Skip to main content
NCBI home page
Journal of Virology logoLink to Journal of Virology
. 1994 Sep;68(9):5548–5555. doi: 10.1128/jvi.68.9.5548-5555.1994

A protease-sensitive hinge linking the two domains of the hepatitis B virus core protein is exposed on the viral capsid surface.

M Seifer 1, D N Standring 1
PMCID: PMC236955  PMID: 7520091

Abstract

Core particles of hepatitis B virus are assembled from dimers of a single 185-residue (subtype adw) viral capsid or core protein (p21.5) which possesses two distinct domains: residues 1 to 144 form a minimal capsid assembly domain, and the arginine-rich, carboxyl-terminal residues 150 to 185 form a protamine-like domain that mediates nucleic acid binding. Little is known about the topography of the p21.5 polypeptide within either the p21.5 capsids or dimers. Here, using site-specific proteases and monoclonal antibodies, we have defined the accessibility of p21.5 residues in dimers and capsids assembled from wild-type and mutant hepatitis B virus core proteins in Xenopus oocytes and in vitro. The data reveal the protamine region to be accessible to external reagents in p21.5 dimers but largely cryptic in wild-type capsids. Strikingly, in capsids the only protease target region was a 9-residue peptide covering p21.5 residues Glu-145 to Asp-153, which falls largely between the two core protein domains. By analogy with protease-sensitive interdomain regions in other proteins, we propose that this peptide constitutes a hinge between the assembly and nucleic acid binding domains of p21.5. We further found that deletion or replacement of the terminal Cys-185 residue greatly increased surface exposure of the protamine tails in capsids, suggesting that a known disulfide linkage involving this residue tethers the protamine region inside the core particles. We propose that disruption of this disulfide linkage allows the protamine region to appear transiently on the surface of the core particle.

Full text

PDF
5548

Images in this article

5550Image
on p.5550
5550Image
on p.5550
5551Image
on p.5551
5552Image
on p.5552
5554Image
on p.5554

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Argos P., Fuller S. D. A model for the hepatitis B virus core protein: prediction of antigenic sites and relationship to RNA virus capsid proteins. EMBO J. 1988 Mar;7(3):819–824. doi: 10.1002/j.1460-2075.1988.tb02880.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bichko V., Schödel F., Nassal M., Gren E., Berzinsh I., Borisova G., Miska S., Peterson D. L., Gren E., Pushko P. Epitopes recognized by antibodies to denatured core protein of hepatitis B virus. Mol Immunol. 1993 Feb;30(3):221–231. doi: 10.1016/0161-5890(93)90051-c. [DOI] [PubMed] [Google Scholar]
  3. Birnbaum F., Nassal M. Hepatitis B virus nucleocapsid assembly: primary structure requirements in the core protein. J Virol. 1990 Jul;64(7):3319–3330. doi: 10.1128/jvi.64.7.3319-3330.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Clarke B. E., Brown A. L., Grace K. G., Hastings G. Z., Brown F., Rowlands D. J., Francis M. J. Presentation and immunogenicity of viral epitopes on the surface of hybrid hepatitis B virus core particles produced in bacteria. J Gen Virol. 1990 May;71(Pt 5):1109–1117. doi: 10.1099/0022-1317-71-5-1109. [DOI] [PubMed] [Google Scholar]
  5. Davies D. R., Metzger H. Structural basis of antibody function. Annu Rev Immunol. 1983;1:87–117. doi: 10.1146/annurev.iy.01.040183.000511. [DOI] [PubMed] [Google Scholar]
  6. Edman J. C., Hallewell R. A., Valenzuela P., Goodman H. M., Rutter W. J. Synthesis of hepatitis B surface and core antigens in E. coli. Nature. 1981 Jun 11;291(5815):503–506. doi: 10.1038/291503a0. [DOI] [PubMed] [Google Scholar]
  7. Gallina A., Bonelli F., Zentilin L., Rindi G., Muttini M., Milanesi G. A recombinant hepatitis B core antigen polypeptide with the protamine-like domain deleted self-assembles into capsid particles but fails to bind nucleic acids. J Virol. 1989 Nov;63(11):4645–4652. doi: 10.1128/jvi.63.11.4645-4652.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Ganem D. Assembly of hepadnaviral virions and subviral particles. Curr Top Microbiol Immunol. 1991;168:61–83. doi: 10.1007/978-3-642-76015-0_4. [DOI] [PubMed] [Google Scholar]
  9. Godley L., Pfeifer J., Steinhauer D., Ely B., Shaw G., Kaufmann R., Suchanek E., Pabo C., Skehel J. J., Wiley D. C. Introduction of intersubunit disulfide bonds in the membrane-distal region of the influenza hemagglutinin abolishes membrane fusion activity. Cell. 1992 Feb 21;68(4):635–645. doi: 10.1016/0092-8674(92)90140-8. [DOI] [PubMed] [Google Scholar]
  10. Hatton T., Zhou S., Standring D. N. RNA- and DNA-binding activities in hepatitis B virus capsid protein: a model for their roles in viral replication. J Virol. 1992 Sep;66(9):5232–5241. doi: 10.1128/jvi.66.9.5232-5241.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Imai M., Nomura M., Gotanda T., Sano T., Tachibana K., Miyamoto H., Takahashi K., Toyama S., Miyakawa Y., Mayumi M. Demonstration of two distinct antigenic determinants on hepatitis B e antigen by monoclonal antibodies. J Immunol. 1982 Jan;128(1):69–72. [PubMed] [Google Scholar]
  12. Kniskern P. J., Hagopian A., Montgomery D. L., Burke P., Dunn N. R., Hofmann K. J., Miller W. J., Ellis R. W. Unusually high-level expression of a foreign gene (hepatitis B virus core antigen) in Saccharomyces cerevisiae. Gene. 1986;46(1):135–141. doi: 10.1016/0378-1119(86)90177-0. [DOI] [PubMed] [Google Scholar]
  13. Lanford R. E., Notvall L. Expression of hepatitis B virus core and precore antigens in insect cells and characterization of a core-associated kinase activity. Virology. 1990 May;176(1):222–233. doi: 10.1016/0042-6822(90)90247-o. [DOI] [PubMed] [Google Scholar]
  14. Machida A., Ohnuma H., Takai E., Tsuda F., Tanaka T., Naito M., Munekata E., Miyakawa Y., Mayumi M. Antigenic sites on the arginine-rich carboxyl-terminal domain of the capsid protein of hepatitis B virus distinct from hepatitis B core or e antigen. Mol Immunol. 1989 Apr;26(4):413–421. doi: 10.1016/0161-5890(89)90130-2. [DOI] [PubMed] [Google Scholar]
  15. Machida A., Ohnuma H., Tsuda F., Yoshikawa A., Hoshi Y., Tanaka T., Kishimoto S., Akahane Y., Miyakawa Y., Mayumi M. Phosphorylation in the carboxyl-terminal domain of the capsid protein of hepatitis B virus: evaluation with a monoclonal antibody. J Virol. 1991 Nov;65(11):6024–6030. doi: 10.1128/jvi.65.11.6024-6030.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Nassal M., Rieger A., Steinau O. Topological analysis of the hepatitis B virus core particle by cysteine-cysteine cross-linking. J Mol Biol. 1992 Jun 20;225(4):1013–1025. doi: 10.1016/0022-2836(92)90101-o. [DOI] [PubMed] [Google Scholar]
  17. Nassal M. 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. 1992 Jul;66(7):4107–4116. doi: 10.1128/jvi.66.7.4107-4116.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Pugh J., Zweidler A., Summers J. Characterization of the major duck hepatitis B virus core particle protein. J Virol. 1989 Mar;63(3):1371–1376. doi: 10.1128/jvi.63.3.1371-1376.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Salfeld J., Pfaff E., Noah M., Schaller H. Antigenic determinants and functional domains in core antigen and e antigen from hepatitis B virus. J Virol. 1989 Feb;63(2):798–808. doi: 10.1128/jvi.63.2.798-808.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Schlicht H. J., Bartenschlager R., Schaller H. 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. 1989 Jul;63(7):2995–3000. doi: 10.1128/jvi.63.7.2995-3000.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Schödel F., Moriarty A. M., Peterson D. L., Zheng J. A., Hughes J. L., Will H., Leturcq D. J., McGee J. S., Milich D. R. The position of heterologous epitopes inserted in hepatitis B virus core particles determines their immunogenicity. J Virol. 1992 Jan;66(1):106–114. doi: 10.1128/jvi.66.1.106-114.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Seifer M., Heermann K. H., Gerlich W. H. Expression pattern of the hepatitis B virus genome in transfected mouse fibroblasts. Virology. 1990 Nov;179(1):287–299. doi: 10.1016/0042-6822(90)90297-5. [DOI] [PubMed] [Google Scholar]
  23. Seifer M., Standring D. N. Stability governs the apparent expression of "particulate" hepatitis B e antigen by mutant hepatitis B virus core particles. Virology. 1993 Sep;196(1):70–78. doi: 10.1006/viro.1993.1455. [DOI] [PubMed] [Google Scholar]
  24. Seifer M., Zhou S., Standring D. N. A micromolar pool of antigenically distinct precursors is required to initiate cooperative assembly of hepatitis B virus capsids in Xenopus oocytes. J Virol. 1993 Jan;67(1):249–257. doi: 10.1128/jvi.67.1.249-257.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Standring D. N., Ou J. H., Masiarz F. R., Rutter W. J. 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 U S A. 1988 Nov;85(22):8405–8409. doi: 10.1073/pnas.85.22.8405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sällberg M., Rudén U., Magnius L. O., Harthus H. P., Noah M., Wahren B. Characterisation of a linear binding site for a monoclonal antibody to hepatitis B core antigen. J Med Virol. 1991 Apr;33(4):248–252. doi: 10.1002/jmv.1890330407. [DOI] [PubMed] [Google Scholar]
  27. Takahashi K., Machida A., Funatsu G., Nomura M., Usuda S., Aoyagi S., Tachibana K., Miyamoto H., Imai M., Nakamura T. Immunochemical structure of hepatitis B e antigen in the serum. J Immunol. 1983 Jun;130(6):2903–2907. [PubMed] [Google Scholar]
  28. Zheng J., Schödel F., Peterson D. L. The structure of hepadnaviral core antigens. Identification of free thiols and determination of the disulfide bonding pattern. J Biol Chem. 1992 May 5;267(13):9422–9429. [PubMed] [Google Scholar]
  29. Zhou S. L., Standring D. N. Production of hepatitis B virus nucleocapsidlike core particles in Xenopus oocytes: assembly occurs mainly in the cytoplasm and does not require the nucleus. J Virol. 1991 Oct;65(10):5457–5464. doi: 10.1128/jvi.65.10.5457-5464.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Zhou S., Standring D. N. Cys residues of the hepatitis B virus capsid protein are not essential for the assembly of viral core particles but can influence their stability. J Virol. 1992 Sep;66(9):5393–5398. doi: 10.1128/jvi.66.9.5393-5398.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Zhou S., Standring D. N. Hepatitis B virus capsid particles are assembled from core-protein dimer precursors. Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10046–10050. doi: 10.1073/pnas.89.21.10046. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Zhou S., Yang S. Q., Standring D. N. Characterization of hepatitis B virus capsid particle assembly in Xenopus oocytes. J Virol. 1992 May;66(5):3086–3092. doi: 10.1128/jvi.66.5.3086-3092.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES