Proteomic Analysis of HepaRG Cells: A Novel Cell Line That

Proteomic Analysis of HepaRG Cells: A Novel Cell Line That Supports Hepatitis B Virus Infection Ramamurthy Narayan,*,†,# Bevin Gangadharan,†,# Olivier Hantz,‡ Robin Antrobus,† ´ ngel Garcı´a,†,§ Raymond A. Dwek,† and Nicole Zitzmann† A Oxford Antiviral Drug Discovery Unit, Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, United Kingdom, INSERM, U871, 69003 Lyon, France, and Departamento de Farmacoloxı´a, Universidade de Santiago de Compostela, Santiago de Compostela, Spain Received July 24, 2008

Abstract: The first proteomic characterization of the HepaRG cell line, the only cell line that is susceptible to hepatitis B virus (HBV) infection and supports a complete virus life cycle, is reported. Differential analysis of naı¨ve and HBV-infected HepaRG cells by two-dimensional gel electrophoresis revealed 19 differentially regulated features, 7 increasing and 12 decreasing with HBV infection. The proteins identified in these features were involved in various cellular pathways including apoptosis, DNA/RNA processing, and hepatocellular impairment. Similar expression changes in a number of the identified proteins have already been reported for other virus systems. Identification of these expression changes is a validation of the proteomics approach and contributes to an understanding of host cellular response to HBV infection. Keywords: Hepatitis B virus • HepaRG cell line • Two dimensional electrophoresis • Hepatocyte

Introduction Hepatitis B virus (HBV) is a small enveloped DNA virus of the family Hepadnaviridae. Hepadnaviruses are species specific and target parenchymal liver cells in their host.1 Infection with HBV is a major public health problem with chronic carriers developing liver cirrhosis which can progress to hepatocellular carcinoma.2-4 The integration of HBV DNA into the host genome is associated with the late phase of infection and precedes the onset of liver cancer.5 Chimpanzees, recombinant mice, tree shrews, marmosets, woodchucks and ducks have been used as animal models for the study of HBV infection.6 HBV replication can be initiated in cell culture by transfection into HepG2,7,8 Huh7,9 HepAD3810 and primary hepatocytes.11 However, HBV infection in vitro has only been possible in differentiated primary human hepatocytes.12 HepaRG is a recently described cell line obtained from liver tumor tissue of a patient suffering from hepatitis C virus (HCV) * To whom correspondence should be addressed. Ramamurthy Narayan, Oxford Antiviral Drug Discovery Unit, Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, U.K. Phone: +44-1865-275749. Fax: +44-1865-275716. E-mail: [email protected]. † University of Oxford. ‡ INSERM, U871. § Universidade de Santiago de Compostela. # These authors contributed equally to this work.

118 Journal of Proteome Research 2009, 8, 118–122 Published on Web 12/03/2008

associated hepatocellular carcinoma (HCC).13,14 It was subsequently shown to be HCV-free and upon induction of differentiation using dimethyl sulfoxide (DMSO), insulin and hydrocortisone hemisuccinate,13 the cells express specific hepatocyte function, displaying both hepatocyte-like and biliary-like epithelial phenotypes and expressing oval cell specific markers. It has been possible to experimentally infect HepaRG with HBV at infection rates comparable to those achieved with primary hepatocyte cultures.1,13,15 Tong and co-workers performed a proteomics study comparing the HBV producing cell line HepG2.2.15 and its parental cell line HepG2 to identify alterations in cellular protein expression.16 Established in 1987, the HepG2.2.15 cell line is able to support the assembly and secretion of replicative intermediates of HBV DNA.8 While HepG2.2.15 cells constitutively secrete HBV, HepaRG is the only cell line able to be infected by the virus; it therefore provides a better representation of natural infection in liver cells and is appropriate for looking at the mechanism of HBV entry. Reported here is the first proteomic characterization of the HepaRG cell line, the only cell line susceptible to HBV infection, revealing novel changes within its proteome on infection with HBV.

Experimental Section HepaRG Cell Culture. Two to three weeks prior to infection, cell differentiation was induced by adding 2% (v/v) DMSO to the medium. To obtain infected cells, four flasks of differentiated HepaRG cells were incubated with an infectious HBV inoculum (obtained from cultures of HepG2 clone 2.2.15 cells)8 diluted in culture medium in the presence of 4% (v/v) PEG 8000 for 17-20 h at 37 °C.13 The cells were then washed with medium and cultured in the presence of 2% (v/v) DMSO, 5 µg/mL insulin and 50 mM hydrocortisone. Four control flasks were treated in the same way but omitting the infectious inoculum. Confirmation of HBV Infection. Two weeks postinfection, the supernatant was tested for the presence of Hepatitis B surface (HBs) antigen presence by ELISA (monolisa HBsAgBioRad) according to manufacturer’s instruction. Infection was also confirmed by the detection of viral RNA in infected cells by Northern blot analysis. Immunofluorescence analysis using anti-HBs antibody showed that at least 15-20% of cells strongly expressed viral antigen and were infected (data not shown). 10.1021/pr800562j CCC: $40.75

 2009 American Chemical Society

communications Two-Dimensional Gel Electrophoresis (2-DE). 2-DE was performed essentially as described earlier.17,18 Eight gels were run using cells from each of the eight flasks described earlier containing uninfected and infected cells. HepaRG cells were lysed by sonication in sample buffer (5 M urea, 2 M thiourea, 2 mM tributyl phosphine, 65 mM DTT, 4% (w/v) CHAPS, 150 mM nondetergent sulfobetaine 256 (NDSB-256), 1 mM sodium orthovanadate, 1 mM sodium fluoride and 1 mM benzamidine). The lysate was centrifuged at 13 000g for 5 min and 500 µg of protein was made up in carrier ampholytes (0.9% (v/v) pH 3-10, 0.45% (v/v) pH 2-4 and 0.45% (v/v) pH 9-11; SERVALYT, SERVA, Heidelberg, Germany) and 0.0012% (w/v) bromophenol blue up to 375 µL in sample buffer and left overnight to be rehydrated on 18 cm pH 3-10 nonlinear immobilized pH gradient DryStrips (GE Healthcare, Bucks, U.K.). Isoelectric focusing was carried out for 75 kVh at 17 °C. Strips were incubated in equilibration solution (4 M urea, 2 M thiourea, 50 mM Tris-HCl (pH 6.8), 30% (v/v) glycerol, 2% (w/v) SDS, 130 mM DTT, 0.002% (w/v) bromophenol blue) for 15 min. Proteins were separated by 9-16% (w/v) SDS-PAGE gradient gels using 20 mA per gel for 1 h, followed by 40 mA per gel for 4 h at 10 °C. Following electrophoresis, gels were fixed in 40% (v/v) ethanol and 10% (v/v) acetic acid and stained with the fluorescent dye OGT 1238.18 The 16-bit images were obtained at 200 µm resolution by scanning gels with an Apollo II linear fluorescence scanner (Oxford Glycosciences, Abingdon, U.K.). Differential Image Analysis. Scanned images were processed with a custom version of Melanie II (Oxford Glycosciences, Abingdon, U.K.).18 For image analysis, four replicate gels from separate uninfected HepaRG cell cultures were compared with four replicate gels from separate HBV-infected HepaRG cell cultures. A synthetic image was created using accurate spot matching which showed all features in the gels for uninfected and infected HepaRG cells. The optical density of each feature was determined by summing pixels within the feature boundary and the volume was determined by integrating this optical density over the area of the feature. All statistical calculations were based on the percentage volume of the features and changes in protein expression were determined as a ratio of the mean percentages of feature volumes. Only features present in at least three of four individual gels belonging to either the uninfected or infected group of gels were considered. Features which changed by at least 2-fold in percentage spot volume were considered as differentially expressed. Changes were statistically validated using a rank-sum test on percentage spot volumes with P e 0.05 (95% confidence) as previously described.18,19 All features were further validated by visualizing the features across all gels in a montage format. These differentially expressed features were excised from the gels for mass spectrometric analysis. In-Gel Digestion and Peptide Extraction. Differentially expressed features assigned for mass spectrometric analysis were excised from gels using a software-driven robotic cutter (Oxford Glycosciences, Abingdon, U.K.). Recovered gel pieces were dried in a SpeedVac followed by in-gel trypsin digestion and peptide extraction using the automated DigestPro workstation (Intavis, Koeln, Germany) according to the protocol of Shevchenko and co-workers.20 Combined fractions were lyophilized and dissolved in 0.1% (v/v) formic acid prior to mass spectrometric analysis. Mass Spectrometric Analysis. Tryptic peptides were analyzed using a Q-TOF 1 mass spectrometer coupled with a CapLC (Waters, Hertfordshire, U.K.). Peptides were concen-

trated and desalted on a 300 µm i.d./5 mm C18 precolumn and resolved on a 75 µm i.d./25 cm C18 PepMap analytical column (LC Packings, CA) with a 45 min 5-95% (v/v) acetonitrile gradient containing 0.1% (v/v) formic acid at a flow rate of 200 nL/min. Spectra were acquired in positive mode. MS to MS/ MS switching was controlled in an automatic data-dependent fashion with a 1 s survey scan followed by three 1 s MS/MS scans. Ions selected for MS/MS were excluded from further fragmentation for 2 min. Raw MS/MS spectra were smoothed and centered using ProteinLynx Global server 2.1.5; spectra were not deisotoped. Processed peak list (.pkl) files were searched against the Swiss-Prot database (release 54.4) using MASCOT (Matrix Science, London, U.K.). Searches were restricted to the human (17 565 sequences) and virus (11 132 sequences) taxonomies. Carbamidomethyl cysteine was defined as a fixed modification and oxidized methionine as a variable modification. Data were searched allowing 0.5 Da error to accommodate calibration drift and up to 2 missed tryptic cleavage sites. All data were checked for consistent error distribution and all positive identifications were checked manually. Ingenuity Pathways Analysis. The Ingenuity Pathways Analysis software (Ingenuity Systems, CA) was used to investigate possible interactions between all the identified proteins. Interactive pathways were generated to observed potential direct and indirect relations among the differentially expressed proteins.

Results and Discussion Approximately 1300 spots were detected in each gel (uninfected 1213 ( SD18; infected 1400 ( SD13). A synthetic gel image representative of all the features in the differential analysis comparing gels from uninfected and infected cells is shown in Figure 1. Original gel images for all eight gels are shown in Figure 1 of the Supporting Information. In total, 19 differentially expressed features (7 increased and 12 decreased in HBV-infected cells) were selected for LC-MS/MS analysis resulting in the identification of 44 proteins (Supporting Information Table 1 and Table 1). Identified proteins were related to various biological processes such as apoptosis (annexins and elongation factor 2), DNA/RNA processing (APEX nuclease and hnRNP proteins) and hepatocellular impairment (glutathione proteins). Possible interactions between the differentially expressed proteins were analyzed using the Ingenuity Pathways Analysis software and three related pathways are shown in Figure 2 of the Supporting Information. These interaction pathways cover various biological processes including hepatic disease, cancer, cell cycle and cellular assembly and organization. Many of the identified proteins have already been seen to change in other viral systems. The potential role of these proteins are discussed below. Elongation factor 2 (EF2) is a translation factor essential for protein synthesis. In our study, this protein was up-regulated in infected cells. EF2 has been identified as one of the host proteins which interact with HBV core in a yeast two-hybrid screen.21 Recently, EF2 levels were found to be elevated in serum of patients with HCC compared to that of chronic hepatitis patients and normal controls.22 The HepaRG cell line used in our study was derived from a HCC source, and therefore, one may expect to observe elevated EF2 levels. However, we observed a further increase post HBV infection, the reason for which remains unclear. Journal of Proteome Research • Vol. 8, No. 1, 2009 119

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Figure 1. Synthetic 2D-PAGE image representing all protein spots present in the uninfected versus HBV-infected cell analysis. Gels were run using pH 3-10 nonlinear immobilized pH gradient DryStrips with 9-16% (w/v) SDS-PAGE gradient gels and were stained using the fluorescent dye OGT 1238. The synthetic image shown was created using accurate spot matching as previously described.18,19 Differentially expressed features are identified with arrows and the numbers in parentheses refer to the Swiss-Prot accession numbers of the proteins. The names of selected proteins are shown in Table 1 and a full list of all proteins shown on this image can be found in Supporting Information Table 1. U, feature present only in gels representing uninfected cells; I, feature present only in gels representing infected cells; *, features present in both uninfected and infected cells but expressed to a higher extent in the group indicated. For complete gel figures, see Figure 1 of the Supporting Information.

Apoptosis can provide protection against viral infection by inducing premature death of virally infected cells. Viruses often inhibit apoptosis of infected cells in order to replicate efficiently. Failure to inhibit the apoptosis can restrict virus growth and consequently a number of viruses encode potent cell death suppressors. Studies with lentiviruses (HIV1, HIV2 and SIV) have shown that EF2 inhibits apoptosis by suppressing the apoptosis-related proteases caspase 9 and caspase 3, which are induced by the viral protein vpr.23 The up-regulation of EF2 in HBV-infected cells may play a similar role in suppressing apoptosis. Annexin A1 was found to be elevated and annexin A2 was observed at a higher molecular weight in HepaRG cells post HBV infection. Annexins are calcium/phospholipid binding proteins that inhibit phospholipase A2 and inflammation. In a recent proteomic study of the HBV producing cell line HepG2.2.15, annexin A3 and A4 were shown to be differentially expressed.16 Salmon annexin 1 has also been found to be overexpressed in fish cells during infectious pancreatic necrosis virus (IPNV) infection and supports viral growth by inhibiting the premature death of virus-infected cells.24 Annexin 2 belongs to a family of Ca2+ dependent membranebinding proteins and is involved in many biological functions, such as the anti-inflammatory effect of glucocorticoid, Ca2+ 120

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dependent exocytosis, immune response and calcium transport. There are several reports implicating annexin 2 in viral infection, including assisting in the assembly of HIV,25 enhancing virus-membrane fusion26 and supporting HIV infection.27 In HBV infection, annexin 2 has been shown to bind S100 A10 (p11), a Ca2+-modulated protein, to form a heterotetrameric complex and HBV polymerase has been shown to interact with p11 which in turn inhibits the activity of HBV pol both in vitro and in vivo.28 In another proteomics study, S100 A10 was found to be decreased in the HBV producing cell line HepG2.2.15 compared to its parental cell line HepG2.16 Annexin 2 functions as a receptor for β2 glycoprotein-I on the membrane surface of a HCC cell line which may aid HBV infection.29 Heterogeneous nuclear ribonucleoproteins (hnRNP) K and A/B were down-regulated in infected HepaRG cells. hnRNP K is a pre-mRNA binding protein that plays a role in nuclear metabolism of hnRNA. In a recent study, HBV has been shown to depend on host hnRNP K to modulate its replication efficacy, and it has been suggested that hnRNP K could be used to suppress HBV infection.30 HCV core protein has been shown to bind host hnRNP K and disrupt the suppressive effect of hnRNP K on the thymidine kinase gene promoter essential for G1-S transition, thus, affecting regulation of cell growth31 and possibly causing apoptosis in HCV-infected cells. hnRNP A/B is also known as APOBEC-1 binding protein 1; different members of the APOBEC family have been shown to interact with various viruses. APOBEC-3G inhibits HBV propagation in both mammalian and nonmammalian cells,32-34 while rat APOBEC-1 can restrict HIV propagation and also aid editing of HIV RNA.33,35 Splicing of the HIV 1 pre-mRNA is highly regulated and dependent on the host splicing machinery. hnRNP A/B proteins are necessary for inhibition of the splicing of HIV I tat exon 2.36,37 The copper chaperone for superoxide dismutase (CCS) which delivers copper to Cu/Zn superoxide dismutase (SOD) was found to decrease in HBV-infected HepaRG cells. In addition, Cu/ZnSOD levels have been shown to significantly increase during hepatitis infection.38 The CCS chaperone identified in our study has been shown by others to activate a Cu/Zn SOD called SOD1 by directly inserting the copper cofactor into the apo form of SOD1.39 In another proteomics study, an increase in manganese binding SOD has been shown in the HBV producing cell line HepG2.2.15 compared to its parental HepG2 cell line.16 Ras GTPase activating protein-binding protein 1 (G3BP1), an effector of Ras and phosphorylation dependent RNase, has been implicated in Ras signaling, NF-κB signaling, the ubiquitin proteosome pathway and RNA processing. G3BP1 has been shown to be involved in HCV replication and may be a component of the HCV replication complex.40 PDZ domain containing-protein decreased in HepaRG cells following HBV infection. It is a scaffolding protein that connects membrane proteins and their regulatory components. In certain types of human papilloma virus (HPV), the E6, HPV16 and HPV18 proteins target different regions of the PDZ domain for proteosome mediated degradation.41 Glutathione S-transferases A1/A2 were found to be upregulated in infected HepaRG cells. Glutathione S-transferases are enzymes involved in the detoxification of electrophilic compounds, including carcinogens, therapeutic drugs, environmental toxins and products of oxidative stress. The concentration of glutathione-S-transferase alpha 1-1 has recently been suggested as a marker for hepatocellular impairment.42

communications Table 1. Selected Differentially Expressed Proteins Identified in the Uninfected Versus HBV-Infected Analysis protein name [AN]

fold

Annexin A1 [P04083]

I

Annexin A2 [P07355]

-

Copper chaperone for superoxide dismutase [O14618] Elongation factor 2 [P13639]

U* I

Glutathione S-transferase A1 [P08263] or A2 [P09210]

I

Heterogeneous nuclear ribonucleoprotein A/B (hnRNP A/B) (APOBEC-1 binding protein 1) [Q99729]

U*

Heterogeneous nuclear ribonucleoprotein K (hnRNP K) [P61978] U

PDZ domain containing-protein [O60450]

U*

Ras GTPase-activating protein-binding protein 1 [Q13283]

U

function

Calcium/phospholipid-binding protein. Promotes membrane fusion. Involved in exocytosis. Regulates phospholipase A2 activity Calcium-regulated membrane-binding protein. Affinity for calcium enhanced by anionic phospholipids Delivers copper to copper zinc superoxide dismutase Promotes GTP-dependent translocation of nascent protein from A- to P-site of ribosome Conjugation of reduced glutathione to exogenous and endogenous hydrophobic electrophiles Involved with pre-mRNA processing. Forms complexes with other hnRNP and heterogeneous nuclear RNA in nucleous. Binds to APOB mRNA transcripts around RNA editing site Major pre-mRNA-binding protein. Binds to poly(C). May play a role in nuclear metabolism of hnRNAs. Also binds poly(C) single-stranded DNA Scaffold protein connecting membrane proteins and regulatory components. May be involved in coordination of regulatory processes for ion transport and second messenger cascades. May connect SRB1 with cholesterol transport/metabolism Effector of stress granule assembly. Phosphorylation-dependent sequence-specific endoribonuclease in vitro. ATP- and magnesium- dependent helicase. Unwinds DNA & RNA duplexes. Moves in 5′ to 3′ direction along bound single-stranded DNA

* Proteins shown were differentially expressed by 2-fold or more when comparing uninfected and HBV-infected gels. AN, accession number; U, feature present only in uninfected gels; I, feature present only in infected gels; *, features present in both uninfected and infected but expressed to a higher extent in the indicated group,-, present in different locations in infected and uninfected. For a full list of all proteins identified, see Supporting information Table 1.

Glutathione-S-transferase P has been shown to be downregulated in another proteomic study comparing the HBV producing cell line HepG2.2.15 and its parental clone.16 This other proteomics study has shown transaldolase increased in HepG2.2.15 and we have also seen this protein increased in HBV-infected HepaRG cells, although the reason for this change is unclear. The proteins identified in this study may help to understand more about the mechanism HBV uses in infection. Interestingly, only differentiated HepaRG cells are susceptible to this hepatic virus.13 To investigate why only differentiated HepaRG cells are susceptible to HBV, we are currently using proteomics to compare undifferentiated cells with differentiated cells. The combination of data for both of these proteomics studies may help to further understand HBV infection. This is the first proteomic characterization of the HepaRG cell line identifying novel differentially expressed cellular proteins in HBV infection. Some of the proteins discussed above have already been seen to change in HBV infection (EF2, annexin A2, hnRNP K) or are closely related to other proteins changing in HBV infection (CCS, hnRNP A/B, annexin A1, glutathione-S-transferase A1/A2). However, many of the identified cellular protein changes have only been seen in other viral systems (G3BP1, PDZ domain containing protein) and therefore, along with other proteins in Supporting Information Table 1 which appear to have no association with any viruses, these proteins are novel to HBV infection.

Conclusion This study represents the first proteomic analysis of virally infected HepaRG cells, the only cell line susceptible to HBV infection. Of the 19 differentially expressed features found, 12

were decreased and 7 increased in infected cells revealing novel changes within the HepaRG proteome on infection with HBV. While others have shown differentially expressed proteins in cells constitutively secreting HBV, this is the first proteomic characterization of HepaRG cells which are most appropriate for looking at the mechanism of HBV entry. Interestingly, many of the identified proteins discussed have already been observed in other viral infections with similar changes in expression. These differentially expressed proteins which are related to other viral diseases may help to further understand the host cellular response and mechanism of HBV infection.

Acknowledgment. This work was supported by the Oxford Glycobiology Endowment and grants from the ANRS (Agence Nationale de Recherches sur le Sida et les he´patites virales, France). A.G. is a Ramo´n y Cajal Research Fellow (Spanish Ministry of Education and Science, Spain). N.Z. is a Glycobiology Career Development Fellow and Senior Research Fellow of Linacre College, Oxford. We thank Prof. Christian Trepo for providing the HepaRG cell line. We acknowledge Bettina Kampa, David Giles and David Chittenden for practical assistance, Dr. Stephen Woodhouse for advice on using the Ingenuity Pathways Analysis software and Dr. Jo O’Leary for the critical review of the manuscript. Supporting Information Available: Labeled images of all gels, a complete table of identified proteins and potential protein interactions between the identified proteins are shown in the online Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org. Journal of Proteome Research • Vol. 8, No. 1, 2009 121

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