Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1988 Apr 1;106(4):1093–1104. doi: 10.1083/jcb.106.4.1093

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

PMCID: PMC2114996  PMID: 3283145

Abstract

The major hepatitis B virus (HBV) core protein is a viral structural protein involved in nucleic acid binding. Its coding sequence contains an extension of 29 codons (the "precore" region) at the amino terminus of the protein which is present in a fraction of the viral transcripts. This region is evolutionarily conserved among mammalian and avian HBVs, suggesting it has functional importance, although at least for duck HBV it has been shown to be nonessential for replication of infectious virions. Using in vitro assays for protein translocation across the endoplasmic reticulum membrane, we found that the precore region of the HBV genome encodes a signal sequence. This signal sequence was recognized by signal recognition particle, which targeted the nascent precore protein to the endoplasmic reticulum membrane with efficiencies comparable to those of other mammalian secretory proteins. A 19-amino acid signal peptide was removed by signal peptidase on the lumenal side of the microsomal membrane, generating a protein similar to the HBV major core protein, but containing 10 additional amino acids from the precore region at its amino terminus. Surprisingly, we found that 70- 80% of this signal peptidase-cleaved product was localized on the cytoplasmic side of the microsomal vesicles and was not associated with the membranes. We conclude that translocation was aborted by an unknown mechanism, then the protein disengaged from the translocation machinery and was released back into the cytoplasm. Thus, a cytoplasmically disposed protein was created whose amino terminus resulted from signal peptidase cleavage. The remaining 20-30% appeared to be completely translocated into the lumen of the microsomes. A deletion mutant lacking the carboxy-terminal nucleic acid binding domain of the precore protein was similarly partitioned between the lumen of the microsomes and the cytoplasmic compartment, indicating that this highly charged domain is not responsible for the aborted translocation. We discuss the implications of our findings for the protein translocation process and suggest a possible role in the virus life cycle.

Full Text

The Full Text of this article is available as a PDF (2.0 MB).

Selected References

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

  1. Blobel G. Intracellular protein topogenesis. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1496–1500. doi: 10.1073/pnas.77.3.1496. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. Eble B. E., Lingappa V. R., Ganem D. Hepatitis B surface antigen: an unusual secreted protein initially synthesized as a transmembrane polypeptide. Mol Cell Biol. 1986 May;6(5):1454–1463. doi: 10.1128/mcb.6.5.1454. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Enders G. H., Ganem D., Varmus H. E. 5'-terminal sequences influence the segregation of ground squirrel hepatitis virus RNAs into polyribosomes and viral core particles. J Virol. 1987 Jan;61(1):35–41. doi: 10.1128/jvi.61.1.35-41.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Enders G. H., Ganem D., Varmus H. Mapping the major transcripts of ground squirrel hepatitis virus: the presumptive template for reverse transcriptase is terminally redundant. Cell. 1985 Aug;42(1):297–308. doi: 10.1016/s0092-8674(85)80125-2. [DOI] [PubMed] [Google Scholar]
  6. Erickson A. H., Blobel G. Cell-free translation of messenger RNA in a wheat germ system. Methods Enzymol. 1983;96:38–50. doi: 10.1016/s0076-6879(83)96007-x. [DOI] [PubMed] [Google Scholar]
  7. Garcia P. D., Ghrayeb J., Inouye M., Walter P. Wild type and mutant signal peptides of Escherichia coli outer membrane lipoprotein interact with equal efficiency with mammalian signal recognition particle. J Biol Chem. 1987 Jul 15;262(20):9463–9468. [PubMed] [Google Scholar]
  8. Hunkapiller M. W., Lujan E., Ostrander F., Hood L. E. Isolation of microgram quantities of proteins from polyacrylamide gels for amino acid sequence analysis. Methods Enzymol. 1983;91:227–236. doi: 10.1016/s0076-6879(83)91019-4. [DOI] [PubMed] [Google Scholar]
  9. Krieg P. A., Melton D. A. In vitro RNA synthesis with SP6 RNA polymerase. Methods Enzymol. 1987;155:397–415. doi: 10.1016/0076-6879(87)55027-3. [DOI] [PubMed] [Google Scholar]
  10. McLachlan A., Milich D. R., Raney A. K., Riggs M. G., Hughes J. L., Sorge J., Chisari F. V. Expression of hepatitis B virus surface and core antigens: influences of pre-S and precore sequences. J Virol. 1987 Mar;61(3):683–692. doi: 10.1128/jvi.61.3.683-692.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Ohno-Iwashita Y., Wolfe P., Ito K., Wickner W. Processing of preproteins by liposomes bearing leader peptidase. Biochemistry. 1984 Dec 4;23(25):6178–6184. doi: 10.1021/bi00320a044. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Roossinck M. J., Jameel S., Loukin S. H., Siddiqui A. Expression of hepatitis B viral core region in mammalian cells. Mol Cell Biol. 1986 May;6(5):1393–1400. doi: 10.1128/mcb.6.5.1393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Roossinck M. J., Siddiqui A. In vivo phosphorylation and protein analysis of hepatitis B virus core antigen. J Virol. 1987 Apr;61(4):955–961. doi: 10.1128/jvi.61.4.955-961.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Standring D. N., Rutter W. J. The molecular analysis of hepatitis B virus. Prog Liver Dis. 1986;8:311–333. [PubMed] [Google Scholar]
  16. 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]
  17. Tiollais P., Pourcel C., Dejean A. The hepatitis B virus. Nature. 1985 Oct 10;317(6037):489–495. doi: 10.1038/317489a0. [DOI] [PubMed] [Google Scholar]
  18. Walter P., Blobel G. Preparation of microsomal membranes for cotranslational protein translocation. Methods Enzymol. 1983;96:84–93. doi: 10.1016/s0076-6879(83)96010-x. [DOI] [PubMed] [Google Scholar]
  19. Walter P., Blobel G. Purification of a membrane-associated protein complex required for protein translocation across the endoplasmic reticulum. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7112–7116. doi: 10.1073/pnas.77.12.7112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Walter P., Blobel G. Signal recognition particle: a ribonucleoprotein required for cotranslational translocation of proteins, isolation and properties. Methods Enzymol. 1983;96:682–691. doi: 10.1016/s0076-6879(83)96057-3. [DOI] [PubMed] [Google Scholar]
  21. Walter P., Blobel G. Translocation of proteins across the endoplasmic reticulum III. Signal recognition protein (SRP) causes signal sequence-dependent and site-specific arrest of chain elongation that is released by microsomal membranes. J Cell Biol. 1981 Nov;91(2 Pt 1):557–561. doi: 10.1083/jcb.91.2.557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Walter P., Ibrahimi I., Blobel G. Translocation of proteins across the endoplasmic reticulum. I. Signal recognition protein (SRP) binds to in-vitro-assembled polysomes synthesizing secretory protein. J Cell Biol. 1981 Nov;91(2 Pt 1):545–550. doi: 10.1083/jcb.91.2.545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Watson M. E. Compilation of published signal sequences. Nucleic Acids Res. 1984 Jul 11;12(13):5145–5164. doi: 10.1093/nar/12.13.5145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Yost C. S., Hedgpeth J., Lingappa V. R. A stop transfer sequence confers predictable transmembrane orientation to a previously secreted protein in cell-free systems. Cell. 1983 Oct;34(3):759–766. doi: 10.1016/0092-8674(83)90532-9. [DOI] [PubMed] [Google Scholar]
  25. Zimmermann R., Mollay C. Import of honeybee prepromelittin into the endoplasmic reticulum. Requirements for membrane insertion, processing, and sequestration. J Biol Chem. 1986 Sep 25;261(27):12889–12895. [PubMed] [Google Scholar]
  26. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded DNA template. DNA. 1984 Dec;3(6):479–488. doi: 10.1089/dna.1.1984.3.479. [DOI] [PubMed] [Google Scholar]
  27. von Heijne G. How signal sequences maintain cleavage specificity. J Mol Biol. 1984 Feb 25;173(2):243–251. doi: 10.1016/0022-2836(84)90192-x. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES