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. 1996 Sep;70(9):5741–5750. doi: 10.1128/jvi.70.9.5741-5750.1996

Evidence for activation of the hepatitis B virus polymerase by binding of its RNA template.

J E Tavis 1, D Ganem 1
PMCID: PMC190587  PMID: 8709189

Abstract

The hepatitis B viruses replicate by reverse transcription of an RNA pregenome by using a virally encoded polymerase. A key early step in replication is binding of the polymerase to an RNA stem-loop (epsilon) of the pregenome; epsilon is both the RNA encapsidation signal and the origin of reverse transcription. Here we provide evidence that this interaction is also key to the development of enzymatic activity during biosynthesis of the polymerase. Duck hepatitis B virus polymerase expressed in Saccharomyces cerevisiae can synthesize DNA from epsilon-containing RNAs and can also end label other small RNAs. Expression of functional polymerase in S. cerevisiae requires interaction between the polymerase and epsilon during or shortly after translation for it to develop any enzymatic activity; if epsilon is absent during expression, the polymerase is inactive on RNAs both with and without epsilon. Functional duck polymerase can also be produced by in vitro translation, and synthesis of the polymerase in the presence of epsilon induces resistance in the polymerase to proteolysis by papain, trypsin, and bromelain. Induction of the resistance is specific for epsilon sequences that can support RNA encapsidation and initiation of DNA synthesis. Induction of the resistance precedes initiation of DNA synthesis and is reversible by degradation of epsilon. These two sets of data (i) support a model in which binding of epsilon to the polymerase induces a structural alteration of the polymerase prior to the development of enzymatic activity and (ii) suggest that this alteration may be required for the polymerase to mature to an active form.

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Selected References

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  1. Bohen S. P., Kralli A., Yamamoto K. R. Hold 'em and fold 'em: chaperones and signal transduction. Science. 1995 Jun 2;268(5215):1303–1304. doi: 10.1126/science.7761850. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Clark J. M. Novel non-templated nucleotide addition reactions catalyzed by procaryotic and eucaryotic DNA polymerases. Nucleic Acids Res. 1988 Oct 25;16(20):9677–9686. doi: 10.1093/nar/16.20.9677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Gething M. J., Sambrook J. Protein folding in the cell. Nature. 1992 Jan 2;355(6355):33–45. doi: 10.1038/355033a0. [DOI] [PubMed] [Google Scholar]
  6. Hartl F. U., Hlodan R., Langer T. Molecular chaperones in protein folding: the art of avoiding sticky situations. Trends Biochem Sci. 1994 Jan;19(1):20–25. doi: 10.1016/0968-0004(94)90169-4. [DOI] [PubMed] [Google Scholar]
  7. Hendrick J. P., Hartl F. U. Molecular chaperone functions of heat-shock proteins. Annu Rev Biochem. 1993;62:349–384. doi: 10.1146/annurev.bi.62.070193.002025. [DOI] [PubMed] [Google Scholar]
  8. Hu J., Seeger C. Hsp90 is required for the activity of a hepatitis B virus reverse transcriptase. Proc Natl Acad Sci U S A. 1996 Feb 6;93(3):1060–1064. doi: 10.1073/pnas.93.3.1060. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Huovila A. P., Eder A. M., Fuller S. D. Hepatitis B surface antigen assembles in a post-ER, pre-Golgi compartment. J Cell Biol. 1992 Sep;118(6):1305–1320. doi: 10.1083/jcb.118.6.1305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Jakob U., Buchner J. Assisting spontaneity: the role of Hsp90 and small Hsps as molecular chaperones. Trends Biochem Sci. 1994 May;19(5):205–211. doi: 10.1016/0968-0004(94)90023-x. [DOI] [PubMed] [Google Scholar]
  11. Kimura Y., Yahara I., Lindquist S. Role of the protein chaperone YDJ1 in establishing Hsp90-mediated signal transduction pathways. Science. 1995 Jun 2;268(5215):1362–1365. doi: 10.1126/science.7761857. [DOI] [PubMed] [Google Scholar]
  12. Kolodziej P. A., Young R. A. Epitope tagging and protein surveillance. Methods Enzymol. 1991;194:508–519. doi: 10.1016/0076-6879(91)94038-e. [DOI] [PubMed] [Google Scholar]
  13. Patzer E. J., Nakamura G. R., Simonsen C. C., Levinson A. D., Brands R. Intracellular assembly and packaging of hepatitis B surface antigen particles occur in the endoplasmic reticulum. J Virol. 1986 Jun;58(3):884–892. doi: 10.1128/jvi.58.3.884-892.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Radziwill G., Zentgraf H., Schaller H., Bosch V. The duck hepatitis B virus DNA polymerase is tightly associated with the viral core structure and unable to switch to an exogenous template. Virology. 1988 Mar;163(1):123–132. doi: 10.1016/0042-6822(88)90239-5. [DOI] [PubMed] [Google Scholar]
  16. Seifer M., Standring D. N. Recombinant human hepatitis B virus reverse transcriptase is active in the absence of the nucleocapsid or the viral replication origin, DR1. J Virol. 1993 Aug;67(8):4513–4520. doi: 10.1128/jvi.67.8.4513-4520.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Simon K., Lingappa V. R., Ganem D. Secreted hepatitis B surface antigen polypeptides are derived from a transmembrane precursor. J Cell Biol. 1988 Dec;107(6 Pt 1):2163–2168. doi: 10.1083/jcb.107.6.2163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. Tavis J. E., Ganem D. RNA sequences controlling the initiation and transfer of duck hepatitis B virus minus-strand DNA. J Virol. 1995 Jul;69(7):4283–4291. doi: 10.1128/jvi.69.7.4283-4291.1995. [DOI] [PMC free article] [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]
  24. Wang G. H., Zoulim F., Leber E. H., Kitson J., Seeger C. Role of RNA in enzymatic activity of the reverse transcriptase of hepatitis B viruses. J Virol. 1994 Dec;68(12):8437–8442. doi: 10.1128/jvi.68.12.8437-8442.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Weber M., Bronsema V., Bartos H., Bosserhoff A., Bartenschlager R., Schaller H. Hepadnavirus P protein utilizes a tyrosine residue in the TP domain to prime reverse transcription. J Virol. 1994 May;68(5):2994–2999. doi: 10.1128/jvi.68.5.2994-2999.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Zoulim F., Seeger C. Reverse transcription in hepatitis B viruses is primed by a tyrosine residue of the polymerase. J Virol. 1994 Jan;68(1):6–13. doi: 10.1128/jvi.68.1.6-13.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]

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