILAC and LTQ-FT mass spectrometry allowed for a larger number of protein identifications and quantifications. In addition, HCV proteins were identified by the SILAC approach but not by the 2-D PAGE approach. With a significantly greater number of identified and quantified proteins, the SILAC approach provided a more comprehensive proteomics analysis of the detergent-insoluble fractions from HCV replicon cells.

Expression of Selected Proteins in Lipid Rafts and Total Cellular Lysates—
We reported previously that the levels of N-Ras were increased in detergent-insoluble fractions of HCV replicon cells compared with control cells due to the protein subcellular redistribution (10). We therefore assessed levels of selected proteins in detergent-insoluble fractions as well as in total lysates of SFL-2, SFL-3, and control cells by Western blot. In agreement with the 2-D PAGE and SILAC experiments, levels of RhoA, Rab9A, Bax, Cdc42, Sar1b, SR-BI, and syntaxin 7 were increased by 4.8-fold ± 0.5, 3.8-fold ± 0.1, 4.7-fold ± 0.8, 2.3-fold ± 0.4, 3.4-fold ± 1, 2.1-fold ± 0.05, and 1.7-fold ± 0.1 (mean ± S.E.), respectively, in detergent-insoluble fractions of HCV replicon cells compared with control T1 cells (Fig. 3). Similar results were obtained when compared with control Huh7 cells (data not shown). The amounts of these proteins in the total lysates were comparable (RhoA, Rab9A, Bax, Cdc42, and Sar1b) or only slightly increased (1.4-fold ± 0.05 and 1.3-fold ± 0.05 for SR-BI and syntaxin 7, respectively) (Fig. 3), suggesting as previously observed for N-Ras that these proteins were redistributed into lipid rafts upon HCV replication.


Figure 3
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  		FIG. 3. Western blot analysis of selected proteins. Proteins isolated from detergent-insoluble fractions and from total cell lysates of HCV cell lines SFL-2 and SFL-3 and control cell line were resolved by SDS-PAGE. RhoA, Rab9A, Bax, Cdc42, SR-BI, syntaxin 7, and Sar1b were detected with respective antibodies.

 
Effects of Selected Proteins on HCV Replication—
We used a siRNA approach to study the effects of reduced expression of selected modified proteins on HCV replication. SFL-2 and SFL-3 cells were transfected with siRNA against RhoA, Rab9A, syntaxin 7, Bax, or Cdc42 or with non-targeting siRNA. The levels of targeted proteins were reduced by 63–90% as demonstrated by Western blot (Fig. 4A). Levels of HCV RNA were assessed 40–70 h post-transfection by real time PCR analysis with the use of minus strand-specific primer for cDNA synthesis (Fig. 4B). Each experiment was performed four times independently. Reduction of syntaxin 7 induced a reproducible decrease (1.6-fold ± 0.3, mean ± S.E. (p = 0.05)) in HCV replication. In contrast, reduction of Cdc42 and of RhoA levels resulted in increased amounts of HCV RNA (1.7-fold ± 0.2 (p = 0.01) and 1.4-fold ± 0.1 (p = 0.03), respectively). Reduction of Rab9A and Bax levels had no significant effects on HCV replication.


Figure 4
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  		FIG. 4. Effects of reduced expression of selected proteins on HCV replication. SFL-2 and SFL-3 cells were transfected with siRNA specific to Rab9A, Bax, RhoA, Cdc42, or syntaxin 7 or with non-targeting siRNA. Forty to 70 h post-transfection, cells were lysed, and reduction of specific target proteins was verified by Western blot (A). In parallel, total RNA was isolated, and levels of HCV RNA were assessed by real time PCR using an HCV RNA minus strand-specific primer for cDNA synthesis (B).

 

   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
A limiting factor for protein identification in complex mixtures is the resolution method used for separation and the sensitivity of the mass spectrometer. Using 2-D PAGE, we detected ~100 protein spots in lipid rafts. Using a similar approach, ~70–100 proteins were identified in lipid rafts from Jurkat or human endothelial cells (16–18). Other techniques led to the identification of ~70 proteins and 380 proteins in lipid rafts from a monocytic cell line (19, 20) and from Vero cells (21), respectively. The most comprehensive analysis to date was reported by Foster et al. (22). This study identified 703 proteins in lipid rafts of HeLa cells by using SILAC, SDS-PAGE separation of proteins, and LC-MS/MS analysis with Q-TOF-MS. We used a similar approach and identified 1036 proteins, including 413 proteins identified by at least two unique peptides. It has to be noted that, based on the criteria used for the protein identification, one could expect approximately one protein misidentification for 400 proteins. In addition, the SILAC approach allowed us to quantify modifications occurring upon HCV replication for 268 proteins. Importantly the relative quantification data obtained by the 2-D gel approach were highly concordant with the data obtained by the SILAC approach.

In agreement with previously reported proteomics analysis of lipid rafts (16–22), identified proteins consisted of proteins that regulate vesicular transport and protein trafficking, signal transduction, and protein processing as well as cytoskeleton-associated proteins and proteins involved in cell metabolism. In particular, the large number of small GTPases we identified is concordant with the study of Foster et al. (22) who showed enrichment of G-proteins in the detergent-insoluble membrane domains when compared with total membranes. However, because of the nature of our sample preparation, the possibility of contamination from other subcellular organelles such as mitochondria that may lead to protein identifications irrelevant to this study cannot be excluded. We found a significant number of mitochondrial proteins (including Bax, metaxin 2, ATP synthase ß chain, prohibitin, voltage-dependent anion channel 1, and dehydrogenase/reductase SDR 7) that were strongly up-regulated in the detergent-insoluble domains of Huh7 cells bearing HCV replicons.

Several Rab GTPases were up-regulated in the detergent-insoluble membrane domains upon HCV replication. Rab proteins regulate vesicular transport by recruiting specific effectors that mediate tethering and docking of vesicles, facilitating fusion between membrane compartments (23). Recently a close connection between Rab protein functions and signal transduction pathways has also been revealed (24). It was demonstrated that Rab9 is required for the life cycle of human immunodeficiency virus type 1, filoviruses, and measles virus (25). The authors suggested that Rab9-mediated transport may be essential for egress of those enveloped viruses because Rab9 facilitates the pathway from late endosomes to the trans-Golgi network whence the cargo is transported to the plasma membrane (26). We observed a redistribution of Rab9A into detergent-insoluble domains in HCV replicon cells by both 2-D PAGE and SILAC. siRNA-mediated Rab9A reduction had no effect on HCV replication. Infectious HCV particles are not produced in Huh7 cells bearing HCV replicons; therefore a role of Rab9A on HCV assembly and egress cannot be investigated in this cell model. Other regulated Rab members identified in our study play a role in the early endocytic pathway (Rab21 and Rab22), the transport between the endoplasmic reticulum and the Golgi (Rab1), or the late endocytic compartment (Rab7). Interestingly Rab1, Rab7, and Rab18 were also shown to be associated with lipid bodies (27). Moreover Rab18 has been shown to induce apposition of the ER and lipid droplet membranes and to be recruited into lipid droplet membranes in response to lipid metabolism regulation (28, 29). Therefore, we can speculate that the up-regulation of these specific Rab proteins in the detergent-insoluble membrane domains of HCV cells observed in our study may reflect a complex modulation of vesicle formation and traffic by HCV and may involve accumulation of lipid bodies. We detected a 60% decrease in HCV replication upon reduction of syntaxin 7. This effect is significant and similar in magnitude to the effects of interferon, an antiviral cytokine known as a potent inhibitor of HCV replication and used to treat HCV patients, on HCV replication in this cell system. Syntaxins, like Rabs, mediate membrane fusion and have a role in determining specificity of vesicle traffic (30).

We observed an increase in HCV replication upon reduced expression of Cdc42 and RhoA, which belong to the Rho family of small GTPases. These proteins play a role in cell migration, membrane traffic, and actin cytoskeleton reorganization (31). It has also been shown that Cdc42 stimulates PI3K and mTOR pathway (31, 32). We demonstrated previously that inhibition of PI3K or inhibition of mTOR increases HCV replication (10). Therefore, modulation of HCV replication by Cdc42 and RhoA may be mediated by the PI3K-mTOR pathway.

Other proteins of interest uncovered in this study include scavenger receptor SR-BI, which has been shown to be a co-receptor for HCV infection of hepatocytes (33). SR-BI is responsible for selective uptake of high density lipoproteins, and localization of SR-BI in lipid rafts is important for cholesterol metabolism signaling in hepatic cells (34). hnRNP K is a component of the heterogeneous nuclear ribonucleoprotein complex and is implicated in chromatin remodeling, transcription, splicing, and translation (35). It has been reported that hnRNP K interacts with HCV core protein (36) and binds to and modulates replication of hepatitis B virus (37). Derlin-1 and transitional endoplasmic reticulum ATPase p97 form a complex in membranes of the ER and are involved in ER-associated protein degradation (38). This pathway has been co-opted by certain viruses to destroy cellular proteins required for the immune defense of the host selectively (38, 39). Glypican 3 is a membrane heparan sulfate proteoglycan expressed in fetal liver and hepatocellular carcinoma (40). Finally it was also reported that levels of lipid raft-associated heat shock protein 70 are increased in respiratory syncytial virus-infected cells and that heat shock protein 70 interacts with the virus polymerase complex (41).

In conclusion, we undertook a comprehensive, in-depth quantitative proteomics analysis of the detergent-insoluble subcellular fraction and identified protein modifications in this subcellular domain occurring upon HCV replication. The majority of the modified proteins were proteins involved in vesicular transport, protein trafficking, and cell signaling. Interestingly this study uncovered extensive subcellular relocalization of proteins upon HCV replication. We also obtained evidence that some of these protein changes may directly affect HCV replication.


  FOOTNOTES
 
Received, April 6, 2006, and in revised form, August 19, 2006.

Published, MCP Papers in Press, August 29, 2006, DOI 10.1074/mcp.M600121-MCP200

1 The abbreviations used are: HCV, hepatitis C virus; DMEM, Dulbecco’s modified Eagle’s medium; 2-D, two-dimensional; LTQ, linear ion trap quadrupole; SILAC, stable isotope labeling by amino acids in cell culture; siRNA, small interfering RNA; mTOR, mammalian target of rapamycin; SR-BI, scavenger receptor class B type 1; hnRNP K, heterogeneous nuclear ribonucleoprotein K; ER, endoplasmic reticulum; PI3K, phosphatidylinositol 3-kinase. Back

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

S The on-line version of this article (available at http://www.mcponline.org) contains supplemental material. Back

{ddagger} To whom correspondence should be addressed: Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N., M5-A864, Box 19024, Seattle, WA 98109. Tel.: 206-667-7080; Fax: 206-667-2537; E-mail: lberetta{at}fhcrc.org


   REFERENCES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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