Proteomic analysis of cellular protein alterations using a hepatitis B virus-producing cellular model

Hepatitis B virus (HBV) is one of the major etiological factors responsible for acute and chronic liver disease and for the development of hepatocellular carcinoma (HCC). To determine the effects of HBV replication on host cell-protein expression, we utilized 2-DE and MS/MS analysis to compare and identify differentially expressed proteins between an HBV-producing cell line HepG2.2.15 and its parental cell line HepG2. Of the 66 spots identified as differentially expressed (+/- over twofold, p <0.05) between the two cell lines, 62 spots (corresponding to 61 unique proteins) were positively identified by MS/MS analysis. These proteins could be clearly divided into three major groups by cluster and metabolic/signaling pathway analysis: proteins involved in retinol metabolism pathway, calcium ion-binding proteins, and proteins associated with protein degradation pathways. Other proteins identified include those that function in diverse biological processes such as signal transduction, immune regulation, molecular chaperone, electron transport/redox regulation, cell proliferation/differentiation, and mRNA splicing. In summary, we profiled proteome alterations between HepG2.2.15 and HepG2 cells. The proteins identified in this study would be useful in revealing the mechanisms underlying HBV-host cell interactions and the development of HCC. This study can also provide some useful clues for antiviral research.     

Protein profile in hepatitis B virus replicating rat primary hepatocytes and HepG2 cells by iTRAQ‐coupled 2‐D LC‐MS/MS analysis: Insights on liver angiogenesis

Hepatitis B virus (HBV) infection and in particular Hepatitis B Virus X Protein have been shown to modulate angiogenesis. However, a comprehensive and coordinated mechanism in the HBV‐induced angiogenesis remains to be established. In this study, transient transfection of replicative HBV genome was carried out in rat primary hepatocytes (RPHs) as well as HepG2 cells. Angiogenesis was assessed by tube formation assay. 2‐D LC‐MS/MS analysis was used to detect differentially expressed proteins in cells, supporting HBV replication compared with those transfected with the empty vector. A cell‐based HBV replication was established in both RPHs and HepG2 cells. HBV replication‐induced angiogenesis was indicated by tube formation of endothelial cells cultured in condition medium from RPHs or HepG2 cells supporting HBV replication. Enzymes associated with angiogenesis, namely fumarate hydratase and tryptophanyl‐tRNA synthetase, were identified by 2‐D LC‐MS/MS analysis in HBV replicating RPHs and HepG2 cells. Our results indicated that the application of quantitative proteomics based on iTRAQ can be an effective approach to evaluate the effects of HBV replication on liver angiogenesis. The angiogenesis‐associated proteins identified in our study may eventually lead to novel anti‐angiogenic hepatocellular carcinoma cancer therapy based on tumor vascular targeting or be the markers for hepatocellular carcinoma diagnosis.     

Protein alteration of HepG2.2.15 cells induced by iron overload

Hepatitis B can progress into hepatocellular carcinoma. Body irons may interfere with the clearance of hepatitis B virus (HBV) and contribute to genesis of tumor. To investigate the role of iron played in HBV-related pathogenesis, here we studied the effect of iron with different concentrations and valence states on growth of HepG2.2.15 cells and secretion of virus proteins. A strong tolerance of HepG2.2.15 cells to iron challenge was found. The concentration of hepatitis B surface antigen in cell culture medium was decreased after iron stimulation. Lower concentrations of iron facilitated hepatitis B e-antigen (HBeAg) secretion. Fe(2+) appeared more effective on HBeAg secretion than Fe(3+) did. In parallel, the differential protein profiles in HepG2.2.15 cells were studied by iTRAQ and LC-MS/MS. The differentially expressed proteins were mainly involved in stress response, signal transduction, apoptosis, etc. Four proteins (14-3-3 β/α, VCP, migration inhibitory factor, and Nup153) were verified by Western-blotting and found to be consistent with the iTRAQ data. Interestingly, nuclear import of Nuclear factor kappa B (NFκB) and its activity were found to be affected by the decreased Nup153 in iron stimulated HepG2.2.15 cells. The results may indicate possible molecular mechanism how the synergism of HBV and iron stimulation damages host liver cells.     

Proteome responses to stable hepatitis B virus transfection and following interferon alpha treatment in human liver cell line HepG2

Hepatitis B virus (HBV) infection is a worldwide health problem and may develop to liver fibrosis, cirrhosis, and even hepatocellular carcinoma. To investigate the global proteome responses of liver‐derived cells to HBV infection and IFNα treatment, 2‐DE and MS‐based analysis were performed to compare the proteome changes between HBV stably transfected cell line HepG2.2.15 and its parental cell line HepG2, as well as HepG2.2.15 before and after IFNα treatment (5000 IU/mL for 72 h). Compared to HepG2, 12 of 18 down‐regulated and 27 of 32 up‐regulated proteins were identified in HepG2.2.15. After IFNα treatment, 6 of 7 down‐regulated and 11 of 14 up‐regulated proteins were identified. Differentially expressed proteins caused by HBV infection were involved with cytoskeletal matrix, heat shock stress, kinases/signal transduction, protease/proteasome components, etc. Prohibitin showed a dose‐dependent up‐regulation during IFNα treatment and might play a potent role in anti‐HBV activities of IFNα by enhancing the crossbinding p53 expression to achieve the apoptosis of HBV infected liver cells. Down‐regulation of interferon‐stimulated gene 15 (ISG15) in HepG2.2.15 and recovery by IFNα suggested its relationship with IFNα's anti‐HBV effect.     

iTRAQ‐coupled 2‐D LC‐MS/MS analysis of protein profile associated with HBV‐modulated DNA methylation

The development of hepatocellular carcinoma (HCC) is believed to be associated with multiple risk factors, including the infection of hepatitis B virus (HBV). Based on the analysis of individual genes, evidence has indicated the association between HCC and HBV and has also been expanded to epigenetic regulation, with an involvement of HBV in the DNA methylation of the promoter of cellular target genes leading to changes in their expression. Proteomic study has been widely used to map a comprehensive protein profile, which in turn could provide a better understanding of underlying mechanisms of disease onset. In the present study, we performed a proteomic profiling by using iTRAQ‐coupled 2‐D LC/MS‐MS analysis to identify cellular genes down‐regulated in HBV‐producing HepG2.2.15 cells compared with HepG2 cells. A total of 15 proteins including S100A6 and Annexin A2 were identified by our approach. The significance of these cellular proteins as target of HBV‐mediated epigenetic regulation was supported by our validation assays, including their reactivation in cells treated with 5‐aza‐2′‐deoxycytidine (a DNA methyltransferase inhibitor) by real‐time RT‐PCR and Western blot analysis, as well as the DNA methylation status analysis by bisulfite genome sequencing. Our approach provides a comprehensive analysis of cellular target proteins to HBV‐mediated epigenetic regulation and further analysis should facilitate a better understanding of its involvement in HCC development.     

Two-dimensional blue native/SDS-PAGE analysis reveals heat shock protein chaperone machinery involved in hepatitis B virus production in HepG2.2.15 cells

Hepatitis B virus (HBV) infection is a major health concern with more than two billion individuals currently infected worldwide. Despite the prevalence of infection, gaining a complete understanding of the molecular mechanisms of HBV infection has been difficult because HBV cannot infect common immortalized cell lines. HepG2.2.15, however, is a well established version of the HepG2 cell line that constitutively expresses HBV. Therefore, comparative proteomics analysis of HepG2.2.15 and HepG2 may provide valuable clues for understanding the HBV virus life cycle. In this study, two-dimensional blue native/SDS-PAGE was utilized to characterize different multiprotein complexes from whole cell lysates between HepG2.2.15 and HepG2. These results demonstrate that two unique protein complexes existed in HepG2.2.15 cells. When these complexes were excised from the gel and subjected to the second dimension separation and the proteins were sequenced by mass spectrometry, 20 non-redundant proteins were identified. Of these proteins, almost 20% corresponded to heat shock proteins, including HSP60, HSP70, and HSP90. Antibody-based supershift assays were used to verify the validity of the distinct protein complexes. Co-immunoprecipitation assays confirmed that HSP60, HSP70, and HSP90 proteins physically interacted in HepG2.2.15 but not HepG2 cells. We further demonstrated that down-regulation of HSP70 or HSP90 by small interfering RNA significantly inhibited HBV viral production but did not influence cellular proliferation or apoptosis. Consistent with these results, a significant reduction in HepG2.2.15 HBV secretion was observed when the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin was used to treat HepG2.2.15 cells. Collectively these results suggest that the interaction of HSP90 with HSP70/HSP60 contributes to the HBV life cycle by forming a multichaperone machine that may constitute therapeutic targets for HBV-associated diseases.     

Differential cellular gene expression induced by hepatitis B and C viruses

Hepatitis B virus (HBV) is a hepatotropic virus that causes acute and chronic hepatocellular injury and hepatocellular carcinoma. To clarify how HBV proteins regulate host cellular gene expression, we used our in-house cDNA microarray and HepG2.2.15 cells, which are derived from HepG2 cells and produce all HBV proteins. Of 2304 genes investigated, several genes were differentially expressed in HepG2.2.15 cells compared with HepG2 cells. These genes included insulin-like growth factor II and alpha-fetoprotein, consistent with previous reports. Furthermore, we previously performed similar microarray analyses to clarify the effects of hepatitis C virus (HCV) proteins on host cells, using a HepG2-derivative cell line, which produces all HCV proteins. Using these two microarray results, we compared the differences in cellular gene expression induced by HBV and HCV proteins. The expression of the majority of genes investigated differed only slightly between HBV and HCV protein-producing cells. However, HBV and HCV proteins clearly regulated several genes in a reciprocal manner. Combined, these microarray results shed new light on the effects of HBV proteins on cellular gene expression and on the differences in the pathogenic activities of these two hepatitis viruses.     

Proteomic investigation of 5-fluorouracil resistance in a human hepatocellular carcinoma cell line

Multi-drug resistance (MDR) is a major obstacle towards a successful treatment of hepatocellular carcinoma (HCC). The mechanisms of MDR are intricate and have not been fully understood. Therefore, we employed a cell-line model consisting of the 5-fluorouracil (5-FU) resistant BEL7402/5-FU cell line and its parental BEL7402 cell line. Using relative and absolute quantification (iTRAQ)-coupled 2D LC-MS/MS, a successfully exploited high-throughput proteomic technology, in total, 660 unique proteins were identified and 52 proteins showed to be differentially expressed in BEL7402/5-FU compared with BEL7402. Several differentially expressed proteins were further validated by Western blot and real-time quantitative RT-PCR analysis. Furthermore, the association of MDR with ANXA3, one of the highly expressed proteins in BEL7402/5-FU, was verified. Our study represents the first successful application of iTRAQ technology for MDR mechanisms analysis in HCC. Many of the differentially expressed proteins identified had not been linked to MDR in HCC before, which provide valuable information for further understanding of MDR.     

Glucose-regulated Protein 78 Is an Intracellular Antiviral Factor against Hepatitis B Virus

Hepatitis B virus (HBV) infection is a global public health problem that plays a crucial role in the pathogenesis of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. However, the pathogenesis of HBV infection and the mechanisms of host-virus interactions are still elusive. In this study, two-dimensional gel electrophoresis and mass spectrometry-based comparative proteomics were applied to analyze the host response to HBV using an inducible HBV-producing cell line, HepAD38. Twenty-three proteins were identified as differentially expressed with glucose-regulated protein 78 (GRP78) as one of the most significantly up-regulated proteins induced by HBV replication. This induction was further confirmed in both HepAD38 and HepG2 cells transfected with HBV-producing plasmids by real time RT-PCR and Western blotting as well as in HBV-infected human liver biopsies by immunohistochemistry. Knockdown of GRP78 expression by RNA interference resulted in a significant increase of both intracellular and extracellular HBV virions in the transient HBV-producing HepG2 cells concomitant with enhanced levels of hepatitis B surface antigen and e antigen in the culture medium. Conversely overexpression of GRP78 in HepG2 cells led to HBV suppression concomitant with induction of the positive regulatory circuit of GRP78 and interferon-β1 (IFN-β1). In this connection, the IFN-β1-mediated 2′,5′-oligoadenylate synthetase and RNase L signaling pathway was noted to be activated in GRP78-overexpressing HepG2 cells. Moreover GRP78 was significantly down-regulated in the livers of chronic hepatitis B patients after effective anti-HBV treatment (p = 0.019) as compared with their counterpart pretreatment liver biopsies. In conclusion, the present study demonstrates for the first time that GRP78 functions as an endogenous anti-HBV factor via the IFN-β1-2′,5′-oligoadenylate synthetase-RNase L pathway in hepatocytes. Induction of hepatic GRP78 may provide a novel therapeutic approach in treating HBV infection.Hepatitis B virus infection is a global public health problem. An estimated 2 billion (one-third of the world''s population) people are infected with HBV¹ worldwide, and more than 400 million are chronic hepatitis B (CHB) carriers (1). Epidemiological studies have shown that HBV infection is one of the major risk factors for chronic hepatitis, liver fibrosis, and hepatocellular carcinoma (HCC). Every year, over 1 million people die of HBV-related liver diseases, 30–50% of which are attributed to HCC (2). In China, more than 130 million (10% of the national population) people are suffering from CHB (3), and HCC has been ranked as the second major cause of cancer-related death since 1990 (4). However, the limited efficacy of antiviral therapies, high rates of post-treatment HBV relapse, and the emergence of drug-resistant viral mutants have greatly hindered the effective management of CHB infection. Therefore, it is of prime importance to understand the mechanisms of HBV-host interactions during malignant transformation in CHB infection to identify novel therapeutic anti-HBV targets.Because human HBV is incapable of infecting hepatocytes in vitro efficiently and the availability of reliable in vitro culture systems that favor HBV replication is limited, the pathogenetic studies of HBV and the development of anti-HBV drugs have long been hampered. HepAD38 and HepG2.2.15, both of which are derived from HepG2 cells and integrated with a greater than 1-unit-length HBV genome, have been widely accepted and are well established cell lines for the study of the HBV life cycle and screening potential HBV inhibitors since the late 1990s (5, 6). Recently comparative proteomics analysis of the HBV-expressing HepG2.2.15 cells and the parental HepG2 cells has been performed in two independent laboratories to characterize the altered proteome profile induced by HBV (7, 8). However, the different genetic backgrounds of HepG2.2.15 and HepG2 may lead to an inaccurate evaluation of the impact of HBV replication on host genes. When compared with HepG2.2.15 cells, which produce HBV particles in a continuous manner, HepAD38 cells produce higher levels of HBV DNA in a controllable and inducible way (5). HBV production in HepAD38 is under the strict control of a tetracycline-responsive promoter; therefore, a direct comparison of cellular characteristics with or without HBV replication in HepAD38 is easily achieved. To date, changes in the proteome profile of HepAD38 induced by HBV replication have not been reported. In this study, we performed comparative proteomics to globally analyze the host response to HBV by using an inducible HBV-producing cell line, HepAD38. The combination of two-dimensional gel electrophoresis (2-DE) and MALDI-TOF MS revealed that 23 cellular proteins were differentially expressed when HBV replicated. Among them, GRP78, which was one of the most highly up-regulated proteins, was further selected for functional assessment.     

Quantitative proteome analysis of ovarian cancer tissues using a iTRAQ approach

Quantitative proteomics can be used as a screening tool for identification of differentially expressed proteins as potential biomarkers for cancers. Here, we comparatively analyzed the proteome profiles of ovarian cancer tissues and normal ovarian epithelial tissues. Using the high‐throughput proteomic technology of isobaric tags for relative and absolute quantitation (iTRAQ)‐coupled with two‐dimensional‐liquid chromatography‐tandem mass spectrometry, 1,259 unique proteins were identified. Of those, 205 were potentially differentially expressed between ovarian cancer and normal ovarian tissues. Several of the potentially differentially expressed proteins were validated by Western blotting and real‐time quantitative RT‐PCR analyses. Furthermore, up‐regulation of KRT8, PPA1, IDH2, and S100A11 were validated in ovarian tissue microarrays by immunohistochemistry. Silencing of S100A11 expression suppressed the migration and invasion properties of ovarian cancer cells in vitro. Our study represents the successful application of iTRAQ technology to an investigation of ovarian cancer. Many of the potentially differentially expressed proteins identified had not been linked to ovarian cancer before, and provide valuable novel insights into the underlying mechanisms of carcinogenesis in human ovarian cancer. J. Cell. Biochem. 113: 3762–3772, 2012. © 2012 Wiley Periodicals, Inc.     

Antisense oligonucleotides targeting abhydrolase domain containing 2 block human hepatitis B virus propagation

Hepatitis B virus (HBV) infection is a major health concern worldwide and only a minority of treated patients develop a sustained protective response following a short course of therapy, and most patients require prolonged treatment to suppress viral replication. However, several recent reports showed that inhibition of certain host cell proteins prevented viral infection, specifically the human abhydrolase domain containing 2 (ABHD2) has been confirmed by our previous study to be upregulated in HepG2.2.15 cells but downregulated by lamivudine. These observations suggested that ABHD2 was important for HBV propagation and could be a target of novel anti-HBV drugs. To assess the importance of ABHD2 to the HBV infection process, antisense oligonucleotides (ASODNs) were used to downregulate ABHD2 expression in HepG2.2.15 cells. From 5 ASODNS candidates tested, AB3 significantly downregulated ABHD2 mRNA and protein expression levels. Further, AB3 significantly reduced HBV DNA, hepatitis B surface antigen, and hepatitis B "e" antigen protein expression levels in cell medium without affecting cell viability. These results suggest that downregulation of ABHD2 using ASODNs blocked HBV replication and expression without affecting host cell physiology. Further, data demonstrated an essential role of ABHD2 in HBV propagation, suggesting it can serve as a novel target for anti-HBV drug development.     

Quantitative Proteomics Analysis of the Hepatitis C Virus Replicon High-Permissive and Low-Permissive Cell Lines

Chronic hepatitis C virus (HCV) infection is one of the leading causes of severe hepatitis. The molecular mechanisms underlying HCV replication and pathogenesis remain unclear. The development of the subgenome replicon model system significantly enhanced study of HCV. However, the permissiveness of the HCV subgenome replicon greatly differs among different hepatoma cell lines. Proteomic analysis of different permissive cell lines might provide new clues in understanding HCV replication. In this study, to detect potential candidates that might account for the differences in HCV replication. Label-free and iTRAQ labeling were used to analyze the differentially expressed protein profiles between Huh7.5.1 wt and HepG2 cells. A total of 4919 proteins were quantified in which 114 proteins were commonly identified as differentially expressed by both quantitative methods. A total of 37 differential proteins were validated by qRT-PCR. The differential expression of Glutathione S-transferase P (GSTP1), Ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1), carboxylesterase 1 (CES1), vimentin, Proteasome activator complex subunit1 (PSME1), and Cathepsin B (CTSB) were verified by western blot. And over-expression of CTSB or knock-down of vimentin induced significant changes to HCV RNA levels. Additionally, we demonstrated that CTSB was able to inhibit HCV replication and viral protein translation. These results highlight the potential role of CTSB and vimentin in virus replication.     

2’,3’-Cyclic Nucleotide 3’-Phosphodiesterases Inhibit Hepatitis B Virus Replication

2’,3’-cyclic nucleotide 3’-phosphodiesterase (CNP) is a member of the interferon-stimulated genes, which includes isoforms CNP1 and CNP2. CNP1 is locally expressed in the myelin sheath but CNP2 is additionally expressed at low levels outside the nervous system. CNPs regulate multiple cellular functions and suppress protein production by association with polyadenylation of mRNA. Polyadenylation of Hepatitis B virus (HBV) RNAs is crucial for HBV replication. Whether CNPs interact with polyadenylation signal of HBV RNAs and interfere HBV replication is unknown. In this study, we evaluated expressions of CNP isoforms in hepatoma cell lines and their effects on HBV replication. We found that CNP2 is moderately expressed and gently responded to interferon treatment in HepG2, but not in Huh7 cells. The CNP1 and CNP2 potently inhibited HBV production by blocking viral proteins synthesis and reducing viral RNAs, respectively. In chronic hepatitis B patients, CNP was expressed in most of HBV-infected hepatocytes of liver specimens. Knockdown of CNP expression moderately improved viral production in the HepG2.2.15 cells treated with IFN-α. In conclusion, CNP might be a mediator of interferon-induced response against HBV.     

Stable expression and integrated hepatitis B virus genome in a human hepatoma cell line

HepG2.2.15 cell is a widely used cell model for studying HBV (hepatitis B virus) in vitro. In these cells, the HBV genome is integrated in several sites of HepG2 cellular DNA. These multiple copies may have some influence on the cellular processes. We constructed a new plasmid, pSEH-Flag-HBV, and transfected it into HepG2 cells, and then screened it with hygromycin. We then used ELISA, PCR, and RT-PCR to detect the expression of HBV in these cell lines. A cell line that stably expressed hepatitis B e antigen (HBeAg) and hepatitis B surface antigen (HBsAg) was established. Using Southern blotting analysis, we found that the HBV genome was integrated as a single copy in the cellular DNA. This cell line will be a useful alternative model for HBV studies.     

Two-dimensional liquid chromatography-tandem mass spectrometry coupled with isobaric tags for relative and absolute quantification (iTRAQ) labeling approach revealed first proteome profiles of pulmonary alveolar macrophages infected with porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome (PRRS) has devastated the pig industry worldwide for almost 25 years, and its virus (PRRSV) preferentially infects and replicates in pulmonary alveolar macrophages (PAMs). To discover cellular protein responses in PRRSV-infected PAMs, two-dimensional liquid chromatography-tandem mass spectrometry coupled with isobaric tags for relative and absolute quantification (iTRAQ) labeling was employed to quantitatively identify the differentially expressed proteins between the PRRSV-infected groups and the controls. A total of 160 cellular proteins in PAMs that were significantly altered post-infection were identified. These differentially expressed proteins are related to the biological processes of virus binding, cell structure, signal transduction, cell adhesion, etc., and their interactions. This is the first report that analyzed the cellular protein profile of PRRSV-infected PAMs using iTRAQ technology, and this data provides important information to help understand the host response to PRRSV and to define the cellular requirements for the underlying mechanism of PRRSV replication and pathogenesis.     

Proteome analysis of porcine epidemic diarrhea virus (PEDV)‐infected Vero cells

Porcine epidemic diarrhea virus (PEDV) causes an acute, highly contagious, and devastating viral enteric disease with a high mortality rate in suckling pigs. A large‐scale outbreak of PED occurred in China in 2010, with PEDV emerging in the United States in 2013 and spreading rapidly, posing significant economic and public health concerns. In this study, LC–MS/MS coupled to iTRAQ labeling was used to quantitatively identify differentially expressed cellular proteins in PEDV‐infected Vero cells. We identified 49 differentially expressed cellular proteins, of which 8 were upregulated and 41 downregulated. These differentially expressed proteins were involved in apoptosis, signal transduction, and stress responses. Based on these differentially expressed proteins, we propose that PEDV might utilize apoptosis and extracellular signal regulated kinases pathways for maximum viral replication. Our study is the first attempt to analyze the protein profile of PEDV‐infected cells by quantitative proteomics, and we believe our findings provide valuable information with respect to better understanding the host response to PEDV infection.