Abstract
Hepadnaviruses replicate by reverse transcription of an RNA pregenome. Reverse transcription initiates within the stem-loop (SL) of the epsilon RNA packaging signal and is discontinuous: the nascent minus-polarity DNA is transferred to direct repeat 1 (DR1) at the 3' end of the pregenomic RNA prior to extensive elongation. In this study we analyzed the initiation and transfer of duck hepatitis B virus minus-strand DNA by using functional viral polymerase expressed in yeast cells. We extensively mutagenized both DR1 and the SL and observed the effects on reverse transcription initiation and on the transfer and subsequent extension of minus-strand DNA. Our results indicate that sequences throughout the SL affect initiation and that minus-strand DNAs initiated at three locations within the SL are competent for transfer to DR1. A short region of homology between the 5' end of minus-strand DNA and DR1 was necessary but not sufficient to direct the transfer and subsequent extension reactions. This homology was tolerant of minor substitutions, and 2 nucleotides of homology mediated transfer accurately. Mutations had greater detrimental effects on transfer and subsequent extension of minus-strand DNA when they were placed in DR1 than when they were placed in the SL. Efficient transfer of minus-strand DNA from a mutant SL to DR2 was observed in the yeast system. The hexanucleotide AAUUAC was identified as the primary cis element of the transfer acceptor, but this element was also insufficient to independently specify the acceptor location. Therefore, additional information, possibly positional context or unrecognized RNA secondary structure, is required.
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Selected References
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- 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]
- Ganem D., Pollack J. R., Tavis J. Hepatitis B virus reverse transcriptase and its many roles in hepadnaviral genomic replication. Infect Agents Dis. 1994 Apr-Jun;3(2-3):85–93. [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]
- 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]
- Seeger C., Maragos J. Identification and characterization of the woodchuck hepatitis virus origin of DNA replication. J Virol. 1990 Jan;64(1):16–23. doi: 10.1128/jvi.64.1.16-23.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Tavis J. E., Ganem D. Expression of functional hepatitis B virus polymerase in yeast reveals it to be the sole viral protein required for correct initiation of reverse transcription. Proc Natl Acad Sci U S A. 1993 May 1;90(9):4107–4111. doi: 10.1073/pnas.90.9.4107. [DOI] [PMC free article] [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]