Abstract
A characteristic of all hepadnaviruses is the relaxed-circular conformation of the DNA genome within an infectious virion. Synthesis of the relaxed-circular genome by reverse transcription requires three template switches. These template switches, as for the template switches or strand transfers of other reverse-transcribing genetic elements, require repeated sequences (the donor and acceptor sites) between which a complementary strand of nucleic acid is transferred. The mechanism for each of the template switches in hepadnaviruses is poorly understood. To determine whether sequences other than the donor and acceptor sites are involved in the template switches of duck hepatitis B virus (DHBV), a series of molecular clones which express viral genomes bearing deletion mutations were analyzed. We found that three regions of the DHBV genome, which are distinct from the donor and acceptor sites, are required for the synthesis of relaxed-circular DNA. One region, located near the 3' end of the minus-strand template, is required for the template switch that circularizes the genome. The other two regions, located in the middle of the genome and near DR2, appear to be required for plus-strand primer translocation. We speculate that these cis-acting sequences may play a role in the organization of the minus-strand DNA template within the capsid particle so that it supports efficient template switching during plus-strand DNA synthesis.
Full Text
The Full Text of this article is available as a PDF (1.3 MB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- 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]
- Chang L. J., Hirsch R. C., Ganem D., Varmus H. E. Effects of insertional and point mutations on the functions of the duck hepatitis B virus polymerase. J Virol. 1990 Nov;64(11):5553–5558. doi: 10.1128/jvi.64.11.5553-5558.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Chen Y., Robinson W. S., Marion P. L. Selected mutations of the duck hepatitis B virus P gene RNase H domain affect both RNA packaging and priming of minus-strand DNA synthesis. J Virol. 1994 Aug;68(8):5232–5238. doi: 10.1128/jvi.68.8.5232-5238.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Church G. M., Gilbert W. Genomic sequencing. Proc Natl Acad Sci U S A. 1984 Apr;81(7):1991–1995. doi: 10.1073/pnas.81.7.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- 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]
- Hoheisel J., Pohl F. M. Simplified preparation of unidirectional deletion clones. Nucleic Acids Res. 1986 Apr 25;14(8):3605–3605. doi: 10.1093/nar/14.8.3605. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Huang M., Summers J. pet, a small sequence distal to the pregenome cap site, is required for expression of the duck hepatitis B virus pregenome. J Virol. 1994 Mar;68(3):1564–1572. doi: 10.1128/jvi.68.3.1564-1572.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- Lien J. M., Aldrich C. E., Mason W. S. Evidence that a capped oligoribonucleotide is the primer for duck hepatitis B virus plus-strand DNA synthesis. J Virol. 1986 Jan;57(1):229–236. doi: 10.1128/jvi.57.1.229-236.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Loeb D. D., Gulya K. J., Tian R. Sequence identity of the terminal redundancies on the minus-strand DNA template is necessary but not sufficient for the template switch during hepadnavirus plus-strand DNA synthesis. J Virol. 1997 Jan;71(1):152–160. doi: 10.1128/jvi.71.1.152-160.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Loeb D. D., Hirsch R. C., Ganem D. Sequence-independent RNA cleavages generate the primers for plus strand DNA synthesis in hepatitis B viruses: implications for other reverse transcribing elements. EMBO J. 1991 Nov;10(11):3533–3540. doi: 10.1002/j.1460-2075.1991.tb04917.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Loeb D. D., Tian R., Gulya K. J. Mutations within DR2 independently reduce the amount of both minus- and plus-strand DNA synthesized during duck hepatitis B virus replication. J Virol. 1996 Dec;70(12):8684–8690. doi: 10.1128/jvi.70.12.8684-8690.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Loeb D. D., Tian R. Transfer of the minus strand of DNA during hepadnavirus replication is not invariable but prefers a specific location. J Virol. 1995 Nov;69(11):6886–6891. doi: 10.1128/jvi.69.11.6886-6891.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mandart E., Kay A., Galibert F. Nucleotide sequence of a cloned duck hepatitis B virus genome: comparison with woodchuck and human hepatitis B virus sequences. J Virol. 1984 Mar;49(3):782–792. doi: 10.1128/jvi.49.3.782-792.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mueller-Hill K., Loeb D. D. Previously unsuspected cis-acting sequences for DNA replication revealed by characterization of a chimeric heron/duck hepatitis B virus. J Virol. 1996 Dec;70(12):8310–8317. doi: 10.1128/jvi.70.12.8310-8317.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Radziwill G., Tucker W., Schaller H. Mutational analysis of the hepatitis B virus P gene product: domain structure and RNase H activity. J Virol. 1990 Feb;64(2):613–620. doi: 10.1128/jvi.64.2.613-620.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]