1887

Abstract

The G1862T mutation, which occurs most frequently in subgenotype A1 of the hepatitis B virus (HBV), results in a valine to phenylalanine substitution at the −3 position of the signal peptide cleavage site at the amino end of the precore/core (preC/C) precursor protein. The objective of this study was to functionally characterize the G1862T mutation relative to its wild-type counterpart in subgenotype A1. Huh7 cells were transfected with subgenotype A1 replication-competent plasmids, with and without G1862T. Secretion of HBsAg and HBeAg, preC/C/HBeAg expression in the secretory pathway, activation of the unfolded protein response (UPR) and subsequent activation of apoptosis were monitored. The introduction of G1862T did not affect HBsAg expression. Cells transfected with the G1862T subgenotype A1 plasmid showed decreased expression of intracellular HBcAg and of nuclear preC/C/HBeAg and extracellular HBeAg, when compared to cells transfected with its wild-type counterpart as a result of the accumulation of the mutant protein in the endoplasmic reticulum (ER) and ER–Golgi intermediate compartment (ERGIC) . This accumulation of preC/C/HBeAg protein in the ER led to the earlier activation of the three UPR pathways, but not to an increase in apoptosis. Therefore, it is evident that the presence of G1862T in subgenotype A1 does not completely abolish HBeAg expression, but affects the rate of HBeAg maturation, its passage through the secretory pathway and activation of the UPR. Increase in ER stress can result in liver damage, which has been shown to be a contributing factor to hepatocarcinogenesis and may explain why G1862T is frequently found in subgenotype A1 from liver disease patients.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/jgv.0.000793
2017-06-01
2020-01-26
Loading full text...

Full text loading...

/deliver/fulltext/jgv/98/6/1422.html?itemId=/content/journal/jgv/10.1099/jgv.0.000793&mimeType=html&fmt=ahah

References

  1. Marion PL, Robinson WS. Hepadna viruses: hepatitis B and related viruses. Curr Top Microbiol Immunol 1983;105:99–121[PubMed]
    [Google Scholar]
  2. Summers J, Mason WS. Replication of the genome of a hepatitis B-like virus by reverse transcription of an RNA intermediate. Cell 1982;29:403–415 [CrossRef][PubMed]
    [Google Scholar]
  3. Mphahlele M, Francois G, Kew M, van Damme P, Hoosen A et al. Epidemiology and control of hepatitis B: implications for eastern and southern Africa. South Afr J Epidemiol Infect 2002;17:12–17
    [Google Scholar]
  4. Kimbi GC, Kramvis A, Kew MC. Distinctive sequence characteristics of subgenotype A1 isolates of hepatitis B virus from South Africa. J Gen Virol 2004;85:1211–1220 [CrossRef][PubMed]
    [Google Scholar]
  5. Kramvis A, Weitzmann L, Owiredu WK, Kew MC. Analysis of the complete genome of subgroup A' hepatitis B virus isolates from South Africa. J Gen Virol 2002;83:835–839 [CrossRef][PubMed]
    [Google Scholar]
  6. Gopalakrishnan D, Keyter M, Shenoy KT, Leena KB, Thayumanavan L et al. Hepatitis B virus subgenotype A1 predominates in liver disease patients from Kerala, India. World J Gastroenterol 2013;19:9294–9306 [CrossRef][PubMed]
    [Google Scholar]
  7. Kew MC. Hepatitis viruses and hepatocellular carcinoma. S Afr Med J 1994;84:550–556[PubMed]
    [Google Scholar]
  8. Kramvis A, Bukofzer S, Kew MC, Song E. Nucleic acid sequence analysis of the precore region of hepatitis B virus from sera of southern African black adult carriers of the virus. Hepatology 1997;25:235–240 [CrossRef][PubMed]
    [Google Scholar]
  9. Kramvis A, Kew MC, Bukofzer S. Hepatitis B virus precore mutants in serum and liver of Southern African Blacks with hepatocellular carcinoma. J Hepatol 1998;28:132–141 [CrossRef][PubMed]
    [Google Scholar]
  10. Sugauchi F, Kumada H, Acharya SA, Shrestha SM, Gamutan MT et al. Epidemiological and sequence differences between two subtypes (Ae and Aa) of hepatitis B virus genotype A. J Gen Virol 2004;85:811–820 [CrossRef][PubMed]
    [Google Scholar]
  11. Song E, Dusheiko GM, Bowyer S, Kew MC. Hepatitis B virus replication in southern Africa blacks with HBsAg-positive hepatocellular carcinoma. Hepatology 1984;4:608–610 [CrossRef][PubMed]
    [Google Scholar]
  12. Sugiyama M, Tanaka Y, Kato T, Orito E, Ito K et al. Influence of hepatitis B virus genotypes on the intra- and extracellular expression of viral DNA and antigens. Hepatology 2006;44:915–924 [CrossRef][PubMed]
    [Google Scholar]
  13. Tanaka Y, Hasegawa I, Kato T, Orito E, Hirashima N et al. A case-control study for differences among hepatitis B virus infections of genotypes A (subtypes Aa and Ae) and D. Hepatology 2004;40:747–755 [CrossRef][PubMed]
    [Google Scholar]
  14. Günther S, Fischer L, Pult I, Sterneck M, Will H. Naturally occurring variants of hepatitis B virus. Adv Virus Res 1999;52:25–137[PubMed][CrossRef]
    [Google Scholar]
  15. Parekh S, Zoulim F, Ahn SH, Tsai A, Li J et al. Genome replication, virion secretion, and e antigen expression of naturally occurring hepatitis B virus core promoter mutants. J Virol 2003;77:6601–6612 [CrossRef][PubMed]
    [Google Scholar]
  16. Ahn SH, Kramvis A, Kawai S, Spangenberg HC, Li J et al. Sequence variation upstream of precore translation initiation codon reduces hepatitis B virus e antigen production. Gastroenterology 2003;125:1370–1378 [CrossRef][PubMed]
    [Google Scholar]
  17. Chen CY, Crowther C, Kew MC, Kramvis A. A valine to phenylalanine mutation in the precore region of hepatitis B virus causes intracellular retention and impaired secretion of HBe-antigen. Hepatol Res 2008;38:580–592 [CrossRef][PubMed]
    [Google Scholar]
  18. Guarnieri M, Kim KH, Bang G, Li J, Zhou Y et al. Point mutations upstream of hepatitis B virus core gene affect DNA replication at the step of core protein expression. J Virol 2006;80:587–595 [CrossRef][PubMed]
    [Google Scholar]
  19. Messageot F, Salhi S, Eon P, Rossignol JM. Proteolytic processing of the hepatitis B virus e antigen precursor. Cleavage at two furin consensus sequences. J Biol Chem 2003;278:891–895 [CrossRef][PubMed]
    [Google Scholar]
  20. Lainé S, Thouard A, Derancourt J, Kress M, Sitterlin D et al. In vitro and in vivo interactions between the hepatitis B virus protein P22 and the cellular protein gC1qR. J Virol 2003;77:12875–12880 [CrossRef][PubMed]
    [Google Scholar]
  21. Salhi S, Messageot F, Carlier D, Jean-Jean O, Rossignol JM. Identification of a cellular protein specifically interacting with the precursor of the hepatitis B e antigen. J Viral Hepat 2001;8:169–173 [CrossRef][PubMed]
    [Google Scholar]
  22. Milich DR, Chen MK, Hughes JL, Jones JE. The secreted hepatitis B precore antigen can modulate the immune response to the nucleocapsid: a mechanism for persistence. J Immunol 1998;160:2013–2021[PubMed]
    [Google Scholar]
  23. Nassal M, Rieger A. A bulged region of the hepatitis B virus RNA encapsidation signal contains the replication origin for discontinuous first-strand DNA synthesis. J Virol 1996;70:2764–2773[PubMed]
    [Google Scholar]
  24. Rieger A, Nassal M. Distinct requirements for primary sequence in the 5'- and 3'-part of a bulge in the hepatitis B virus RNA encapsidation signal revealed by a combined in vivo selection/in vitro amplification system. Nucleic Acids Res 1995;23:3909–3915 [CrossRef][PubMed]
    [Google Scholar]
  25. Fallows DA, Goff SP. Mutations in the epsilon sequences of human hepatitis B virus affect both RNA encapsidation and reverse transcription. J Virol 1995;69:3067–3073[PubMed]
    [Google Scholar]
  26. Loriot MA, Marcellin P, Talbodec N, Guigonis V, Gigou M et al. Low frequency of precore hepatitis B virus mutants in anti-hepatitis B e-positive reactivation after loss of hepatitis B e antigen in patients with chronic hepatitis B. Hepatology 1995;21:627–631[PubMed]
    [Google Scholar]
  27. Santantonio T, Jung MC, Miska S, Pastore G, Pape GR et al. Prevalence and type of pre-C HBV mutants in anti-HBe positive carriers with chronic liver disease in a highly endemic area. Virology 1991;183:840–844 [CrossRef][PubMed]
    [Google Scholar]
  28. Valliammai T, Thyagarajan SP, Zuckerman AJ, Harrison TJ. Precore and core mutations in HBV from individuals in India with chronic infection. J Med Virol 1995;45:321–325 [CrossRef][PubMed]
    [Google Scholar]
  29. Bruss V, Gerlich WH. Formation of transmembraneous hepatitis B e-antigen by cotranslational in vitro processing of the viral precore protein. Virology 1988;163:268–275 [CrossRef][PubMed]
    [Google Scholar]
  30. Nielsen H, Engelbrecht J, Brunak S, von Heijne G. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 1997;10:1–6 [CrossRef][PubMed]
    [Google Scholar]
  31. von Heijne G. Patterns of amino acids near signal-sequence cleavage sites. Eur J Biochem 1983;133:17–21 [CrossRef][PubMed]
    [Google Scholar]
  32. Karamyshev AL, Karamysheva ZN, Kajava AV, Ksenzenko VN, Nesmeyanova MA. Processing of Escherichia coli alkaline phosphatase: role of the primary structure of the signal peptide cleavage region. J Mol Biol 1998;277:859–870 [CrossRef][PubMed]
    [Google Scholar]
  33. Hou J, Lin Y, Waters J, Wang Z, Min J et al. Detection and significance of a G1862T variant of hepatitis B virus in Chinese patients with fulminant hepatitis. J Gen Virol 2002;83:2291–2298 [CrossRef][PubMed]
    [Google Scholar]
  34. Hammond C, Helenius A. Quality control in the secretory pathway: retention of a misfolded viral membrane glycoprotein involves cycling between the ER, intermediate compartment, and Golgi apparatus. J Cell Biol 1994;126:41–52 [CrossRef][PubMed]
    [Google Scholar]
  35. Kostova Z, Wolf DH. For whom the bell tolls: protein quality control of the endoplasmic reticulum and the ubiquitin-proteasome connection. Embo J 2003;22:2309–2317 [CrossRef][PubMed]
    [Google Scholar]
  36. Ellgaard L, Molinari M, Helenius A. Setting the standards: quality control in the secretory pathway. Science 1999;286:1882–1888 [CrossRef][PubMed]
    [Google Scholar]
  37. Bukau B, Weissman J, Horwich A. Molecular chaperones and protein quality control. Cell 2006;125:443–451 [CrossRef][PubMed]
    [Google Scholar]
  38. Ma Y, Hendershot LM. Delineation of a negative feedback regulatory loop that controls protein translation during endoplasmic reticulum stress. J Biol Chem 2003;278:34864–34873 [CrossRef][PubMed]
    [Google Scholar]
  39. Malhotra JD, Kaufman RJ. The endoplasmic reticulum and the unfolded protein response. Semin Cell Dev Biol 2007;18:716–731 [CrossRef][PubMed]
    [Google Scholar]
  40. Rutkowski DT, Kaufman RJ. A trip to the ER: coping with stress. Trends Cell Biol 2004;14:20–28 [CrossRef][PubMed]
    [Google Scholar]
  41. Ron D, Walter P. Signal integration in the endoplasmic reticulum unfolded protein response. Nat Rev Mol Cell Biol 2007;8:519–529 [CrossRef][PubMed]
    [Google Scholar]
  42. Schröder M, Kaufman RJ. The mammalian unfolded protein response. Annu Rev Biochem 2005;74:739–789 [CrossRef][PubMed]
    [Google Scholar]
  43. Shore GC, Papa FR, Oakes SA. Signaling cell death from the endoplasmic reticulum stress response. Curr Opin Cell Biol 2011;23:143–149 [CrossRef][PubMed]
    [Google Scholar]
  44. Kaufman RJ. Orchestrating the unfolded protein response in health and disease. J Clin Invest 2002;110:1389–1398 [CrossRef][PubMed]
    [Google Scholar]
  45. Leers MP, Kölgen W, Björklund V, Bergman T, Tribbick G et al. Immunocytochemical detection and mapping of a cytokeratin 18 neo-epitope exposed during early apoptosis. J Pathol 1999;187:567–572 [CrossRef][PubMed]
    [Google Scholar]
  46. Hägg M, Bivén K, Ueno T, Rydlander L, Björklund P et al. A novel high-through-put assay for screening of pro-apoptotic drugs. Invest New Drugs 2002;20:253–259 [CrossRef][PubMed]
    [Google Scholar]
  47. Bivén K, Erdal H, Hägg M, Ueno T, Zhou R et al. A novel assay for discovery and characterization of pro-apoptotic drugs and for monitoring apoptosis in patient sera. Apoptosis 2003;8:263–268 [CrossRef][PubMed]
    [Google Scholar]
  48. Knaus T, Nassal M. The encapsidation signal on the hepatitis B virus RNA pregenome forms a stem-loop structure that is critical for its function. Nucleic Acids Res 1993;21:3967–3975 [CrossRef][PubMed]
    [Google Scholar]
  49. Kimbi GC, Kew MC, Kramvis A. The effect of the G1888A mutation of subgenotype A1 of hepatitis B virus on the translation of the core protein. Virus Res 2012;163:334–340 [CrossRef][PubMed]
    [Google Scholar]
  50. Ma XK, Ding GZ, Shi CH, Zuo P, Lu HY et al. Structure in the precore region of hepatitis B core gene affecting its expression in E. coli. Sci Sin B 1987;30:1190–1198[PubMed]
    [Google Scholar]
  51. Shi CH, Song YM, Li AL, Ma XK, Huang CF, Al L, Xk M. The effect of upstream sequences to initiator on the expression of gene coding for hepatitis B core antigen. Sci Sin B 1987;30:625–629[PubMed]
    [Google Scholar]
  52. 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][PubMed]
    [Google Scholar]
  53. Chu CM, Liaw YF. Immunohistological study of intrahepatic expression of hepatitis B core and E antigens in chronic type B hepatitis. J Clin Pathol 1992;45:791–795 [CrossRef][PubMed]
    [Google Scholar]
  54. Naoumov NV, Portmann BC, Tedder RS, Ferns B, Eddleston AL et al. Detection of hepatitis B virus antigens in liver tissue. A relation to viral replication and histology in chronic hepatitis B infection. Gastroenterology 1990;99:1248–1253[PubMed][CrossRef]
    [Google Scholar]
  55. Yamada G, Takaguchi K, Matsueda K, Nishimoto H, Takahashi M et al. Immunoelectron microscopic observation of intrahepatic HBeAg in patients with chronic hepatitis B. Hepatology 1990;12:133–140 [CrossRef][PubMed]
    [Google Scholar]
  56. Ou JH, Yeh CT, Yen TS. Transport of hepatitis B virus precore protein into the nucleus after cleavage of its signal peptide. J Virol 1989;63:5238–5243[PubMed]
    [Google Scholar]
  57. Standring DN, Ou JH, Masiarz FR, Rutter WJ. A signal peptide encoded within the precore region of hepatitis B virus directs the secretion of a heterogeneous population of e antigens in Xenopus oocytes. Proc Natl Acad Sci USA 1988;85:8405–8409 [CrossRef][PubMed]
    [Google Scholar]
  58. Garcia PD, Ou JH, Rutter WJ, Walter P. Targeting of the hepatitis B virus precore protein to the endoplasmic reticulum membrane: after signal peptide cleavage translocation can be aborted and the product released into the cytoplasm. J Cell Biol 1988;106:1093–1104 [CrossRef][PubMed]
    [Google Scholar]
  59. Duriez M, Rossignol JM, Sitterlin D. The hepatitis B virus precore protein is retrotransported from endoplasmic reticulum (ER) to cytosol through the ER-associated degradation pathway. J Biol Chem 2008;283:32352–32360 [CrossRef][PubMed]
    [Google Scholar]
  60. Eckhardt SG, Milich DR, Mclachlan A. Hepatitis B virus core antigen has two nuclear localization sequences in the arginine-rich carboxyl terminus. J Virol 1991;65:575–582[PubMed]
    [Google Scholar]
  61. Rutkowski DT, Kaufman RJ. That which does not kill me makes me stronger: adapting to chronic ER stress. Trends Biochem Sci 2007;32:469–476 [CrossRef][PubMed]
    [Google Scholar]
  62. Huang CF, Lin SS, Ho YC, Chen FL, Yang CC. The immune response induced by hepatitis B virus principal antigens. Cell Mol Immunol 2006;3:97–106[PubMed]
    [Google Scholar]
  63. 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][PubMed]
    [Google Scholar]
  64. Chen MT, Billaud JN, Sällberg M, Guidotti LG, Chisari FV et al. A function of the hepatitis B virus precore protein is to regulate the immune response to the core antigen. Proc Natl Acad Sci USA 2004;101:14913–14918 [CrossRef][PubMed]
    [Google Scholar]
  65. Kramvis A, Kew MC. Molecular characterization of subgenotype A1 (subgroup Aa) of hepatitis B virus. Hepatol Res 2007;37:S27–S32 [CrossRef][PubMed]
    [Google Scholar]
  66. Bhoola NH, Reumann K, Kew MC, Will H, Kramvis A. Construction of replication competent plasmids of hepatitis B virus subgenotypes A1, A2 and D3 with authentic endogenous promoters. J Virol Methods 2014;203:54–64 [CrossRef][PubMed]
    [Google Scholar]
  67. Chan JY, Kwong M. Impaired expression of glutathione synthetic enzyme genes in mice with targeted deletion of the Nrf2 basic-leucine zipper protein. Biochim Biophys Acta 2000;1517:19–26 [CrossRef][PubMed]
    [Google Scholar]
  68. Cybulsky AV, Takano T, Papillon J, Khadir A, Liu J et al. Complement C5b-9 membrane attack complex increases expression of endoplasmic reticulum stress proteins in glomerular epithelial cells. J Biol Chem 2002;277:41342–41351 [CrossRef][PubMed]
    [Google Scholar]
  69. Guo YL, Baysal K, Kang B, Yang LJ, Williamson JR. Correlation between sustained c-Jun N-terminal protein kinase activation and apoptosis induced by tumor necrosis factor-α in rat mesangial cells. J Biol Chem 1998;273:4027–4034 [CrossRef][PubMed]
    [Google Scholar]
  70. Xu Y, Bradham C, Brenner DA, Czaja MJ. Hydrogen peroxide-induced liver cell necrosis is dependent on AP-1 activation. Am J Physiol 1997;273:G795–G803[PubMed]
    [Google Scholar]
  71. Walsh R, Nuttall S, Revill P, Colledge D, Cabuang L et al. Targeting the hepatitis B virus precore antigen with a novel IgNAR single variable domain intrabody. Virology 2011;411:132–141 [CrossRef][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/jgv.0.000793
Loading
/content/journal/jgv/10.1099/jgv.0.000793
Loading

Data & Media loading...

Most cited articles

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