1887

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Volume 96, Issue 1

Expression of viral polymerase and phosphorylation of core protein determine core and capsid localization of the human hepatitis B virus Free

Abstract

Biopsies from patients show that hepadnaviral core proteins and capsids – collectively called core – are found in the nucleus and cytoplasm of infected hepatocytes. In the majority of studies, cytoplasmic core localization is related to low viraemia while nuclear core localization is associated with high viral loads. In order to better understand the molecular interactions leading to core localization, we analysed transfected hepatoma cells using immune fluorescence microscopy. We observed that expression of core protein in the absence of other viral proteins led to nuclear localization of core protein and capsids, while expression of core in the context of the other viral proteins resulted in a predominantly cytoplasmic localization. Analysis of which viral partner was responsible for cytoplasmic retention indicated that the HBx, surface proteins and HBeAg had no impact but that the viral polymerase was the major determinant. Further analysis revealed that ϵ, an RNA structure to which the viral polymerase binds, was essential for cytoplasmic retention. Furthermore, we showed that core protein phosphorylation at Ser 164 was essential for the cytoplasmic core localization phenotype, which is likely to explain differences observed between individual cells.

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© 2015 CNRS, UMR 5234
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2015-01-01
2024-04-25
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References

  1. Abraham T. M., Loeb D. D. 2006; Base pairing between the 5′ half of ϵ and a cis-acting sequence, ϕ, makes a contribution to the synthesis of minus-strand DNA for human hepatitis B virus. J Virol 80:4380–4387 [View Article][PubMed]
     [Google Scholar]
  2. Akiba T., Nakayama H., Miyazaki Y., Kanno A., Ishii M., Ohori H. 1987; Relationship between the replication of hepatitis B virus and the localization of virus nucleocapsid antigen (HBcAg) in hepatocytes. J Gen Virol 68:871–877 [View Article][PubMed]
     [Google Scholar]
  3. Bartenschlager R., Schaller H. 1992; Hepadnaviral assembly is initiated by polymerase binding to the encapsidation signal in the viral RNA genome. EMBO J 11:3413–3420[PubMed]
     [Google Scholar]
  4. Beck J., Nassal M. 2003; Efficient Hsp90-independent in vitro activation by Hsc70 and Hsp40 of duck hepatitis B virus reverse transcriptase, an assumed Hsp90 client protein. J Biol Chem 278:36128–36138 [View Article][PubMed]
     [Google Scholar]
  5. Beck J., Nassal M. 2007; Hepatitis B virus replication. World J Gastroenterol 13:48–64 [View Article][PubMed]
     [Google Scholar]
  6. Belnap D. M., Watts N. R., Conway J. F., Cheng N., Stahl S. J., Wingfield P. T., Steven A. C. 2003; Diversity of core antigen epitopes of hepatitis B virus. Proc Natl Acad Sci U S A 100:10884–10889 [View Article][PubMed]
     [Google Scholar]
  7. Blum H. E., Zhang Z. S., Galun E., von Weizsäcker F., Garner B., Liang T. J., Wands J. R. 1992; Hepatitis B virus X protein is not central to the viral life cycle in vitro. J Virol 66:1223–1227[PubMed]
     [Google Scholar]
  8. Bruss V. 2004; Envelopment of the hepatitis B virus nucleocapsid. Virus Res 106:199–209 [View Article][PubMed]
     [Google Scholar]
  9. Bruss V., Lu X., Thomssen R., Gerlich W. H. 1994; Post-translational alterations in transmembrane topology of the hepatitis B virus large envelope protein. EMBO J 13:2273–2279[PubMed]
     [Google Scholar]
  10. Cao F., Tavis J. E. 2004; Detection and characterization of cytoplasmic hepatitis B virus reverse transcriptase. J Gen Virol 85:3353–3360 [View Article][PubMed]
     [Google Scholar]
  11. Chang L. J., Pryciak P., Ganem D., Varmus H. E. 1989; Biosynthesis of the reverse transcriptase of hepatitis B viruses involves de novo translational initiation not ribosomal frameshifting. Nature 337:364–368 [View Article][PubMed]
     [Google Scholar]
  12. Chen S. Y., Kao C. F., Chen C. M., Shih C. M., Hsu M. J., Chao C. H., Wang S. H., You L. R., Lee Y. H. 2003; Mechanisms for inhibition of hepatitis B virus gene expression and replication by hepatitis C virus core protein. J Biol Chem 278:591–607 [View Article][PubMed]
     [Google Scholar]
  13. Chu C. M., Liaw Y. F. 1987; Intrahepatic distribution of hepatitis B surface and core antigens in chronic hepatitis B virus infection. Hepatocyte with cytoplasmic/membranous hepatitis B core antigen as a possible target for immune hepatocytolysis. Gastroenterology 92:220–225[PubMed]
     [Google Scholar]
  14. Chu C. M., Liaw Y. F. 1997; Natural history of chronic hepatitis B virus infection: an immunopathological study. J Gastroenterol Hepatol 12:S218–S222 [View Article][PubMed]
     [Google Scholar]
  15. Chu C. M., Yeh C. T., Sheen I. S., Liaw Y. F. 1995; Subcellular localization of hepatitis B core antigen in relation to hepatocyte regeneration in chronic hepatitis B. Gastroenterology 109:1926–1932 [View Article][PubMed]
     [Google Scholar]
  16. Chu C. M., Yeh C. T., Chien R. N., Sheen I. S., Liaw Y. F. 1997; The degrees of hepatocyte nuclear but not cytoplasmic expression of hepatitis B core antigen reflect the level of viral replication in chronic hepatitis B virus infection. J Clin Microbiol 35:102–105[PubMed]
     [Google Scholar]
  17. Deres K., Schröder C. H., Paessens A., Goldmann S., Hacker H. J., Weber O., Krämer T., Niewöhner U., Pleiss U.other authors 2003; Inhibition of hepatitis B virus replication by drug-induced depletion of nucleocapsids. Science 299:893–896 [View Article][PubMed]
     [Google Scholar]
  18. Diao J., Garces R., Richardson C. D. 2001; X protein of hepatitis B virus modulates cytokine and growth factor related signal transduction pathways during the course of viral infections and hepatocarcinogenesis. Cytokine Growth Factor Rev 12:189–205 [View Article][PubMed]
     [Google Scholar]
  19. Duriez M., Rossignol J.-M., Sitterlin D. 2008; The hepatitis B virus precore protein is retrotransported from endoplasmic reticulum (ER) to cytosol through the ER-associated degradation pathway. J Biol Chem 283:32352–32360 [View Article][PubMed]
     [Google Scholar]
  20. Fouillot N., Tlouzeau S., Rossignol J. M., Jean-Jean O. 1993; Translation of the hepatitis B virus P gene by ribosomal scanning as an alternative to internal initiation. J Virol 67:4886–4895[PubMed]
     [Google Scholar]
  21. Garcia P. D., Ou J. H., Rutter W. J., Walter P. 1988; 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 106:1093–1104 [View Article][PubMed]
     [Google Scholar]
  22. Gazina E. V., Fielding J. E., Lin B., Anderson D. A. 2000; Core protein phosphorylation modulates pregenomic RNA encapsidation to different extents in human and duck hepatitis B viruses. J Virol 74:4721–4728 [View Article][PubMed]
     [Google Scholar]
  23. Gerelsaikhan T., Tavis J. E., Bruss V. 1996; Hepatitis B virus nucleocapsid envelopment does not occur without genomic DNA synthesis. J Virol 70:4269–4274[PubMed]
     [Google Scholar]
  24. Gerlich W. H., Goldmann U., Müller R., Stibbe W., Wolff W. 1982; Specificity and localization of the hepatitis B virus-associated protein kinase. J Virol 42:761–766[PubMed]
     [Google Scholar]
  25. Gripon P., Rumin S., Urban S., Le Seyec J., Glaise D., Cannie I., Guyomard C., Lucas J., Trepo C., Guguen-Guillouzo C. 2002; Infection of a human hepatoma cell line by hepatitis B virus. Proc Natl Acad Sci U S A 99:15655–15660 [View Article][PubMed]
     [Google Scholar]
  26. Guidotti L. G., Martinez V., Loh Y. T., Rogler C. E., Chisari F. V. 1994; Hepatitis B virus nucleocapsid particles do not cross the hepatocyte nuclear membrane in transgenic mice. J Virol 68:5469–5475[PubMed]
     [Google Scholar]
  27. Hu J., Flores D., Toft D., Wang X., Nguyen D. 2004; Requirement of heat shock protein 90 for human hepatitis B virus reverse transcriptase function. J Virol 78:13122–13131 [View Article][PubMed]
     [Google Scholar]
  28. Hwang W. L., Su T. S. 1998; Translational regulation of hepatitis B virus polymerase gene by termination-reinitiation of an upstream minicistron in a length-dependent manner. J Gen Virol 79:2181–2189[PubMed]
     [Google Scholar]
  29. Jean-Jean O., Weimer T., de Recondo A. M., Will H., Rossignol J. M. 1989; Internal entry of ribosomes and ribosomal scanning involved in hepatitis B virus P gene expression. J Virol 63:5451–5454[PubMed]
     [Google Scholar]
  30. Jones S. A., Boregowda R., Spratt T. E., Hu J. 2012; In vitro epsilon RNA-dependent protein priming activity of human hepatitis B virus polymerase. J Virol 86:5134–5150 [View Article][PubMed]
     [Google Scholar]
  31. Kang H. Y., Lee S., Park S. G., Yu J., Kim Y., Jung G. 2006; Phosphorylation of hepatitis B virus Cp at Ser87 facilitates core assembly. Biochem J 398:311–317 [View Article][PubMed]
     [Google Scholar]
  32. Kann M., Sodeik B., Vlachou A., Gerlich W. H., Helenius A. 1999; Phosphorylation-dependent binding of hepatitis B virus core particles to the nuclear pore complex. J Cell Biol 145:45–55 [View Article][PubMed]
     [Google Scholar]
  33. Kaul G., Pattan G., Rafeequi T. 2011; Eukaryotic elongation factor-2 (eEF2): its regulation and peptide chain elongation. Cell Biochem Funct 29:227–234 [View Article][PubMed]
     [Google Scholar]
  34. Kim T. H., Cho E. Y., Oh H. J., Choi C. S., Kim J. W., Moon H. B., Kim H. C. 2006; The degrees of hepatocyte cytoplasmic expression of hepatitis B core antigen correlate with histologic activity of liver disease in the young patients with chronic hepatitis B infection. J Korean Med Sci 21:279–283 [View Article][PubMed]
     [Google Scholar]
  35. Li H. C., Huang E. Y., Su P. Y., Wu S. Y., Yang C. C., Lin Y. S., Chang W. C., Shih C. 2010; Nuclear export and import of human hepatitis B virus capsid protein and particles. PLoS Pathog 6:e1001162 [View Article][PubMed]
     [Google Scholar]
  36. Liao W., Ou J. H. 1995; Phosphorylation and nuclear localization of the hepatitis B virus core protein: significance of serine in the three repeated SPRRR motifs. J Virol 69:1025–1029[PubMed]
     [Google Scholar]
  37. Liu C.-J., Jeng Y.-M., Chen C.-L., Cheng H.-R., Chen P.-J., Chen T.-C., Liu C.-H., Lai M.-Y., Chen D.-S., Kao J.-H. 2009; Hepatitis B virus basal core promoter mutation and DNA load correlate with expression of hepatitis B core antigen in patients with chronic hepatitis B. J Infect Dis 199:742–749 [View Article][PubMed]
     [Google Scholar]
  38. Lucifora J., Arzberger S., Durantel D., Belloni L., Strubin M., Levrero M., Zoulim F., Hantz O., Protzer U. 2011; Hepatitis B virus X protein is essential to initiate and maintain virus replication after infection. J Hepatol 55:996–1003 [View Article][PubMed]
     [Google Scholar]
  39. Michalak T., Nowosławski A. 1982; Crystalline aggregates of hepatitis B core particles in cytoplasm of hepatocytes. Intervirology 17:247–252 [View Article][PubMed]
     [Google Scholar]
  40. Naoumov N. V., Portmann B. C., Tedder R. S., Ferns B., Eddleston A. L., Alexander G. J., Williams R. 1990; Detection of hepatitis B virus antigens in liver tissue. A relation to viral replication and histology in chronic hepatitis B infection. Gastroenterology 99:1248–1253[PubMed]
     [Google Scholar]
  41. Oropeza C. E., McLachlan A. 2007; Complementarity between epsilon and phi sequences in pregenomic RNA influences hepatitis B virus replication efficiency. Virology 359:371–381 [View Article][PubMed]
     [Google Scholar]
  42. Park Y. N., Han K. H., Kim K. S., Chung J. P., Kim S., Park C. 1999; Cytoplasmic expression of hepatitis B core antigen in chronic hepatitis B virus infection: role of precore stop mutants. Liver 19:199–205 [View Article][PubMed]
     [Google Scholar]
  43. Petit M. A., Pillot J. 1985; HBc and HBe antigenicity and DNA-binding activity of major core protein P22 in hepatitis B virus core particles isolated from the cytoplasm of human liver cells. J Virol 53:543–551[PubMed]
     [Google Scholar]
  44. Rabe B., Vlachou A., Panté N., Helenius A., Kann M. 2003; Nuclear import of hepatitis B virus capsids and release of the viral genome. Proc Natl Acad Sci U S A 100:9849–9854 [View Article][PubMed]
     [Google Scholar]
  45. Rabe B., Delaleau M., Bischof A., Foss M., Sominskaya I., Pumpens P., Cazenave C., Castroviejo M., Kann M. 2009; Nuclear entry of hepatitis B virus capsids involves disintegration to protein dimers followed by nuclear reassociation to capsids. PLoS Pathog 5:e1000563 [View Article][PubMed]
     [Google Scholar]
  46. Schmitz A., Schwarz A., Foss M., Zhou L., Rabe B., Hoellenriegel J., Stoeber M., Panté N., Kann M. 2010; Nucleoporin 153 arrests the nuclear import of hepatitis B virus capsids in the nuclear basket. PLoS Pathog 6:e1000741–e1000741 [View Article][PubMed]
     [Google Scholar]
  47. Seifer M., Standring D. N. 1995; Assembly and antigenicity of hepatitis B virus core particles. Intervirology 38:47–62[PubMed]
     [Google Scholar]
  48. Sells M. A., Zelent A. Z., Shvartsman M., Acs G. 1988; Replicative intermediates of hepatitis B virus in HepG2 cells that produce infectious virions. J Virol 62:2836–2844[PubMed]
     [Google Scholar]
  49. Sharma R. R., Dhiman R. K., Chawla Y., Vasistha R. K. 2002; Immunohistochemistry for core and surface antigens in chronic hepatitis. Trop Gastroenterol 23:16–19[PubMed]
     [Google Scholar]
  50. Skabkin M. A., Skabkina O. V., Hellen C. U., Pestova T. V. 2013; Reinitiation and other unconventional posttermination events during eukaryotic translation. Mol Cell 51:249–264 [View Article][PubMed]
     [Google Scholar]
  51. Stahl M., Beck J., Nassal M. 2007; Chaperones activate hepadnavirus reverse transcriptase by transiently exposing a C-proximal region in the terminal protein domain that contributes to ϵ RNA binding. J Virol 81:13354–13364 [View Article][PubMed]
     [Google Scholar]
  52. Su H., Yee J. K. 1992; Regulation of hepatitis B virus gene expression by its two enhancers. Proc Natl Acad Sci U S A 89:2708–2712 [View Article][PubMed]
     [Google Scholar]
  53. Tavis J. E., Ganem D. 1996; Evidence for activation of the hepatitis B virus polymerase by binding of its RNA template. J Virol 70:5741–5750[PubMed]
     [Google Scholar]
  54. Tavis J. E., Massey B., Gong Y. 1998; The duck hepatitis B virus polymerase is activated by its RNA packaging signal, epsilon. J Virol 72:5789–5796[PubMed]
     [Google Scholar]
  55. von Weizsäcker F., Köck J., Wieland S., Beck J., Nassal M., Blum H. E. 2002; Cis-preferential recruitment of duck hepatitis B virus core protein to the RNA/polymerase preassembly complex. Hepatology 35:209–216 [View Article][PubMed]
     [Google Scholar]
  56. Wang Z., Wu L., Cheng X., Liu S., Li B., Li H., Kang F., Wang J., Xia H.other authors 2013; Replication-competent infectious hepatitis B virus vectors carrying substantially sized transgenes by redesigned viral polymerase translation. PLoS ONE 8:e60306 [View Article][PubMed]
     [Google Scholar]
  57. Watanabe T., Sorensen E. M., Naito A., Schott M., Kim S., Ahlquist P. 2007; Involvement of host cellular multivesicular body functions in hepatitis B virus budding. Proc Natl Acad Sci U S A 104:10205–10210 [View Article][PubMed]
     [Google Scholar]
  58. Watts N. R., Vethanayagam J. G., Ferns R. B., Tedder R. S., Harris A., Stahl S. J., Steven A. C., Wingfield P. T. 2010; Molecular basis for the high degree of antigenic cross reactivity between hepatitis B virus capsids (HBcAg) and subunits: insights into the enigmatic nature of e-antigen. J Mol Biol 398:530–541 [View Article][PubMed]
     [Google Scholar]
  59. Whalley S. A., Murray J. M., Brown D., Webster G. J., Emery V. C., Dusheiko G. M., Perelson A. S. 2001; Kinetics of acute hepatitis B virus infection in humans. J Exp Med 193:847–854 [View Article][PubMed]
     [Google Scholar]
  60. Wittkop L., Schwarz A., Cassany A., Grün-Bernhard S., Delaleau M., Rabe B., Cazenave C., Gerlich W., Glebe D., Kann M. 2010; Inhibition of protein kinase C phosphorylation of hepatitis B virus capsids inhibits virion formation and causes intracellular capsid accumulation. Cell Microbiol 12:962–975 [View Article][PubMed]
     [Google Scholar]
  61. Xu C., Guo H., Pan X.-B., Mao R., Yu W., Xu X., Wei L., Chang J., Block T. M., Guo J.-T. 2010; Interferons accelerate decay of replication-competent nucleocapsids of hepatitis B virus. J Virol 84:9332–9340 [View Article][PubMed]
     [Google Scholar]
  62. Yaginuma K., Shirakata Y., Kobayashi M., Koike K. 1987; Hepatitis B virus (HBV) particles are produced in a cell culture system by transient expression of transfected HBV DNA. Proc Natl Acad Sci U S A 84:2678–2682 [View Article][PubMed]
     [Google Scholar]
  63. Zhang Z., Tavis J. E. 2006; The duck hepatitis B virus reverse transcriptase functions as a full-length monomer. J Biol Chem 281:35794–35801 [View Article][PubMed]
     [Google Scholar]
  64. Zlotnick A., Johnson J. M., Wingfield P. W., Stahl S. J., Endres D. 1999; A theoretical model successfully identifies features of hepatitis B virus capsid assembly. Biochemistry 38:14644–14652 [View Article][PubMed]
     [Google Scholar]
  65. Zoulim F., Saputelli J., Seeger C. 1994; Woodchuck hepatitis virus X protein is required for viral infection in vivo. J Virol 68:2026–2030[PubMed]
     [Google Scholar]
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Expression of viral polymerase and phosphorylation of core protein determine core and capsid localization of the human hepatitis B virus
J. Gen. Virol. 96, 183 (2015); https://doi.org/10.1099/vir.0.064816-0
/content/journal/jgv/10.1099/vir.0.064816-0
/content/journal/jgv/10.1099/vir.0.064816-0
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