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. 1997 Jan;71(1):345–353. doi: 10.1128/jvi.71.1.345-353.1997

Posttranscriptional regulation of hepatitis B virus replication by the precore protein.

P P Scaglioni 1, M Melegari 1, J R Wands 1
PMCID: PMC191057  PMID: 8985356

Abstract

Hepadnaviruses encode two core-related open reading frames. One directs the synthesis of the p21 core protein, which subsequently becomes a structural component of the viral nucleocapsid. The other produces a p25 precore protein that is targeted by a signal peptide to a cell secretory pathway where N-terminal processing will create a p22 species. This molecule will be further modified at the C-terminal region to generate p17, and the truncated protein is secreted from the cell as hepatitis B e antigen (HBeAg). The function of the precore gene in the biology of hepadnaviruses is unknown. We found that ablation of the precore gene resulted in the generation of a hepatitis B virus (HBV) species with a high-replication-level phenotype. More important, expression in trans of physiologic levels of p25 restored viral replication to wild-type levels. Moreover, transient or stable overexpression of the precore gene resulted in striking inhibition of HBV replication. The molecular species responsible for this viral inhibitory effect was identified as the p22 nonsecreted HBeAg precursor protein. By sucrose gradient sedimentation analysis, we determined that expression of p22 leads to the formation of nucleocapsids similar to those made with wild-type p21 core protein. Immunoprecipitation experiments revealed that the p21 and p22 physically interact and form hybrid nucleocapsid structures devoid of pregenomic viral RNA. These experiments suggest that expression of the precore gene may be important in the regulation of HBV replication and describe a possible molecular mechanism(s) for this effect.

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Selected References

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  1. Acs G., Sells M. A., Purcell R. H., Price P., Engle R., Shapiro M., Popper H. Hepatitis B virus produced by transfected Hep G2 cells causes hepatitis in chimpanzees. Proc Natl Acad Sci U S A. 1987 Jul;84(13):4641–4644. doi: 10.1073/pnas.84.13.4641. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bartenschlager R., Schaller H. Hepadnaviral assembly is initiated by polymerase binding to the encapsidation signal in the viral RNA genome. EMBO J. 1992 Sep;11(9):3413–3420. doi: 10.1002/j.1460-2075.1992.tb05420.x. [DOI] [PMC free article] [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. Bruss V., Gerlich W. H. Formation of transmembraneous hepatitis B e-antigen by cotranslational in vitro processing of the viral precore protein. Virology. 1988 Apr;163(2):268–275. doi: 10.1016/0042-6822(88)90266-8. [DOI] [PubMed] [Google Scholar]
  5. Buckwold V. E., Xu Z., Chen M., Yen T. S., Ou J. H. Effects of a naturally occurring mutation in the hepatitis B virus basal core promoter on precore gene expression and viral replication. J Virol. 1996 Sep;70(9):5845–5851. doi: 10.1128/jvi.70.9.5845-5851.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Carman W. F., Fagan E. A., Hadziyannis S., Karayiannis P., Tassopoulos N. C., Williams R., Thomas H. C. Association of a precore genomic variant of hepatitis B virus with fulminant hepatitis. Hepatology. 1991 Aug;14(2):219–222. [PubMed] [Google Scholar]
  7. Carman W. F., Jacyna M. R., Hadziyannis S., Karayiannis P., McGarvey M. J., Makris A., Thomas H. C. Mutation preventing formation of hepatitis B e antigen in patients with chronic hepatitis B infection. Lancet. 1989 Sep 9;2(8663):588–591. doi: 10.1016/s0140-6736(89)90713-7. [DOI] [PubMed] [Google Scholar]
  8. Chang C., Enders G., Sprengel R., Peters N., Varmus H. E., Ganem D. Expression of the precore region of an avian hepatitis B virus is not required for viral replication. J Virol. 1987 Oct;61(10):3322–3325. doi: 10.1128/jvi.61.10.3322-3325.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chang L. J., Ganem D., Varmus H. E. Mechanism of translation of the hepadnaviral polymerase (P) gene. Proc Natl Acad Sci U S A. 1990 Jul;87(13):5158–5162. doi: 10.1073/pnas.87.13.5158. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chen H. S., Kew M. C., Hornbuckle W. E., Tennant B. C., Cote P. J., Gerin J. L., Purcell R. H., Miller R. H. The precore gene of the woodchuck hepatitis virus genome is not essential for viral replication in the natural host. J Virol. 1992 Sep;66(9):5682–5684. doi: 10.1128/jvi.66.9.5682-5684.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fallows D. A., Goff S. P. Mutations in the epsilon sequences of human hepatitis B virus affect both RNA encapsidation and reverse transcription. J Virol. 1995 May;69(5):3067–3073. doi: 10.1128/jvi.69.5.3067-3073.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Galibert F., Mandart E., Fitoussi F., Tiollais P., Charnay P. Nucleotide sequence of the hepatitis B virus genome (subtype ayw) cloned in E. coli. Nature. 1979 Oct 25;281(5733):646–650. doi: 10.1038/281646a0. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Garcia P. D., Ou J. H., Rutter W. J., 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 Apr;106(4):1093–1104. doi: 10.1083/jcb.106.4.1093. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Graham F. L., Smiley J., Russell W. C., Nairn R. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol. 1977 Jul;36(1):59–74. doi: 10.1099/0022-1317-36-1-59. [DOI] [PubMed] [Google Scholar]
  16. Guidotti L. G., Matzke B., Pasquinelli C., Shoenberger J. M., Rogler C. E., Chisari F. V. The hepatitis B virus (HBV) precore protein inhibits HBV replication in transgenic mice. J Virol. 1996 Oct;70(10):7056–7061. doi: 10.1128/jvi.70.10.7056-7061.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Hirsch R. C., Lavine J. E., Chang L. J., Varmus H. E., Ganem D. Polymerase gene products of hepatitis B viruses are required for genomic RNA packaging as wel as for reverse transcription. Nature. 1990 Apr 5;344(6266):552–555. doi: 10.1038/344552a0. [DOI] [PubMed] [Google Scholar]
  19. Lamberts C., Nassal M., Velhagen I., Zentgraf H., Schröder C. H. Precore-mediated inhibition of hepatitis B virus progeny DNA synthesis. J Virol. 1993 Jul;67(7):3756–3762. doi: 10.1128/jvi.67.7.3756-3762.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Liang T. J., Hasegawa K., Rimon N., Wands J. R., Ben-Porath E. A hepatitis B virus mutant associated with an epidemic of fulminant hepatitis. N Engl J Med. 1991 Jun 13;324(24):1705–1709. doi: 10.1056/NEJM199106133242405. [DOI] [PubMed] [Google Scholar]
  21. Lingappa J. R., Martin R. L., Wong M. L., Ganem D., Welch W. J., Lingappa V. R. A eukaryotic cytosolic chaperonin is associated with a high molecular weight intermediate in the assembly of hepatitis B virus capsid, a multimeric particle. J Cell Biol. 1994 Apr;125(1):99–111. doi: 10.1083/jcb.125.1.99. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Melegari M., Bruno S., Wands J. R. Properties of hepatitis B virus pre-S1 deletion mutants. Virology. 1994 Mar;199(2):292–300. doi: 10.1006/viro.1994.1127. [DOI] [PubMed] [Google Scholar]
  23. Milich D. R., Jones J. E., Hughes J. L., Price J., Raney A. K., McLachlan A. Is a function of the secreted hepatitis B e antigen to induce immunologic tolerance in utero? Proc Natl Acad Sci U S A. 1990 Sep;87(17):6599–6603. doi: 10.1073/pnas.87.17.6599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Miller A. D., Miller D. G., Garcia J. V., Lynch C. M. Use of retroviral vectors for gene transfer and expression. Methods Enzymol. 1993;217:581–599. doi: 10.1016/0076-6879(93)17090-r. [DOI] [PubMed] [Google Scholar]
  25. Morgenstern J. P., Land H. Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line. Nucleic Acids Res. 1990 Jun 25;18(12):3587–3596. doi: 10.1093/nar/18.12.3587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Naoumov N. V., Schneider R., Grötzinger T., Jung M. C., Miska S., Pape G. R., Will H. Precore mutant hepatitis B virus infection and liver disease. Gastroenterology. 1992 Feb;102(2):538–543. doi: 10.1016/0016-5085(92)90101-4. [DOI] [PubMed] [Google Scholar]
  27. Nassal M., Junker-Niepmann M., Schaller H. Translational inactivation of RNA function: discrimination against a subset of genomic transcripts during HBV nucleocapsid assembly. Cell. 1990 Dec 21;63(6):1357–1363. doi: 10.1016/0092-8674(90)90431-d. [DOI] [PubMed] [Google Scholar]
  28. Nassal M., Rieger A. 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. 1993 Jul;67(7):4307–4315. doi: 10.1128/jvi.67.7.4307-4315.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Okamoto H., Tsuda F., Akahane Y., Sugai Y., Yoshiba M., Moriyama K., Tanaka T., Miyakawa Y., Mayumi M. Hepatitis B virus with mutations in the core promoter for an e antigen-negative phenotype in carriers with antibody to e antigen. J Virol. 1994 Dec;68(12):8102–8110. doi: 10.1128/jvi.68.12.8102-8110.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Okamoto H., Yotsumoto S., Akahane Y., Yamanaka T., Miyazaki Y., Sugai Y., Tsuda F., Tanaka T., Miyakawa Y., Mayumi M. Hepatitis B viruses with precore region defects prevail in persistently infected hosts along with seroconversion to the antibody against e antigen. J Virol. 1990 Mar;64(3):1298–1303. doi: 10.1128/jvi.64.3.1298-1303.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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 Jun 13;324(24):1699–1704. doi: 10.1056/NEJM199106133242404. [DOI] [PubMed] [Google Scholar]
  33. Ou J. H., Laub O., Rutter W. J. Hepatitis B virus gene function: the precore region targets the core antigen to cellular membranes and causes the secretion of the e antigen. Proc Natl Acad Sci U S A. 1986 Mar;83(6):1578–1582. doi: 10.1073/pnas.83.6.1578. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Pear W. S., Nolan G. P., Scott M. L., Baltimore D. Production of high-titer helper-free retroviruses by transient transfection. Proc Natl Acad Sci U S A. 1993 Sep 15;90(18):8392–8396. doi: 10.1073/pnas.90.18.8392. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Pugh J. C., Yaginuma K., Koike K., Summers J. Duck hepatitis B virus (DHBV) particles produced by transient expression of DHBV DNA in a human hepatoma cell line are infectious in vitro. J Virol. 1988 Sep;62(9):3513–3516. doi: 10.1128/jvi.62.9.3513-3516.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Roychoudhury S., Faruqi A. F., Shih C. Pregenomic RNA encapsidation analysis of eleven missense and nonsense polymerase mutants of human hepatitis B virus. J Virol. 1991 Jul;65(7):3617–3624. doi: 10.1128/jvi.65.7.3617-3624.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Scaglioni P. P., Melegari M., Wands J. R. Characterization of hepatitis B virus core mutants that inhibit viral replication. Virology. 1994 Nov 15;205(1):112–120. doi: 10.1006/viro.1994.1625. [DOI] [PubMed] [Google Scholar]
  38. Schlicht H. J., Salfeld J., Schaller H. The duck hepatitis B virus pre-C region encodes a signal sequence which is essential for synthesis and secretion of processed core proteins but not for virus formation. J Virol. 1987 Dec;61(12):3701–3709. doi: 10.1128/jvi.61.12.3701-3709.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Schlicht H. J., Schaller H. The secretory core protein of human hepatitis B virus is expressed on the cell surface. J Virol. 1989 Dec;63(12):5399–5404. doi: 10.1128/jvi.63.12.5399-5404.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Schlicht H. J., Wasenauer G. The quaternary structure, antigenicity, and aggregational behavior of the secretory core protein of human hepatitis B virus are determined by its signal sequence. J Virol. 1991 Dec;65(12):6817–6825. doi: 10.1128/jvi.65.12.6817-6825.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Seeger C., Baldwin B., Tennant B. C. Expression of infectious woodchuck hepatitis virus in murine and avian fibroblasts. J Virol. 1989 Nov;63(11):4665–4669. doi: 10.1128/jvi.63.11.4665-4669.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Sells M. A., Chen M. L., Acs G. Production of hepatitis B virus particles in Hep G2 cells transfected with cloned hepatitis B virus DNA. Proc Natl Acad Sci U S A. 1987 Feb;84(4):1005–1009. doi: 10.1073/pnas.84.4.1005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Tong S. P., Li J. S., Vitvitski L., Trépo C. Active hepatitis B virus replication in the presence of anti-HBe is associated with viral variants containing an inactive pre-C region. Virology. 1990 Jun;176(2):596–603. doi: 10.1016/0042-6822(90)90030-u. [DOI] [PubMed] [Google Scholar]
  44. Tong S. P., Li J. S., Vitvitski L., Trépo C. Replication capacities of natural and artificial precore stop codon mutants of hepatitis B virus: relevance of pregenome encapsidation signal. Virology. 1992 Nov;191(1):237–245. doi: 10.1016/0042-6822(92)90185-r. [DOI] [PubMed] [Google Scholar]
  45. Weimer T., Salfeld J., Will H. Expression of the hepatitis B virus core gene in vitro and in vivo. J Virol. 1987 Oct;61(10):3109–3113. doi: 10.1128/jvi.61.10.3109-3113.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Will H., Reiser W., Weimer T., Pfaff E., Büscher M., Sprengel R., Cattaneo R., Schaller H. Replication strategy of human hepatitis B virus. J Virol. 1987 Mar;61(3):904–911. doi: 10.1128/jvi.61.3.904-911.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Yaginuma K., Shirakata Y., Kobayashi M., Koike K. 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. 1987 May;84(9):2678–2682. doi: 10.1073/pnas.84.9.2678. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Zhang P., McLachlan A. Differentiation-specific transcriptional regulation of the hepatitis B virus nucleocapsid gene in human hepatoma cell lines. Virology. 1994 Jul;202(1):430–440. doi: 10.1006/viro.1994.1359. [DOI] [PubMed] [Google Scholar]
  49. 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]

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