Cellular apoptosis induced by replication of hepatitis B virus: possible link between viral genotype and clinical outcome
© Lu et al; licensee BioMed Central Ltd. 2007
Received: 05 September 2007
Accepted: 31 October 2007
Published: 31 October 2007
HBV remains one of the major pathogens of liver diseases but the outcomes as inflammation, cirrhosis and cancer of the liver are greatly related to different viral genotypes. The aim of this study was to assess the pro-apoptotic effect of HBSP from three HBV genotypes on liver derived cells. HepG2 cells were applied in our system and transfected by HBV genotype A, B, and C. Cells were observed under phase contrast microscope, stained by apoptosis marker and analyzed by flow cytometre. HBSP expression was detected by western blot assay. BH3 sequences were aligned and analyzed by Vector NTI. HBV genotypes A, B, and C transfected cells displayed evidence of cell death which was further proved as apoptosis. Natural expression of a pro-apoptotic protein HBSP was detected during genomes transfection. The different apoptotic effects were correlated to the HBSP expression from each genome. Alignment and analysis of the BH3 domains from the three genomes revealed slight variance which might also contribute to the result. Our results suggested that variant HBSP expression and BH3 sequence of HBV genotypes may be involved in differential apoptotic effect in transfected cells. Detailed analysis of the role of HBV genotypes in cellular apoptotic process should provide molecular information on the reported clinical outcome of infection by different HBV genotypes.
Hepatitis B virus (HBV), with eight genotypes (A-H) based on sequence divergence, is one of the global health threats with over 400 million people currently infected .
Outcome of the infection includes viral hepatitis, liver fibrosis or cirrhosis and ultimate hepatocellular carcinoma (HCC). Genotypes with distinct geographic distribution lead to different clinic manifestations. Genotype B is more inclined to develop HCC, whereas genotype A and C cause hepatitis and cirrhosis more that cancer . Viral hepatitis is characterized by diffused inflammatory reaction and associated with cell damage and death . The mechanisms of cell damage are generally defined as the result of a cytotoxic-T lymphocyte (CTL) mediated immune response against the viral infection [4, 5]. Another typical process causing cell death is apoptosis, the programmed cell death . HBV viral proteins, such as HBx and HBSP, have been proved able to induce apoptosis [7, 8]. This regulated apoptosis might be the strategies developed by virus in order to maximize the production of virus progeny and promote the spread to neighboring cells. However, HBV was yet confirmed to directly cause hepatocyte death.
It has been reported that the mitochondria-dependent apoptotic pathway which is governed by Bcl-2 family of proteins is involved in the development of liver diseases [9, 10]. The Bcl-2 family of proteins is defined as the key regulator of apoptosis in the mitochondria-dependent way. They consist of both suppressors and promoters of apoptosis. Four conserved domains within the Bcl-2 family of proteins have been identified through sequence comparisons and named as Bcl2-homology (BH) domains 1–4, particularly, the BH3 domain promotes cell death in most occasions . Recent reports have identified Bcl2-homology domain 3 (BH3) in HBx and HBSP which cast light on how the HBV viral proteins are involved in apoptosis at molecular level [7, 8]. The apoptosis induced by the viral proteins might help the dissemination of viral particles with less host immune neutralization.
In this study we reported evidence of direct cell death caused by HBV genome A, B and C after transfection in HepG2 cells. The transfected cells showed characteristics of cellular apoptosis supported by FACS analysis. Further investigation identified the natural expression of HBSP in HBV genome transfected cells. The observed difference in apoptotic effect caused by the three HBV genotypes revealed different HBSP expression in them. BH3 domain sequence analysis revealed the existence of some variance in the three HBSP proteins which might contribute to the result. The significance of our findings was discussed.
Materials and methods
Cell culture and transfection
HepG2 cells (ATCC, USA) were cultured in DMEM (Gibco Dulbecco, Invitrogen Inc., USA) with 5% fetal bovine serum (Invitrogen Inc., USA) and 5% CO2. Effectene transfection reagent was applied to transiently expressed proteins in HepG2 cells. The cells were tranfected with plasmids when 50% confluency was reached. Transfected cells were maintained at 37°C and examined according to the experiments.
Vector pcDNA3.1(+) containing the replicative HBV genome A, B, and C were transiently transfected into 5 × 105 HepG2 cells, respectively. HepG2 cells transfected with empty vector and cells treated by 50 μM cisplatin for 16 hr were set as (-) and (+) controls. Transfected cells were collected at 24 hr and 48 hr after incubation and analyzed by Apoalert™ annexin-V kit (BD, Biosciences, USA). Cells were rinsed in 100 μl binding buffer and stained with 5 μl annexin-V-FITC and 10 μl propidium iodide (PI). Samples were analyzed on FACS station to determine the apoptotic cell portion after 30 min incubation.
Western blot analysis
HBSP polyclonal anti-serum was acquired by boosting rabbit using an internal HBSP peptide: CDLNLGQDQQQPVRD (Biogenes, Germany). HBSP protein was detected by primary anti-HBSP antibody in 1:1000 dilution and secondary anti-rabbit antibody conjugated with horseradish peroxidase (Pierce, USA) in 1:5000 dilution. Following ECL detection (Pierce, USA) membrane was developed by Kodak E&D system (Kodak, USA).
HBSP amino acid sequences were analyzed by using Vector NTI9. Result was generated and compared with other BH3 domains.
Results and discussion
A cell-based system for HBV genome A, B, and C replication was generated by cloning the linearized genome in the vector pcDNA3.1 . It has been shown to produce replicative viral particles into the culture medium . This system was selected to investigate the effect of HBV genomes on the cells.
Alignment analysis of BH3 domain in HBSPs
This study is first to describe HBV genotype A, B, and C lead to apoptosis in HepG2 cell and the slight difference was related to HBSP expression and its property. During the last two decades, it has been widely believed that HBV does not directly cause cell death in host cells [4, 5]. Our finding raised the idea that HBV can cause apoptosis with its viral proteins [7, 8]. For viruses to avoid the host clearance during the early infection stage they have evolved anti-apoptotic proteins to prevent the host cell from elimination, such as BHRF1 of EBV and E1B19k of Adenovirus [19, 20]. On the other hand, viruses also developed pro-apoptotic mechanisms in the late stage of infection to break host cell and promote the spread of viral progeny, like VPR of HIV . It is therefore not surprising that HBV causes apoptosis. HBV chronic infection is considered the main cause of liver cirrhosis and cancer . HBV Genotype B is more related to HCC, whereas genotype A and C are more inclined to cause cirrhosis . This may be related to the severity of persistent HBV infection which determines the infected cell amount. Our finding of the genotype B expresses more HBSP than the other two genotypes and caused higher apoptotic effect supported this hypothesis since it facilitates the spread of viral progeny to infect more healthy cells. Considering the high regeneration capacity of liver cells, it is also possible that an extensive apoptosis would result in a higher level of liver cell proliferation in a regeneration effort. Such an increase in cell division may perturb the normal cell cycle control, resulting in an accumulation of mutations in the genome of progeny cells which ultimately contribute to HCC development. In conclusion, the present study showed the important role of HBSP in HBV induced apoptosis and it determined the variant outcome of different genotypes which might be related to the clinic outcomes.
This work was supported by grant 03/1/22/18/229 (WN Chen) from the Biomedical Research Council, Agency for Science, Technology and Research, Singapore. YW Lu and TL Tan were recipients of the graduate scholarship from Nanyang Technological University.
- Zuckerman AJ: More than third of world's population has been infected with Hepatitis B virus. BMJ 1999, 318: 1213.PubMedPubMed CentralView ArticleGoogle Scholar
- Enomoto M, Tamori A, Nishiguchi S: Hepatitis B virus genotypes and response to antiviral therapy. Clin Lab 2006,52(1–2):43-47.PubMedGoogle Scholar
- Lau JY, Xie X, Lai MM, Wu PC: Apoptosis and viral hepatitis, Semin. Liver Dis 1998, 18: 169-176.View ArticleGoogle Scholar
- Lowin B, Hahne M, Mattmann C, Tschopp J: Cytolytic T-cell cytotoxicity is mediated through perforin and Fas lytic pathways. Nature 1994, 370: 650-653. 10.1038/370650a0PubMedView ArticleGoogle Scholar
- Brechot C: Pathogenesis of hepatitis B virus-related hepatocellular carcinoma: old and new paradigms. Gastroenterology 2004,127(Suppl 1):S56-S61. 10.1053/j.gastro.2004.09.016PubMedView ArticleGoogle Scholar
- Adams JM, Cory S: The Bcl-2 family: arbiters of cell survival. Science 1998, 281: 1322-1326. 10.1126/science.281.5381.1322PubMedView ArticleGoogle Scholar
- Lu YW, Chen WN: Human hepatitis B virus X protein induces apoptosis in HepG2 cells: role of BH3 domain. Biochem & Biophy Res Com 2005, 338: 1551-1556. 10.1016/j.bbrc.2005.10.117View ArticleGoogle Scholar
- Lu YW, Tan TL, Chan V, Chen WN: The HBSP gene is expressed during HBV replication, and its coded BH-3-containing spliced viral protein induces apoptosis in HepG2 cells. Biochem & Biophy Res Com 2006, 351: 64-70. 10.1016/j.bbrc.2006.10.002View ArticleGoogle Scholar
- Chen GG, Lai PB, Chan PK, Chak EC, Yip JH, Ho RL, Leung BC, Lau WY: Decreased expression of Bid in human hepatocellular carcinoma is related to hepatitis B virus X protein. Eur J Cancer 2001, 37: 1695-1702. 10.1016/S0959-8049(01)00182-4PubMedView ArticleGoogle Scholar
- Ehrmann J, Galuszkova D, Krc I, Jezdinska V, Vojtesek B, Murray PG, Kolao Z: Apoptosis-related proteins, Bcl-2, Bax, Fas, Fas-L and PCNA in liver biopsies of patients with chronic hepatitis B virus infection. Oncol Res 2000, 6: 130-135.View ArticleGoogle Scholar
- Kelekar A, Thomson CB: Bcl-2 family proteins: the role of the BH3 domain in apoptosis. Trends Cell Biol 1998, 8: 324-329. 10.1016/S0962-8924(98)01321-XPubMedView ArticleGoogle Scholar
- Chen WN, Oon CJ, Toh I: Altered antigenicities of hepatitis B virus surface antigen carrying mutations outside the common "a" determinant. Am J Gastroenterol 2000, 95: 1098-1099.PubMedGoogle Scholar
- Oon CJ, Chen WN, Goh KT, Mesenas S, Ng HS, Chiang G, Tan C, Koh S, Teng SW, Toh I, Moh MC, Goo KS, Tan K, Leong AL, Tan GS: Molecular characterization of hepatitis B virus surface antigen mutants in Singapore patients with hepatocellular carcinoma and hepatitis B virus carriers negative for HBsAg but positive for anti-HBs and anti-HBc. J Gastroenterol Hepatol 2002,17(Suppl):S491-S496. 10.1046/j.1440-1746.17.s4.16.xPubMedView ArticleGoogle Scholar
- Lee AT, Ren J, Wong ET, Ban KH, Lee LA, Lee CG: The hepatitis B virus X protein sensitizes HepG2 cells to UV light-induced DNA damage. J Biol Chem 2005, 280: 33525-33535. 10.1074/jbc.M506628200PubMedView ArticleGoogle Scholar
- Huang HL, Jeng KS, Hu CP, Tsai CH, Lo SJ, Chang C: Identification and characterization of a structural protein of hepatitis B virus: a polymerase and surface fusion protein encoded by a spliced RNA. Virology 2000, 275: 398-410. 10.1006/viro.2000.0478PubMedView ArticleGoogle Scholar
- Soussan P, Garreau F, Zylberberg H, Ferray C, Brechot C, Kremsdorf D: In vivo expression of a new hepatitis B virus protein encoded by a spliced RNA. J Clin Invest 2005, 105: 55-60.View ArticleGoogle Scholar
- Soussan P, Tuveri R, Nalpas B, Garreau F, Zavala F, Masson A, PolS , Brechot C, Kremsdorf D: The expression of hepatitis B spliced protein (HBSP) encoded by a spliced hepatitis B virus RNA is associated with viral replication and liver fibrosis. J Hepatol 2003, 38: 343-348. 10.1016/S0168-8278(02)00422-1PubMedView ArticleGoogle Scholar
- Sattler M, Liang H, Nettesheim D, Meadows RP, Harlan JE, Eberstadt M, Yoon HS, Shuker SB, Chang BS, Minn AJ, Thompson CB, Fesik SW: Structure of Bcl-xl-Bak peptide complex: Recognition between regulators of apoptosis. Science 1997, 275: 983-986. 10.1126/science.275.5302.983PubMedView ArticleGoogle Scholar
- Kim ND, Chae HS, Oh ST, Kang JH, Park CH, Park WS, Takada K, Lee JM, Lee WK, Lee SK: Expression of viral microRNAs in Epstein-Barr virus-associated gastric carcinoma. J Virol 2007,81(2):1033-1036. 10.1128/JVI.02271-06PubMed CentralView ArticleGoogle Scholar
- Matsushita T, Okada T, Inaba T, Mizukami H, Ozawa K, Colosi P: The adenovirus E1A and E1B19K genes provide a helper function for transfection-based adeno-associated virus vector production. J Gen Virol 2004,85(Pt 8):2209-2214. 10.1099/vir.0.79940-0PubMedView ArticleGoogle Scholar
- Kaminska M, Francin M, Shalak V, Mirande M: Role of HIV-1 Vpr-induced apoptosis on the release of mitochondrial lysyl-tRNA synthetase. FEBS Lett 2007,581(16):3105-3110. 10.1016/j.febslet.2007.05.076PubMedView ArticleGoogle Scholar
- Liu CJ, Kao JH: Hepatitis B virus-related hepatocellular carcinoma: epidemiology and pathogenic role of viral factors. J Chin Med Assoc 2007,70(4):141-145.PubMedView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.