1887

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Volume 91, Issue 1

Assembly and export determine the intracellular distribution of hepatitis B virus core protein subunits Free

Abstract

Little is known about the parameters and factors that determine the intracellular distribution of the hepatitis B virus core protein (HBc). In order to study HBc in living cells, HBc was tagged with green fluorescent protein (GFP). Being assembly-incompetent, the GFP-fusion protein was distributed equally throughout the cell. Mutational inactivation of known serine-phosphorylation sites within the C-terminal region led to predominantly intranuclear localization. Phosphorylation of these targets, presumably by an SR domain protein kinase, resulted in a predominantly cytoplasmic localization, which suggests active cytoplasmic export or retention. The phosphoserine itself, and not its negative charge, appears essential for the underlying mechanism. In addition, the arginine-rich, protamine-like domain surrounding these phosphorylation sites does not function as the dominant nuclear-localization signal, as had been assumed previously, because neither deleting nor altering these sequences led to a change in intracellular HBc subunit distribution. Restoring the capability of the fusion protein to form capsids by co-assembly with assembly-competent, sterically uncompromised HBc subunits provided a second assay that gave insight into the effects resulting from capsid formation. Assembly was found to be the dominant factor in the cytoplasmic retention of the GFP–HBc fusion protein. Furthermore, the stability of these empty capsids was influenced by the cell-cycle inhibitor nocodazole. Thus, the intracellular distribution of HBc is dominated by cytoplasmic assembly, which is supported by the active nuclear export of HBc subunits, and modulated during the cell cycle by the instability of capsids.

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2010-01-01
2024-04-16
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References

  1. Albin, C. & Robinson, W.(1980). Protein kinase activity in hepatitis B virus. J Virol 34, 297–302. [Google Scholar]
  2. Bartenschlager, R., Junker-Niepmann, M. & Schaller, H.(1990). The P gene product of hepatitis B virus is required as a structural component for genomic RNA encapsidation. J Virol 64, 5324–5332. [Google Scholar]
  3. 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.[CrossRef] [Google Scholar]
  4. Bock, C. T., Schranz, P., Schroder, C. H. & Zentgraf, H.(1994). Hepatitis B virus genome is organized into nucleosomes in the nucleus of the infected cell. Virus Genes 8, 215–229.[CrossRef] [Google Scholar]
  5. Bui, M., Myers, J. & Whittaker, G.(2002). Nucleo-cytoplasmic localization of influenza virus nucleoprotein depends on cell density and phosphorylation. Virus Res 84, 37–44.[CrossRef] [Google Scholar]
  6. Chang, C., Zhou, S., Ganem, D. & Standring, D.(1994). Phenotypic mixing between different hepadnavirus nucleocapsid proteins reveals C protein dimerization to be cis preferential. J Virol 68, 5225–5231. [Google Scholar]
  7. 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.[CrossRef] [Google Scholar]
  8. Daub, H., Blencke, S., Habenberger, P., Kurtenbach, A., Dennenmoser, J., Wissing, J., Ullrich, A. & Cotten, M.(2002). Identification of SRPK1 and SRPK2 as the major cellular protein kinases phosphorylating hepatitis B virus core protein. J Virol 76, 8124–8137.[CrossRef] [Google Scholar]
  9. Eckhardt, S. G., Milich, D. R. & McLachlan, A.(1991). Hepatitis B virus core antigen has two nuclear localization sequences in the arginine-rich carboxyl terminus. J Virol 65, 575–582. [Google Scholar]
  10. Enomoto, M., Sawano, Y., Kosuge, S., Yamano, Y., Kuroki, K. & Ohtsuki, K.(2006). High phosphorylation of HBV core protein by two alpha-type CK2-activated cAMP-dependent protein kinases in vitro. FEBS Lett 580, 894–899.[CrossRef] [Google Scholar]
  11. Falcon, V., Acosta-Rivero, N., Chinea, G., de la Rosa, M. C., Menendez, I., Duenas-Carrera, S., Gra, B., Rodriguez, A., Tsutsumi, V. & other authors(2003). Nuclear localization of nucleocapsid-like particles and HCV core protein in hepatocytes of a chronically HCV-infected patient. Biochem Biophys Res Commun 310, 54–58.[CrossRef] [Google Scholar]
  12. Farza, H., Hadchouel, M., Scotto, J., Tiollais, P., Babinet, C. & Pourcel, C.(1988). Replication and gene expression of hepatitis B virus in a transgenic mouse that contains the complete viral genome. J Virol 62, 4144–4152. [Google Scholar]
  13. Gama-Carvalho, M. & Carmo-Fonseca, M.(2001). The rules and roles of nucleocytoplasmic shuttling proteins. FEBS Lett 498, 157–163.[CrossRef] [Google Scholar]
  14. Ganem, D.(1991). Assembly of hepadnaviral virions and subviral particles. In Current Topics in Microbiology and Immunology, pp. 61–83. Heidelberg: Springer Verlag.
  15. 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.[CrossRef] [Google Scholar]
  16. Gerlich, W. H., Goldmann, U., Muller, R., Stibbe, W. & Wolff, W.(1982). Specificity and localization of the hepatitis B virus-associated protein kinase. J Virol 42, 761–766. [Google Scholar]
  17. Gowans, E. J., Burrell, C. J., Jilbert, A. R. & Marmion, B. P.(1985). Cytoplasmic (but not nuclear) hepatitis B virus (HBV) core antigen reflects HBV DNA synthesis at the level of the infected hepatocyte. Intervirology 24, 220–225.[CrossRef] [Google Scholar]
  18. 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. [Google Scholar]
  19. Kann, M., Sodeik, B., Vlachou, A., Gerlich, W. & Helenius, A.(1999). Phosphorylation-dependent binding of hepatitis B virus core particles to the nuclear pore complex. J Cell Biol 145, 45–55.[CrossRef] [Google Scholar]
  20. Kann, M., Schmitz, A. & Rabe, B.(2007). Intracellular transport of hepatitis B virus. World J Gastroenterol 13, 39–47.[CrossRef] [Google Scholar]
  21. Kingston, R. E., Chen, C. A. & Okayama, H.(2001). Calcium phosphate transfection. Curr Protoc Immunol 10, 10.13 [Google Scholar]
  22. Köck, J., Nassal, M., Deres, K., Blum, H. E. & von Weizsäcker, F.(2004). Hepatitis B virus nucleocapsids formed by carboxy-terminally mutated core proteins contain spliced viral genomes but lack full-size DNA. J Virol 78, 13812–13818.[CrossRef] [Google Scholar]
  23. Kratz, P. A., Böttcher, B. & Nassal, M.(1999). Native display of complete foreign protein domains on the surface of hepatitis B virus capsids. Proc Natl Acad Sci U S A 96, 1915–1920.[CrossRef] [Google Scholar]
  24. Kutay, U., Bischoff, F. R., Kostka, S., Kraft, R. & Gorlich, D.(1997). Export of importin alpha from the nucleus is mediated by a specific nuclear transport factor. Cell 90, 1061–1071.[CrossRef] [Google Scholar]
  25. Larkin, J., Clayton, M., Sun, B., Perchonok, C., Morgan, J., Siracusa, L., Michaels, F. & Feitelson, M.(1999). Hepatitis B virus transgenic mouse model of chronic liver disease. Nat Med 5, 907–912.[CrossRef] [Google Scholar]
  26. 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. [Google Scholar]
  27. Mabit, H., Breiner, K. M., Knaust, A., Zachmann-Brand, B. & Schaller, H.(2001). Signals for bidirectional nucleocytoplasmic transport in the duck hepatitis B virus capsid protein. J Virol 75, 1968–1977.[CrossRef] [Google Scholar]
  28. Mabit, H., Knaust, A., Breiner, K. & Schaller, H.(2003). Nuclear localization of the duck hepatitis B virus capsid protein: detection and functional implications of distinct subnuclear bodies in a compartment associated with RNA synthesis and maturation. J Virol 77, 2157–2164.[CrossRef] [Google Scholar]
  29. 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. [Google Scholar]
  30. Nassal, M.(1992). The arginine-rich domain of the hepatitis B virus core protein is required for pregenome encapsidation and productive viral positive-strand DNA synthesis but not for virus assembly. J Virol 66, 4107–4116. [Google Scholar]
  31. Nassal, M. & Rieger, A.(1993). An intramolecular disulfide bridge between Cys-7 and Cys61 determines the structure of the secretory core gene product (e antigen) of hepatitis B virus. J Virol 67, 4307–4315. [Google Scholar]
  32. Nassal, M., Rieger, A. & Steinau, O.(1992). Topological analysis of the hepatitis B virus core particle by cysteine-cysteine cross-linking. J Mol Biol 225, 1013–1025.[CrossRef] [Google Scholar]
  33. Pante, N. & Kann, M.(2002). Nuclear pore complex is able to transport macromolecules with diameters of ∼39 nm. Mol Biol Cell 13, 425–434.[CrossRef] [Google Scholar]
  34. Protzer, U., Nassal, M., Chiang, P.-W., Kirschfink, M. & Schaller, H.(1999). Interferon gene transfer by a hepatitis B virus vector efficiently suppresses wild-type virus infection. Proc Natl Acad Sci U S A 96, 10818–10823.[CrossRef] [Google Scholar]
  35. Rabe, B., Vlachou, A., Pante, 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.[CrossRef] [Google Scholar]
  36. Rittinger, K., Budman, J., Xu, J., Volinia, S., Cantley, L. C., Smerdon, S. J., Gamblin, S. J. & Yaffe, M. B.(1999). Structural analysis of 14-3-3 phosphopeptide complexes identifies a dual role for the nuclear export signal of 14–3-3 in ligand binding. Mol Cell 4, 153–166.[CrossRef] [Google Scholar]
  37. Sambrook, J., Fritsch, E. F. & Maniatis, T.(1989).Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  38. Schlicht, H. J., Bartenschlager, R. & Schaller, H.(1989). The duck hepatitis B virus core protein contains a highly phosphorylated C terminus that is essential for replication but not for RNA packaging. J Virol 63, 2995–3000. [Google Scholar]
  39. Seeger, C. & Mason, W. S.(2000). Hepatitis B virus biology. Microbiol Mol Biol Rev 64, 51–68.[CrossRef] [Google Scholar]
  40. Seifer, M. & Standring, D.(1995). Assembly and antigenicity of hepatitis B virus core particles. Intervirology 38, 47–62. [Google Scholar]
  41. Serinoz, E., Varli, M., Erden, E., Cinar, K., Kansu, A., Uzunalimoglu, O., Yurdaydin, C. & Bozkaya, H.(2003). Nuclear localization of hepatitis B core antigen and its relation to liver injury, hepatocyte proliferation, and viral load. J Clin Gastroenterol 36, 269–272.[CrossRef] [Google Scholar]
  42. Wang, S. H., Syu, W. J., Huang, K. J., Lei, H. Y., Yao, C. W., King, C. C. & Hu, S. T.(2002). Intracellular localization and determination of a nuclear localization signal of the core protein of dengue virus. J Gen Virol 83, 3093–3102. [Google Scholar]
  43. Wynne, S. A., Crowther, R. A. & Leslie, A. G.(1999). The crystal structure of the human hepatitis B virus capsid. Mol Cell 3, 771–780.[CrossRef] [Google Scholar]
  44. Yaffe, M. B., Rittinger, K., Volinia, S., Caron, P. R., Aitken, A., Leffers, H., Gamblin, S. J., Smerdon, S. J. & Cantley, L. C.(1997). The structural basis for 14-3-3: phosphopeptide binding specificity. Cell 91, 961–971.[CrossRef] [Google Scholar]
  45. Yeh, C.-T., Liaw, Y.-F. & Ou, J.-H.(1990). The arginine-rich domain of hepatitis B virus precore and core proteins contains a signal for nuclear transport. J Virol 64, 6141–6147. [Google Scholar]
  46. Yeh, C.-T., Wong, S. W., Fung, Y.-K. & Ou, J.-H.(1993). Cell cycle regulation of nuclear localization of hepatitis B virus core protein. Proc Natl Acad Sci U S A 90, 6459–6463.[CrossRef] [Google Scholar]
  47. Yoo, J. Y., Kim, H. Y., Park, C. K., Khang, S. K., Jeong, J. W., Chung, W. K., Dibisceglie, A. M. & Hoofnagle, J. H.(1990). Significance of hepatitis B core antigen in the liver in patients with chronic hepatitis B and its relation to hepatitis B virus DNA. J Gastroenterol Hepatol 5, 239–243.[CrossRef] [Google Scholar]
  48. Zhou, S. & Standring, D. N.(1992). Hepatitis B virus capsid particles are assembled from core-protein dimer precursors. Proc Natl Acad Sci U S A 89, 10046–10050.[CrossRef] [Google Scholar]
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Assembly and export determine the intracellular distribution of hepatitis B virus core protein subunits
J. Gen. Virol. 91, 59 (2010); https://doi.org/10.1099/vir.0.013698-0
/content/journal/jgv/10.1099/vir.0.013698-0
/content/journal/jgv/10.1099/vir.0.013698-0
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Distribution patterns and sedimentation positions of the different GFP–HBc fusion proteins (construct 1) expressed alone or in combination with the non-fused proteins (construct 2) [ PDF] (56 KB)

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