Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1995 Nov;69(11):6886–6891. doi: 10.1128/jvi.69.11.6886-6891.1995

Transfer of the minus strand of DNA during hepadnavirus replication is not invariable but prefers a specific location.

D D Loeb 1, R Tian 1
PMCID: PMC189604  PMID: 7474104

Abstract

The current model for replication of duck hepatitis B virus has reverse transcription initiating and copying a UUAC motif within the encapsidation signal, epsilon, near the 5' end of the RNA template. This results in synthesis of four nucleotides of DNA. This short minus-strand DNA product is then transferred to a complementary position, at DR1, near the 3' end of the RNA template. Elongation of minus-strand DNA then ensues. We have examined the transfer of minus-strand DNA during replication of duck hepatitis B virus in cell culture. The initial aim of this work was to examine the effect of mutations at DR1 on the transfer process. We found that when mutations were introduced into the UUAC motif overlapping DR1, the 5' end of minus-DNA no longer mapped to position 2537 but was shifted two or four nucleotides. Mismatches were predicted to exist at the new sites of elongation. Elongation from nucleotide 2537 could be restored in these mutants by making compensatory changes in the UUAC motif within epsilon. This finding led us to examine limitations in the shifting of the site of transfer. When the UUAC motif in epsilon was changed to six different tetranucleotide motifs surrounding position 2537, transfer of minus-strand DNA shifted predictably, albeit inefficiently. Also, when multiple UUAC motifs were introduced near DR1, the UUAC motif at nucleotide 2537 was used preferentially. Overall, our findings confirm the current minus-strand DNA transfer model and demonstrate a marked preference for the site of the transfer.

Full Text

The Full Text of this article is available as a PDF (2.2 MB).

Selected References

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

  1. Bartenschlager R., Junker-Niepmann M., Schaller H. The P gene product of hepatitis B virus is required as a structural component for genomic RNA encapsidation. J Virol. 1990 Nov;64(11):5324–5332. doi: 10.1128/jvi.64.11.5324-5332.1990. [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. Calvert J., Summers J. Two regions of an avian hepadnavirus RNA pregenome are required in cis for encapsidation. J Virol. 1994 Apr;68(4):2084–2090. doi: 10.1128/jvi.68.4.2084-2090.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chang L. J., Pryciak P., Ganem D., Varmus H. E. Biosynthesis of the reverse transcriptase of hepatitis B viruses involves de novo translational initiation not ribosomal frameshifting. Nature. 1989 Jan 26;337(6205):364–368. doi: 10.1038/337364a0. [DOI] [PubMed] [Google Scholar]
  5. Chen C., Okayama H. High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol. 1987 Aug;7(8):2745–2752. doi: 10.1128/mcb.7.8.2745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Condreay L. D., Aldrich C. E., Coates L., Mason W. S., Wu T. T. Efficient duck hepatitis B virus production by an avian liver tumor cell line. J Virol. 1990 Jul;64(7):3249–3258. doi: 10.1128/jvi.64.7.3249-3258.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Condreay L. D., Wu T. T., Aldrich C. E., Delaney M. A., Summers J., Seeger C., Mason W. S. Replication of DHBV genomes with mutations at the sites of initiation of minus- and plus-strand DNA synthesis. Virology. 1992 May;188(1):208–216. doi: 10.1016/0042-6822(92)90751-a. [DOI] [PubMed] [Google Scholar]
  8. Ganem D., Varmus H. E. The molecular biology of the hepatitis B viruses. Annu Rev Biochem. 1987;56:651–693. doi: 10.1146/annurev.bi.56.070187.003251. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Hirsch R. C., Loeb D. D., Pollack J. R., Ganem D. cis-acting sequences required for encapsidation of duck hepatitis B virus pregenomic RNA. J Virol. 1991 Jun;65(6):3309–3316. doi: 10.1128/jvi.65.6.3309-3316.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Junker-Niepmann M., Bartenschlager R., Schaller H. A short cis-acting sequence is required for hepatitis B virus pregenome encapsidation and sufficient for packaging of foreign RNA. EMBO J. 1990 Oct;9(10):3389–3396. doi: 10.1002/j.1460-2075.1990.tb07540.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kawaguchi T., Nomura K., Hirayama Y., Kitagawa T. Establishment and characterization of a chicken hepatocellular carcinoma cell line, LMH. Cancer Res. 1987 Aug 15;47(16):4460–4464. [PubMed] [Google Scholar]
  13. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  14. Pollack J. R., Ganem D. An RNA stem-loop structure directs hepatitis B virus genomic RNA encapsidation. J Virol. 1993 Jun;67(6):3254–3263. doi: 10.1128/jvi.67.6.3254-3263.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Pollack J. R., Ganem D. Site-specific RNA binding by a hepatitis B virus reverse transcriptase initiates two distinct reactions: RNA packaging and DNA synthesis. J Virol. 1994 Sep;68(9):5579–5587. doi: 10.1128/jvi.68.9.5579-5587.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Schlicht H. J., Radziwill G., Schaller H. Synthesis and encapsidation of duck hepatitis B virus reverse transcriptase do not require formation of core-polymerase fusion proteins. Cell. 1989 Jan 13;56(1):85–92. doi: 10.1016/0092-8674(89)90986-0. [DOI] [PubMed] [Google Scholar]
  17. Seeger C., Maragos J. Identification of a signal necessary for initiation of reverse transcription of the hepadnavirus genome. J Virol. 1991 Oct;65(10):5190–5195. doi: 10.1128/jvi.65.10.5190-5195.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Sprengel R., Kuhn C., Will H., Schaller H. Comparative sequence analysis of duck and human hepatitis B virus genomes. J Med Virol. 1985 Apr;15(4):323–333. doi: 10.1002/jmv.1890150402. [DOI] [PubMed] [Google Scholar]
  19. Staprans S., Loeb D. D., Ganem D. Mutations affecting hepadnavirus plus-strand DNA synthesis dissociate primer cleavage from translocation and reveal the origin of linear viral DNA. J Virol. 1991 Mar;65(3):1255–1262. doi: 10.1128/jvi.65.3.1255-1262.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Summers J., Mason W. S. Replication of the genome of a hepatitis B--like virus by reverse transcription of an RNA intermediate. Cell. 1982 Jun;29(2):403–415. doi: 10.1016/0092-8674(82)90157-x. [DOI] [PubMed] [Google Scholar]
  21. Tavis J. E., Perri S., Ganem D. Hepadnavirus reverse transcription initiates within the stem-loop of the RNA packaging signal and employs a novel strand transfer. J Virol. 1994 Jun;68(6):3536–3543. doi: 10.1128/jvi.68.6.3536-3543.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Wang G. H., Seeger C. Novel mechanism for reverse transcription in hepatitis B viruses. J Virol. 1993 Nov;67(11):6507–6512. doi: 10.1128/jvi.67.11.6507-6512.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Wang G. H., Seeger C. The reverse transcriptase of hepatitis B virus acts as a protein primer for viral DNA synthesis. Cell. 1992 Nov 13;71(4):663–670. doi: 10.1016/0092-8674(92)90599-8. [DOI] [PubMed] [Google Scholar]

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

RESOURCES