Evaluation of hepatitis B viral replication and proteomic analysis of HepG2.2.15 cell line after knockdown of HBx

Hangzhou,China

Introduction

Hepatitis B virus (HBV) is one of the major pathogens of human liver disease.Globally,there are more than 360 million people chronically infected with HBV,many of whom willfinally develop severe liver diseases,such as cirrhosis and hepatocellular carcinoma (HCC).[1]It has been reported that chronic HBV carriers have a 100-fold higher risk of developing HCC,and among them,over 500 000 die from liver cirrhosis and HCC annually.[2,3]

HBV belongs to the hepadnavirus family and is composed of an outer envelope formed by HBsAg and an inner nucleocapsid that packages the viral genome and polymerase.The HBV genome is a partially doublestranded relaxed circular DNA molecule containing four partially overlapping open-reading frames encoding viral envelope,core,reverse transcriptase-polymerase,and the X gene.[4]HBx,the product of the X gene,promotes viral gene expression and replication by trans-activating the viral promoters and enhancer/promoter complexes.[5,6]It modulates several signaling pathways of protein kinases related to viral replication.For example,HBx stimulates the Src kinase family,[7]including proline-rich tyrosine kinase (Pyk2),[8]and focal adhesion kinase (FAK);[9]activation of these kinases is dependent on cellular calcium signaling and critical in HBV replication.HBx also promotes viral genome replication via the DDB1-dependent pathway and knockdown of DDB1 reduces HBV transcription and replication.[10]In the case of tumorigenesis,HBx stimulates the activity of TGF-β1 and promotes the tumorigenic properties of this wellknown cytokine.[11,12]It also regulates the expression of several matrix metalloproteinases such asfibronectin and integrin beta-1,thereby remodeling the extracellular matrix,which may promote tumor metastasis.[13,14]A technique for target protein identification,twodimensional electrophoresis (2-DE) in combination with image analysis and mass spectrometry (MS) is commonly used in the area of HBV research.[15,16]Thus,to discover novel anti-HBV targets for attenuating the morbidity and mortality of HBV-related disease,we performed global proteomic profiling using 2-DE to identify the downstream functional proteins of HBx.

The HepG2.2.15 cell line was established by transfecting a hepatoblastoma cell line (HepG2) with plasmids containing two head-to-tail dimers of the HBV genome.The HepG2.2.15 cell line releases high levels of HBsAg and HBeAg into the medium during culture.It supports the full replication cycle of HBV,assembly and secretion of HBV DNA and Dane particles.[17,18]Furthermore,HBV virions produced by the cell line also exhibit high endogenous polymerase activity.[18]Thus,HepG2.2.15 is an appropriate tool to identify the molecular events of the viral replication cycle as well as the secretion of HBV particles into the intracellular environment in vitro.

In this study,we first evaluated the effects of HBx knockdown on HBV replication in vitro.To reveal the underlying mechanisms,proteomic techniques were used to analyze the changes of protein expression profile in HepG2.2.15 cells after RNA interference of HBx by small interfering RNA (siRNA) and identify 12 proteins with altered expression that may play critical roles in HBx-related HBV replication.

Methods

Cell culture

HepG2.2.15 cells were cultured in DMEM medium(HyClone,USA) containing 10% fetal bovine serum(FBS) (Gibco,USA) and 200 mg/L G418 (Sigma,USA) at 37 ℃ in a humidified incubator gassed with 5% CO2.

siRNA transfection

The day before transfection,subcon fluent monolayer HepG2.2.15 cells were harvested from the culture dishes with trypsin and seeded in 6-well plates at 20 000 cells/cm2such that they were about 50% con fluent the next day.Then,the cells were treated with siRNA buffer alone for the control group.Transfection with 40 nmol/L of HBx siRNA (sense:5'-GACCUUGAGGCAUACUUCAdTdT-3'and antisense:5'-UGAAGUAUGCCUCAAGGUCdTdT-3′)or mock siRNA (sense:5'-UUCUCCGAACGUGUCAC GUdTdT-3' and antisense:5'-ACGUGACACGUUCG GAGAAdTdT-3') was performed using oligofectamine transfection reagent in OptiMEMI-reduced serum medium (Invitrogen) in the absence of FBS and antibiotics according to the manufacturer's instructions.Six hours after transfection,the medium was removed and replaced with RPMI-1640 containing 200 μg/mL G418.Supernatant was harvested for further analysis 48 hours after siRNA treatment.

Analysis of HBsAg and HBeAg

HBsAg and HBeAg levels in the supernatant were detected by Abbott AxSYM HBsAg and HBeAg QT assay kits (Abbott Diagnostics Division) following the manufacturer's instructions.The suppression rate of replication of HBsAg and HBeAg in the HBx knockdown group was calculated using the following formula:

Suppression rate=[1-A value (HBx knockdown)/A value (negative control)]×100%

Real-time quantitative PCR (qPCR) analysis of hepatitis B virus DNA

Forty-eight hours after treatment with siRNA,supernatant HBV DNA levels were tested using a realtime PCR kit (Bioselex,Hangzhou,China) according to the manufacturer's instructions on an ABI 7500 real-time PCR system (Applied Biosystems,Foster,CA,USA).This kit was approved by the State Food and Drug Administration of China for in vitro diagnosis with a low detection limit.[19]

2-DE

Cells of each sample were harvested and washed three times with PBS,resuspended in 1 mL lysis buffer containing 7 mol/L urea,2 mol/L thiourea,4% CHAPS,1% DTT,2% IPG buffer (pH 4-7,Amersham Biosciences)and protease inhibitor cocktail (Complete tablets,Roche Diagnostics,Germany),then ultrasonically lysed for 4×10 seconds and incubated on ice for 1 hour.After centrifugation at 23 000 g for 30 minutes,the supernatant was harvested.Protein concentration was determined using the Bradford method.

2-DE was carried out following the instructions from Amersham Biosciences.Brie fly,samples containing 200 μg protein were diluted to 450 μL with rehydration solution (8 mol/L urea,2% CHAPS,20 mmol/L DTT,0.5% IPG buffer pH 4-7,0.002% bromophenol blue).After rehydration with 50 V for 12 hours,the sample proteins were subjected on IPG strips to IEF operating the Amersham Biosciences IPGphor as follows:200 V for 1 hour,500 V for 1 hour,1000-4000 V for 1 hour,4000-8000 V for 1 hour,8000 V for 1 hour,then focused at 8000 V for about 8 hours.After IEF,strips were equilibrated for 15 minutes in SDS equilibration buffer(50 mmol/L Tris-HCl,6 mol/L urea,30% glycerol,2%SDS and 0.002% bromophenol blue) containing 1%DTT and shaken at 120 rpm on an orbital shaker,then transferred to the SDS equilibration buffer containing 2.5% iodoacetamide and shaken for another 15 minutes.After equilibration,the strips were loaded onto vertical SDS PAGE (12.5% T constant).The second dimensional SDS electrophoresis was run using an Ettan DALTsix electrophoresis unit (Amersham Biosciences).Experiments with each group (negative control or HBx knockdown) were performed in triplicate.

Silver staining and image analysis

After electrophoresis,silver staining was done to visualize the gel according to the protocol of Yan et al.[20]Protein patterns on silver-stained gels were scanned using a high-resolution scanner (Amersham Biosciences),and analyzed with ImageMaster 2D software (version 6.0) for spot detection,background subtraction,volume normalization and spot matching.The resulting data were exported to Microsoft Excel,and significantly differentially expressed protein spots matching the threshold of 2.0-fold were selected for MALDI-TOF/TOF MS analysis.

In-gel digestion and MALDI-TOF/TOF MS analysis

Protein spots were excised from wet gels,transferred into Eppendorf tubes and destained.After that,the gel pieces were hydrated in 10 μL proteomics grade trypsin(Sigma) solution (20 ng/μL in 25 mmol/L NH4HCO3)and incubated at 37 ℃ for 15 hours.Peptides were extracted with 50% ACN and 2.5% TFA,and then dried in a lyophilizer and reconstituted in 1.5 μL 0.1% TFA Solution (Virtis,Gardiner,NY,USA).

The dried peptide mixtures were mixed with saturated matrix solution (5 mg/mL CHCA (Sigma,USA) in 0.1% TFA and 50% ACN) and spotted onto the MALDI target plate.Mass analysis was performed on an ABI 4700 Proteomics Analyzer MALDI-TOF/TOF(Applied Biosystems,Foster City,CA,USA) operating in result-dependent acquisition mode.Peptide mass maps were acquired in positive ion re flector mode at 20 kV accelerating voltage with 2000 laser shots per spectrum.When there were monoisotopic peak masses within 900-4000 Da with a minimum signalto-noise ratio (S/N) of 10 and a local noise window width of 250 m/z,the firstfive precursor ions showed the highest intensity excluding common trypsin autolysis peaks and matrix ion signals were selected for fragmentation.The instrument was operated in the MS/MS positive ion mode at 2 kV collision energy with default calibration.Monoisotopic peak masses were automatically determined with a minimum S/N of 5 and a local noise window width of 250 m/z.Combined MS and MS/MS spectra were submitted to GPS Explorer(version 3.6,Applied Biosystems) and MASCOT (version 2.1,Matrix Science) searching against the UniprotKB/SwissProt database.The search parameters were as follows:trypsin cleavage,one missed cleavage allowed,carbamidomethylation of cysteine asfixed modification and methionine oxidation as variable modification,peptide mass tolerance to 150 ppm,fragment tolerance to 0.4 Da,and minimum ion score confidence interval for MS/MS data set to 95%.Significance of the identification was evaluated according to the value of probability and sequence coverage.

Antibodies and Western blotting

Western blotting was performed as previously described.[21]HSP70 (human HSPA1A,Santa Cruz,dilution 1∶1000) was detected in total cell extracts.

Statistical analysis

Data of microparticle enzyme immunoassay (MEIA)results and real-time qPCR are shown as mean±SD.One-way ANOVA analysis of Dunnett's test was used to analyze the significance of differences between the negative control and HBx knockdown groups.The statistical significance in terms of the expression profiles of HepG2.2.15 cells with or without HBx knockdown was estimated by Student's t test.All calculations were done with SPSS 11.5 software,and a P value less than 0.05 was considered statistically significant.

Results

Downregulation of HBx inhibits the expression of HBsAg and HBeAg

To investigate the role of HBx in the production of HBsAg and HBeAg,HepG2.2.15 cells were transfected with HBx siRNA or a mock siRNA as a negative control.The supernatant was collected at 48-hour after the treatment with siRNA and the titer of HBsAg and HBeAg was assessed by HBsAg and HBeAg QT assay.We found that after treatment with HBx siRNA,the suppression rates of HBsAg and HBeAg were 55%and 66% respectively when compared with the mock siRNA-transfected group (Fig.1),indicating that the downregulation of HBx inhibited HBsAg and HBeAg production in vitro.

Downregulation of HBx inhibits HBV DNA replication

We also investigated whether the knockdown of HBx in vitro had a direct effect on HBV DNA replication.The HBV DNA in the supernatant was measured at 48 hours after the treatment with siRNA by real-time qPCR analysis.The HBV DNA replication level was lower in the HBx siRNA group than in the mock siRNA-treated group (P=0.005) (Fig.2),indicating that the downregulation of HBx inhibited HBV DNA replication.

Fig.1.Downregulation of HBx inhibited the expression of HBsAg and HBeAg.HepG2.2.15 cells were treated with HBx siRNA,the culture supernatants were collected for MEIA analysis of HBsAg and HBeAg 48 hours later,and suppression rates of HBsAg and HBeAg were calculated [suppression rate=[1-A value (HBx knockdown)/A value (negative control)]×100%].Each test was repeated 3 times.Results indicated that the relative level of HBsAg(*:P＜0.001) and HBeAg (#:P=0.007) after downregulation of HBx.

Comparative analysis of 2-DE protein profiles and identification of proteins by MALDI-TOF/TOF

To identify key proteins that may play a critical role in HBV replication,comparative 2-DE analysis of HepG2.2.15 cells treated with mock and HBx siRNA was performed.Protein lysate from each group was resolved by 2-DE and visualized by silver staining,then the gels were digitized prior to computer-based matching and quantitative analysis with image analysis software.MALDI-TOF/TOF analysis was performed in order to identify the protein spots with altered intensity (＞2.0-fold) as a result of HBx knockdown.After submitting the combined MS and MS/MS spectra generated by MALDI-TOF/TOF,12 were identified:7 up-regulated proteins and 5 down-regulated proteins.The identified proteins represented a heterogeneous group that included several important molecules relevant to protein folding such as heat shock 70 kDa protein (HSP70) and signal transduction such as calreticulin.The functions of the remaining proteins in HBV pathogenesis are still not clear (Fig.3 and Table ).

Fig.3.Cells were lysed and 200 μg of total protein lysate was subjected to 2-DE,followed by silver staining and image analysis.Results were quantified from three sets of 2-DE.The 2-DE map indicated protein spots changed in volume after knockdown of HBx in HepG2.2.15 cells.The spots of interest were excised from the gels and digested with trypsin.The digested peptides were used for MALDI-TOF/TOF MS analysis.The identified proteins are listed in Table.A:HBx siRNA treated group; B:Mock siRNA treated group.

Table.Differentially-expressed proteins identified after knockdown of HBx in HepG2.2.15 cells

Fig.4.Western blotting analysis of HSP70 (HSPA1A) identified by MS with β-actin as internal reference.Lanes 1,2,3:HepG2.2.15 cells treated with mock siRNA; lanes 4,5,6:HepG2.2.15 treated with siRNA of HBx.The expression level of HSP70 protein was decreased after knockdown of HBx in HepG2.2.15 cells.

Validation of proteins by Western blotting

To validate the 2-DE results and assess the expression changes of several proteins showing differential patterns after treatment with HBx siRNA,Western blotting analysis of protein abundance changes was performed.From the identified candidates,we tested HSP70 by Western blotting analysis using available commercial antibodies.HSP70 protein level was decreased after the knockdown of HBx in HepG2.2.15 cells,which conformed to the 2-DE data (Fig.4).

Discussion

In this study,we first evaluated the effects of knockdown of HBx on HBV replication level by MEIA analysis of HBsAg and HBeAg in the supernatants and real-time qPCR analysis of extracellular HBV DNA.After the knockdown of HBx,the titer of HBsAg and HBeAg as well as the HBV DNA replication level were dramatically decreased,suggesting that HBx plays an important role in HBV replication in this culture system.

The viral HBx protein has been shown to promote transcriptional activation and viral replication in the nucleus and cytoplasm of infected cells.[22]HBx regulates a wide range of genes.It up-regulates the expression of HBV genes by transactivating its own promoters,as well as cellular genes of infected hepatocytes,thereby modifying the environment to facilitate viral replication.[1]Our proteomic screening,which explored global protein changes after the knockdown of HBx,may provide clues to reveal the mechanisms of action of HBx on HBV replication,and possibly identify key factors co-activated with HBx in HBV replication.

In this study,a total of 12 protein spots were identified to exhibit altered expression level after knockdown of HBx with high confidence by peptide massfingerprinting.One of the proteins that decreased after RNA interference of HBx was HSP70,a molecular chaperone involved in the topological reorientation of the HBV large envelope protein.[23]HSP70 plays an important role in viral replication.Gonzalez et al[24]reported that HSP70 coimmunoprecipitates with NS5A,a protein implicated in regulating HCV viral genome replication.[25,26]siRNA-mediated knockdown of HSP70 reduces the augmentation of the HCV internal ribosome entry site,mediates translation induced by NS5A,and modestly suppresses viral protein accumulation.[24]In HBV,HSP70 binds specifically with HBx to form a complex,[27]helping HBx to fold in the right conformation; at the same time,HBx exerts its function by binding to HSP70.[27]It also physically interacts with HSP60 and HSP90 in the HepG2.2.15 cell line to form a multi-chaperone machine that contributes to the HBV life cycle,so down-regulation of HSP70 could significantly inhibit the production of HBV virions.[28]Besides,a carcinogenic role of HSP70 has been reported;[29-31]in the case of HBV-related HCC,expression of HSP70 was increased along with the stepwise progression of hepatocarcinogenesis and there is a positive correlation between HSP70 expression and prognostic factors for HCC.[32]The role of HSP70 in HCC is associated with its function in cell proliferation and apoptosis.HSP70 interacts with p53 as a chaperone,[33]and it has been shown that HSP70 antisense oligomers specifically inhibits tumor cell proliferation by inducing apoptosis.[34]Taken together with these reports,our results strongly suggest that HSP70 plays a pivotal role in HBV replication and HBV-related carcinogenesis.

Calreticulin (CALR),one of the most ubiquitous signaling molecules mainly located in the endoplasmic reticulum (ER),was upregulated after the knockdown of HBx.CALR has important functions in the calcium signaling pathway by homeostatic control of cytosolic and ER calcium levels.[35,36]Ca2+plays critical roles in viral entry,expression of viral genes,posttranslational processing of viral proteins,virion maturation and release.[37]In the case of viral replication,viruses disrupt Ca2+homeostasis and accelerate their replication cycles using Ca2+and cellular Ca2+-binding proteins.[38,39]Modest ER-mitochondrial Ca2+increases activate the Ca2+-dependent Kreb's cycle dehydrogenases to facilitate ATP production,thereby meeting the higher energy demand of viral replication.[40]At the same time,Ca2+overload either in mitochondria or intracellular space has pro-apoptotic effects by several pathways.For example,abnormal increases in ER-mitochondrial Ca2+activates the opening of the permeability transition pore,causes the release of cytochrome C and activation of caspase 9,then triggers apoptosis of the infected cell.[41]Finally,virions are released and viral dissemination is maximized.CALR is also involved in the adaptive immune response; it has been proposed that intracellular viruses subvert this response to pathogeninfected cells through the prevention of cell surface exposure of CALR.[42]In this study,we unveiled a direct link between CALR and HBx,providing a mechanism by which HBx might contribute to HBV replication.

RuvB-like 2 (RUVBL2),which belongs to the ATPase associated with diverse cellular activities family of helicases,was also overexpressed in HepG2.2.15 cells after treatment with HBx siRNA in this study and participated in cellular processes such as cellular transformation,cell signaling,apoptosis,and the DNA damage response.It was reported that RUVBL2 interacts with proteins like β-catenin,c-Myc,and ATF2,[43-45]the former two are believed to contribute to HBV- or HCV-induced liver pathogenesis,including liver cell damage,viral replication and carcinogenesis.[46-49]The mechanism of action of RUVB2 on virus replication remains unclear.As to in fluenza A virus,Kakugawa et al[50]reported that RUVBL2 interacts with the viral nucleoprotein and interrupts its oligomerization,a critical step in the assembly of viral replication complexes,thereby suppressing viral replication in embryonic kidney cells.In this study,our novel finding reveals the association between RUVBL2 and HBx,which may have important anti-HBV functions.RUVBL2 might play this role through the same mechanism.It acts in the in fluenza A virus,but the underlying mechanism is still unclear and needs further investigation.

In summary,we studied the biological functions of HBx protein,and found that HBV replication was significantly inhibited by the knockdown of HBx in vitro.Using a global quantitative proteomic approach,we identified 12 proteins with altered expression after treatment with HBx siRNA.These findings extend our knowledge and deepen understanding of the molecular events of HBV replication mediated by HBx.This is only the first step of our study; more work is needed to reveal the underlying mechanisms of particular proteins in HBV pathogenesis,and identify novel targets for anti-HBV therapeutic intervention.

Funding:This study was supported by grants from the Zhejiang Provincial Natural Science Foundation (Y207465) and the National Basic Research Program of China (973 Program) (2009CB522403).

Ethical approval:Not needed.

Contributors:XHY and ZSS proposed the study.XHY and XCY wrote the first draft.All authors contributed to the design and interpretation of the study and to further drafts.ZSS is the guarantor.

Competing interest:No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

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