1887

Abstract

Infections with Basal Core Promoter (BCP) (A1762T/G1764A) and Pre-Core (PC) (G1896A) hepatitis B virus HBeAg mutants are associated with severe liver injury. We analysed host cell responses in HepG2/C3A, hepatoma cells transfected with infectious clones developed from genotype D wild type (WT) and BCP/PC mutant (MT) viruses isolated from an acute resolved and an acute liver failure hepatitis B case respectively. Cells transfected with MT virus construct showed ~55 % apoptosis and with WT ~30 % apoptosis at 72 h. To determine possible roles of HBe and HBx proteins in apoptosis, we cloned these genes and co-transfected cells with WT+HBe/HBx or MT+HBe/HBx constructs. Co-expression of HBe protein improved cell viability significantly in both WT and MT virus constructs, indicating an important role of HBe in protecting cells. RNA sequencing analysis carried out at 12 and 72 h post-transfection with WT virus construct showed enrichment of innate/adaptive immune response-activating signal transduction, cell survival and amino acid/nucleic acid biosynthetic pathways at 12 and 72 h. By contrast, MT virus construct showed enrichment in host defence pathways and some biosynthetic pathways at the early time point (12 h), and inflammatory response, secretary granule, regulation of membrane potential and stress response regulatory pathways at the late time point (72 h). There was a significant down-regulation of genes involved in endoplasmic reticulum and mitochondrial functions and metabolism with MT construct and this possibly led to induction of apoptosis in cells. Considering rapid apoptotic changes in cells transfected with MT construct, it can be speculated that HBeAg plays a crucial role in cell survival. It enhances induction of metabolic and synthetic pathways and facilitates management of cellular stress that is induced due to hepatitis B virus infection/replication.

Funding
This study was supported by the:
  • KavitaLole , ICMR- National institute of virology , (Award Intramural funds)
Loading

Article metrics loading...

/content/journal/jgv/10.1099/jgv.0.001568
2021-02-17
2021-03-02
Loading full text...

Full text loading...

References

  1. Hepatitis B World Health Organization. https://www.who.int/en/news-room/fact-sheets/detail/hepatitis-b 26th March 2020
  2. Caligiuri P, Cerruti R, Icardi G, Bruzzone B. Overview of hepatitis B virus mutations and their implications in the management of infection. World J Gastroenterol 2016; 22: 145 154 [CrossRef] [PubMed]
    [Google Scholar]
  3. Hunt CM, McGill JM, Allen MI, Condreay LD. Clinical relevance of hepatitis B viral mutations. Hepatology 2000; 31: 1037 1044 [CrossRef] [PubMed]
    [Google Scholar]
  4. Li M-S, Lau TC-K, Chan SK-P, Wong C-H, Ng PK-S et al. The G1613A mutation in the HBV genome affects HBeAg expression and viral replication through altered core promoter activity. PLoS One 2011; 6: e21856 [CrossRef] [PubMed]
    [Google Scholar]
  5. Milich D, Liang TJ. Exploring the biological basis of hepatitis B e antigen in hepatitis B virus infection. Hepatology 2003; 38: 1075 1086 [CrossRef] [PubMed]
    [Google Scholar]
  6. Tong S, Kim KH, Chante C, Wands J, Li J. Hepatitis B virus E antigen variants. Int J Med Sci 2005; 2: 2 7 [CrossRef] [PubMed]
    [Google Scholar]
  7. Liaw YF, Chu CM. Hepatitis B virus infection. Lancet 2009; 373: 582 592 [CrossRef] [PubMed]
    [Google Scholar]
  8. Yang HI, Yeh SH, Chen PJ, Iloeje UH, Jen CL et al. Associations between hepatitis B virus genotype and mutants and the risk of hepatocellular carcinoma. J Natl Cancer Inst 2008; 100: 1134 1143 [CrossRef] [PubMed]
    [Google Scholar]
  9. Scaglioni PP, Melegari M, Wands JR. Biologic properties of hepatitis B viral genomes with mutations in the precore promoter and precore open reading frame. Virology 1997; 233: 374 381 [CrossRef] [PubMed]
    [Google Scholar]
  10. Lamberts C, Nassal M, Velhagen I, Zentgraf H, Schröder CH. Precore-mediated inhibition of hepatitis B virus progeny DNA synthesis. J Virol 1993; 67: 3756 3762 [CrossRef] [PubMed]
    [Google Scholar]
  11. Akarca US, Greene S, Lok AS. Detection of precore hepatitis B virus mutants in asymptomatic HBsAg-positive family members. Hepatology 1994; 19: 1366 1370 [CrossRef] [PubMed]
    [Google Scholar]
  12. Liang TJ. Hepatitis B: the virus and disease. Hepatology 2009; 49: S13 S21 [CrossRef] [PubMed]
    [Google Scholar]
  13. Takeda K, Akahane Y, Suzuki H, Okamoto H, Tsuda F et al. Defects in the precore region of the HBV genome in patients with chronic hepatitis B after sustained seroconversion from HBeAg to anti-HBe induced spontaneously or with interferon therapy. Hepatology 1990; 12: 1284 1289 [CrossRef] [PubMed]
    [Google Scholar]
  14. Carman WF, Jacyna MR, Hadziyannis S, Karayiannis P, McGarvey MJ et al. Mutation preventing formation of hepatitis B e antigen in patients with chronic hepatitis B infection. Lancet 1989; 2: 588 591 [CrossRef] [PubMed]
    [Google Scholar]
  15. Raimondo G, Tanzi E, Brancatelli S, Campo S, Sardo MA et al. Is the course of perinatal hepatitis B virus infection influenced by genetic heterogeneity of the virus?. J Med Virol 1993; 40: 87 90 [CrossRef] [PubMed]
    [Google Scholar]
  16. Mphahlele MJ, Shattock AG, Boner W, Quinn J, McCormick PA et al. Transmission of a homogenous hepatitis B virus population of A1896-containing strains leading to mild resolving acute hepatitis and seroconversion to hepatitis B e antigen antibodies in an adult. Hepatology 1997; 26: 743 746 [CrossRef] [PubMed]
    [Google Scholar]
  17. Chisari FV, Isogawa M, Wieland SF. Pathogenesis of hepatitis B virus infection. Pathol Biol 2010; 58: 258 266 [CrossRef] [PubMed]
    [Google Scholar]
  18. Lamontagne J, Mell JC, Bouchard MJ. Transcriptome-Wide analysis of hepatitis B virus-mediated changes to normal hepatocyte gene expression. PLoS Pathog 2016; 12: e1005438 [CrossRef] [PubMed]
    [Google Scholar]
  19. Arankalle VA, Gandhi S, Lole KS, Chadha MS, Gupte GM. An outbreak of hepatitis B with high mortality in India: association with precore, basal core promoter mutants and improperly sterilized syringes. J Viral Hepat 2011; 18: e20 e28 [CrossRef] [PubMed]
    [Google Scholar]
  20. Tripathy AS, Das R, Chadha MS, Arankalle VA. Epidemic of hepatitis B with high mortality in India: association of fulminant disease with lack of CCl4 and natural killer T cells. J Viral Hepat 2011; 18: e415 e422 [CrossRef] [PubMed]
    [Google Scholar]
  21. Wang H, Kim S, Ryu WS. DDX3 DEAD-box RNA helicase inhibits hepatitis B virus reverse transcription by incorporation into nucleocapsids. J Virol 2009; 83: 5815 5824 [CrossRef] [PubMed]
    [Google Scholar]
  22. Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 2009; 6: 343 345 [CrossRef] [PubMed]
    [Google Scholar]
  23. S Andrew FastQC a quality control tool for high throughput sequence https:// www.bioinformatics.babraham.ac.uk/projects/fastqc/ .
  24. Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for illumina sequence data. Bioinformatics 2014; 30: 2114 2120 [CrossRef] [PubMed]
    [Google Scholar]
  25. Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C et al. Star: ultrafast universal RNA-seq aligner. Bioinformatics 2013; 29: 15 21 [CrossRef] [PubMed]
    [Google Scholar]
  26. Pertea M, Pertea GM, Antonescu CM, Chang TC, Mendell JT et al. StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. Nat Biotechnol 2015; 33: 290 295 [CrossRef] [PubMed]
    [Google Scholar]
  27. Frazee AC, Pertea G, Jaffe AE, Langmead B, Salzberg SL et al. Ballgown bridges the gap between transcriptome assembly and expression analysis. Nat Biotechnol 2015; 33: 243 246 [CrossRef] [PubMed]
    [Google Scholar]
  28. Sun D, Nassal M. Stable HepG2- and Huh7-based human hepatoma cell lines for efficient regulated expression of infectious hepatitis B virus. J Hepatol 2006; 45: 636 645 [CrossRef] [PubMed]
    [Google Scholar]
  29. Yan R, Cai D, Liu Y, Guo H. Detection of hepatitis B virus particles released from cultured cells by particle gel assay. Methods Mol Biol 2017; 1540: 193 202 [CrossRef] [PubMed]
    [Google Scholar]
  30. Lole KS, Arankalle VA. Quantitation of hepatitis B virus DNA by real-time PCR using internal amplification control and dual TaqMan MGB probes. J Virol Methods 2006; 135: 83 90 [CrossRef] [PubMed]
    [Google Scholar]
  31. Devhare PB, Chatterjee SN, Arankalle VA, Lole KS. Analysis of antiviral response in human epithelial cells infected with hepatitis E virus. PLoS One 2013; 8: e63793 [CrossRef] [PubMed]
    [Google Scholar]
  32. Liu N, Zhang J, Yang X, Jiao T, Zhao X et al. Hdm2 promotes neddylation of hepatitis B virus HBx to enhance its stability and function. J Virol 2017; 91: e00340 17 [CrossRef] [PubMed]
    [Google Scholar]
  33. Ferrari C, Penna A, Bertoletti A, Valli A, Antoni AD et al. Cellular immune response to hepatitis B virus-encoded antigens in acute and chronic hepatitis B virus infection. J Immunol 1990; 145: 3442 3449 [PubMed]
    [Google Scholar]
  34. Dunn C, Brunetto M, Reynolds G, Christophides T, Kennedy PT et al. Cytokines induced during chronic hepatitis B virus infection promote a pathway for NK cell-mediated liver damage. J Exp Med 2007; 204: 667 680 [CrossRef] [PubMed]
    [Google Scholar]
  35. Kalinina T, Riu A, Fischer L, Will H, Sterneck M. A dominant hepatitis B virus population defective in virus secretion because of several S-gene mutations from a patient with fulminant hepatitis. Hepatology 2001; 34: 385 394 [CrossRef] [PubMed]
    [Google Scholar]
  36. Laskus T, Rakela J, Nowicki MJ, Persing DH. Hepatitis B virus core promoter sequence analysis in fulminant and chronic hepatitis B. Gastroenterology 1995; 109: 1618 1623 [CrossRef] [PubMed]
    [Google Scholar]
  37. Sato S, Suzuki K, Akahane Y, Akamatsu K, Akiyama K et al. Hepatitis B virus strains with mutations in the core promoter in patients with fulminant hepatitis. Ann Intern Med 1995; 122: 241 248 [CrossRef] [PubMed]
    [Google Scholar]
  38. Hasegawa K, Huang J, Rogers SA, Blum HE, Liang TJ. Enhanced replication of a hepatitis B virus mutant associated with an epidemic of fulminant hepatitis. J Virol 1994; 68: 1651 1659 [CrossRef] [PubMed]
    [Google Scholar]
  39. Omata M, Ehata T, Yokosuka O, Hosoda K, Ohto M. Mutations in the precore region of hepatitis B virus DNA in patients with fulminant and severe hepatitis. N Engl J Med 1991; 324: 1699 1704 [CrossRef] [PubMed]
    [Google Scholar]
  40. Kosaka Y, Takase K, Kojima M, Shimizu M, Inoue K et al. Fulminant hepatitis B: induction by hepatitis B virus mutants defective in the precore region and incapable of encoding E antigen. Gastroenterology 1991; 100: 1087 1094 [CrossRef] [PubMed]
    [Google Scholar]
  41. Liang TJ, Baruch Y, Ben-Porath E, Enat R, Bassan L et al. Hepatitis B virus infection in patients with idiopathic liver disease. Hepatology 1991; 13: 1044 1051 [PubMed]
    [Google Scholar]
  42. Günther S, Sommer G, Von Breunig F, Iwanska A, Kalinina T et al. Amplification of full-length hepatitis B virus genomes from samples from patients with low levels of viremia: frequency and functional consequences of PCR-introduced mutations. J Clin Microbiol 1998; 36: 531 538 [CrossRef] [PubMed]
    [Google Scholar]
  43. Sterneck M, Kalinina T, Otto S, Günther S, Fischer L et al. Neonatal fulminant hepatitis B: structural and functional analysis of complete hepatitis B virus genomes from mother and infant. J Infect Dis 1998; 177: 1378 1381 [CrossRef] [PubMed]
    [Google Scholar]
  44. Wang WH, Grégori G, Hullinger RL, Andrisani OM. Sustained activation of p38 mitogen-activated protein kinase and c-Jun N-terminal kinase pathways by hepatitis B virus X protein mediates apoptosis via induction of Fas/FasL and tumor necrosis factor (TNF) receptor 1/TNF-alpha expression. Mol Cell Biol 2004; 24: 10352 10365 [CrossRef] [PubMed]
    [Google Scholar]
  45. Wang WH, Hullinger RL, Andrisani OM. Hepatitis B virus X protein via the p38MAPK pathway induces E2F1 release and ATR kinase activation mediating p53 apoptosis. J Biol Chem 2008; 283: 25455 25467 [CrossRef] [PubMed]
    [Google Scholar]
  46. Kim HJ, Kim SY, Kim J, Lee H, Choi M et al. Hepatitis B virus X protein induces apoptosis by enhancing translocation of Bax to mitochondria. IUBMB Life 2008; 60: 473 480 [CrossRef] [PubMed]
    [Google Scholar]
  47. Hu L, Chen L, Yang G, Li L, Sun H et al. HBx sensitizes cells to oxidative stress-induced apoptosis by accelerating the loss of Mcl-1 protein via caspase-3 cascade. Mol Cancer 2011; 10: 43 [CrossRef] [PubMed]
    [Google Scholar]
  48. Geng X, Huang C, Qin Y, McCombs JE, Yuan Q et al. Hepatitis B virus X protein targets Bcl-2 proteins to increase intracellular calcium, required for virus replication and cell death induction. Proc Natl Acad Sci U S A 2012; 109: 18471 18476 [CrossRef] [PubMed]
    [Google Scholar]
  49. Elizalde MM, Campos RH, Barbini L. X protein variants of the autochthonous Latin American hepatitis B virus F genotype promotes human hepatocyte death by the induction of apoptosis and autophagy. Virus Res 2017; 242: 156 165 [CrossRef]
    [Google Scholar]
  50. Elizalde MM, Sevic I, González López Ledesma MM, Campos RH, Barbini L et al. Human hepatocytes apoptosis induced by replication of hepatitis B virus subgenotypes F1b and F4: role of basal core promoter and preCore mutations. Virology 2018; 513: 160 167 [CrossRef]
    [Google Scholar]
  51. Bhoola NH, Kramvis A. Hepatitis B e antigen expression by hepatitis B virus subgenotype A1 relative to subgenotypes A2 and D3 in cultured hepatocellular carcinoma (Huh7) cells. Intervirology 2016; 59: 48 59 [CrossRef]
    [Google Scholar]
  52. YW L, Tan TL, Zhang J, Chen WN. Cellular apoptosis induced by replication of hepatitis B virus: possible link between viral genotype and clinical outcome. Virol J. 2007; 4: 117
    [Google Scholar]
  53. YW L, Tan TL, Chan V, Chen WN. The HBSP gene is expressed during HBV replication, and its coded BH3-containing spliced viral protein induces apoptosis in HepG2 cells. Biochem Biophys Res Commun 2006; 351: :64 :70
    [Google Scholar]
  54. Lamontagne J, Mell JC, Bouchard MJ. Transcriptome-Wide analysis of hepatitis B virus-mediated changes to normal hepatocyte gene expression. PLoS Pathog 2016; 12: e1005438 [CrossRef]
    [Google Scholar]
  55. Jagya N, Varma SPK, Thakral D, Joshi P, Durgapal H et al. RNA-seq based transcriptome analysis of hepatitis E virus (HEV) and hepatitis B virus (HBV) replicon transfected Huh-7 cells. PLoS One 2014; 9: e87835 [CrossRef]
    [Google Scholar]
  56. Ryu HM, Park SG, Yea SS, Jang WH, Yang YI. Gene expression analysis of primary normal human hepatocytes infected with human hepatitis B virus. WJG 2006; 12: 4986 4995 [CrossRef]
    [Google Scholar]
  57. Chang C, Enders G, Sprengel R, Peters N, Varmus HE et al. Expression of the precore region of an avian hepatitis B virus is not required for viral replication. J Virol 1987; 61: 3322 3325 [CrossRef]
    [Google Scholar]
  58. Schlicht HJ, Galle P, Schaller H. The hepatitis-B viruses: molecular biology and recent tissue culture systems. J Cell Sci 1987; 1987: 197 212 [CrossRef]
    [Google Scholar]
  59. Chen HS, Kew MC, Hornbuckle WE, Tennant BC, Cote PJ et al. The precore gene of the woodchuck hepatitis virus genome is not essential for viral replication in the natural host. J Virol 1992; 66: 5682 5684 [CrossRef]
    [Google Scholar]
  60. Revill P, Yuen L, Walsh R, Perrault M, Locarnini S et al. Bioinformatic analysis of the hepadnavirus e-antigen and its precursor identifies remarkable sequence conservation in all orthohepadnaviruses. J Med Virol 2010; 82: 104 115 [CrossRef]
    [Google Scholar]
  61. Chen M, Sällberg M, Hughes J, Jones J, Guidotti LG et al. Immune tolerance split between hepatitis B virus precore and core proteins. J Virol 2005; 79: 3016 3027 [CrossRef]
    [Google Scholar]
  62. Milich DR, Jones JE, Hughes JL, Price J, Raney AK et al. Is a function of the secreted hepatitis B e antigen to induce immunologic tolerance in utero?. Proc Natl Acad Sci U S A 1990; 87: 6599 6603 [CrossRef]
    [Google Scholar]
  63. Milich DR, Chen M, Schödel F, Peterson DL, Jones JE et al. Role of B cells in antigen presentation of the hepatitis B core. Proc Natl Acad Sci U S A 1997; 94: 14648 14653 [CrossRef]
    [Google Scholar]
  64. Milich D, Liang TJ. Exploring the biological basis of hepatitis B e antigen in hepatitis B virus infection. Hepatology 2003; 38: 1075 1086 [CrossRef]
    [Google Scholar]
  65. Liu D, Cui L, Wang Y, Yang G, He J et al. Hepatitis B e antigen and its precursors promote the progress of hepatocellular carcinoma by interacting with NUMB and decreasing p53 activity. Hepatology 2016; 64: 390 404 [CrossRef]
    [Google Scholar]
  66. Shirakata Y, Koike K. Hepatitis B virus X protein induces cell death by causing loss of mitochondrial membrane potential. J Biol Chem. 2003; 278: 22071 22078 [CrossRef]
    [Google Scholar]
  67. Rahmani Z, Huh KW, Lasher R, Siddiqui A. Hepatitis B virus X protein colocalizes to mitochondria with a human voltage-dependent anion channel, HVDAC3, and alters its transmembrane potential. J Virol 2000; 74: 2840 2846 [CrossRef]
    [Google Scholar]
  68. Waris G, Huh KW, Siddiqui A. Mitochondrially associated hepatitis B virus X protein constitutively activates transcription factors STAT-3 and NF-κB via oxidative stress. Mol Cell Biol 2001; 21: 7721 7730 [CrossRef]
    [Google Scholar]
  69. Su F, Schneider RJ. Hepatitis B virus HBx protein sensitizes cells to apoptotic killing by tumor necrosis factor alpha. Proc Natl Acad Sci U S A 1997; 94: 8744 8749 [CrossRef]
    [Google Scholar]
  70. Kim SY, Kyaw YY, Cheong J. Functional interaction of endoplasmic reticulum stress and hepatitis B virus in the pathogenesis of liver diseases. World J Gastroenterol 2017; 23: 7657 7665 [CrossRef]
    [Google Scholar]
  71. Kim H, Lee H, Yun Y. X-gene product of hepatitis B virus induces apoptosis in liver cells. J Biol Chem 1998; 273: 381 385 [CrossRef]
    [Google Scholar]
  72. Li G, Mongillo M, Chin KT, Harding H, Ron D et al. Role of ERO1-α–mediated stimulation of inositol 1,4,5-triphosphate receptor activity in endoplasmic reticulum stress–induced apoptosis. J Cell Biol 2009; 186: 783 792 [CrossRef]
    [Google Scholar]
  73. Lee S, Min KT. The interface between ER and mitochondria: molecular compositions and functions. Mol Cells 2018; 41: 1000 1007
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/jgv.0.001568
Loading
/content/journal/jgv/10.1099/jgv.0.001568
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error