Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1991 Nov;10(11):3533–3540. doi: 10.1002/j.1460-2075.1991.tb04917.x

Sequence-independent RNA cleavages generate the primers for plus strand DNA synthesis in hepatitis B viruses: implications for other reverse transcribing elements.

D D Loeb 1, R C Hirsch 1, D Ganem 1
PMCID: PMC453082  PMID: 1915307

Abstract

Reverse transcription of RNA into duplex DNA requires accurate initiation of both minus and plus strand DNA synthesis; this in turn requires the generation of specific primer molecules. We have examined plus strand primer generation in the hepatitis B viruses, small DNA viruses that replicate via reverse transcription. The plus strand primer in these viruses is a short capped RNA derived from the 5' end of the RNA template by cleavage at a specific set of sites. To elucidate the cleavage mechanism we constructed a series of viral mutants bearing alterations in and around the cleavage sites. Our results reveal that the cleavage reaction is sequence-independent and indicate that the cleavage sites are positioned by measurement of the distance from the 5' end of the RNA. Comparison of these findings with what is known about RNase H-mediated primer generation in retroviruses and other retroid elements suggests that, despite many divergent features, some common molecular features are preserved.

Full text

PDF
3533

Images in this article

3534Image
on p.3534
3535Image
on p.3535
3536Image
on p.3536
3537Image
on p.3537
3537Image
on p.3537

Selected References

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

  1. Baltimore D. RNA-dependent DNA polymerase in virions of RNA tumour viruses. Nature. 1970 Jun 27;226(5252):1209–1211. doi: 10.1038/2261209a0. [DOI] [PubMed] [Google Scholar]
  2. Bartenschlager R., Schaller H. The amino-terminal domain of the hepadnaviral P-gene encodes the terminal protein (genome-linked protein) believed to prime reverse transcription. EMBO J. 1988 Dec 20;7(13):4185–4192. doi: 10.1002/j.1460-2075.1988.tb03315.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Boeke J. D., Garfinkel D. J., Styles C. A., Fink G. R. Ty elements transpose through an RNA intermediate. Cell. 1985 Mar;40(3):491–500. doi: 10.1016/0092-8674(85)90197-7. [DOI] [PubMed] [Google Scholar]
  4. Büscher M., Reiser W., Will H., Schaller H. Transcripts and the putative RNA pregenome of duck hepatitis B virus: implications for reverse transcription. Cell. 1985 Mar;40(3):717–724. doi: 10.1016/0092-8674(85)90220-x. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. 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]
  7. 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]
  8. 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]
  9. Finston W. I., Champoux J. J. RNA-primed initiation of Moloney murine leukemia virus plus strands by reverse transcriptase in vitro. J Virol. 1984 Jul;51(1):26–33. doi: 10.1128/jvi.51.1.26-33.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Furfine E. S., Reardon J. E. Reverse transcriptase.RNase H from the human immunodeficiency virus. Relationship of the DNA polymerase and RNA hydrolysis activities. J Biol Chem. 1991 Jan 5;266(1):406–412. [PubMed] [Google Scholar]
  11. 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]
  12. Geballe A. P., Spaete R. R., Mocarski E. S. A cis-acting element within the 5' leader of a cytomegalovirus beta transcript determines kinetic class. Cell. 1986 Sep 12;46(6):865–872. doi: 10.1016/0092-8674(86)90068-1. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. Hizi A., Hughes S. H., Shaharabany M. Mutational analysis of the ribonuclease H activity of human immunodeficiency virus 1 reverse transcriptase. Virology. 1990 Apr;175(2):575–580. doi: 10.1016/0042-6822(90)90444-v. [DOI] [PubMed] [Google Scholar]
  16. Hostomsky Z., Hostomska Z., Hudson G. O., Moomaw E. W., Nodes B. R. Reconstitution in vitro of RNase H activity by using purified N-terminal and C-terminal domains of human immunodeficiency virus type 1 reverse transcriptase. Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1148–1152. doi: 10.1073/pnas.88.4.1148. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Inouye S., Hsu M. Y., Eagle S., Inouye M. Reverse transcriptase associated with the biosynthesis of the branched RNA-linked msDNA in Myxococcus xanthus. Cell. 1989 Feb 24;56(4):709–717. doi: 10.1016/0092-8674(89)90593-x. [DOI] [PubMed] [Google Scholar]
  18. Johnson M. S., McClure M. A., Feng D. F., Gray J., Doolittle R. F. Computer analysis of retroviral pol genes: assignment of enzymatic functions to specific sequences and homologies with nonviral enzymes. Proc Natl Acad Sci U S A. 1986 Oct;83(20):7648–7652. doi: 10.1073/pnas.83.20.7648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Kuiper M. T., Lambowitz A. M. A novel reverse transcriptase activity associated with mitochondrial plasmids of Neurospora. Cell. 1988 Nov 18;55(4):693–704. doi: 10.1016/0092-8674(88)90228-0. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. 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]
  23. Lien J. M., Petcu D. J., Aldrich C. E., Mason W. S. Initiation and termination of duck hepatitis B virus DNA synthesis during virus maturation. J Virol. 1987 Dec;61(12):3832–3840. doi: 10.1128/jvi.61.12.3832-3840.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Molnar-Kimber K. L., Summers J. W., Mason W. S. Mapping of the cohesive overlap of duck hepatitis B virus DNA and of the site of initiation of reverse transcription. J Virol. 1984 Jul;51(1):181–191. doi: 10.1128/jvi.51.1.181-191.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Nassal M., Junker-Niepmann M., Schaller H. Translational inactivation of RNA function: discrimination against a subset of genomic transcripts during HBV nucleocapsid assembly. Cell. 1990 Dec 21;63(6):1357–1363. doi: 10.1016/0092-8674(90)90431-d. [DOI] [PubMed] [Google Scholar]
  26. Oyama F., Kikuchi R., Crouch R. J., Uchida T. Intrinsic properties of reverse transcriptase in reverse transcription. Associated RNase H is essentially regarded as an endonuclease. J Biol Chem. 1989 Nov 5;264(31):18808–18817. [PubMed] [Google Scholar]
  27. Pfeiffer P., Hohn T. Involvement of reverse transcription in the replication of cauliflower mosaic virus: a detailed model and test of some aspects. Cell. 1983 Jul;33(3):781–789. doi: 10.1016/0092-8674(83)90020-x. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. 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]
  30. Resnick R., Omer C. A., Faras A. J. Involvement of retrovirus reverse transcriptase-associated RNase H in the initiation of strong-stop (+) DNA synthesis and the generation of the long terminal repeat. J Virol. 1984 Sep;51(3):813–821. doi: 10.1128/jvi.51.3.813-821.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Schatz O., Mous J., Le Grice S. F. HIV-1 RT-associated ribonuclease H displays both endonuclease and 3'----5' exonuclease activity. EMBO J. 1990 Apr;9(4):1171–1176. doi: 10.1002/j.1460-2075.1990.tb08224.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. Seeger C., Maragos J. Molecular analysis of the function of direct repeats and a polypurine tract for plus-strand DNA priming in woodchuck hepatitis virus. J Virol. 1989 May;63(5):1907–1915. doi: 10.1128/jvi.63.5.1907-1915.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Smith J. K., Cywinski A., Taylor J. M. Specificity of initiation of plus-strand DNA by Rous sarcoma virus. J Virol. 1984 Nov;52(2):314–319. doi: 10.1128/jvi.52.2.314-319.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. 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]
  37. 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]
  38. Tanese N., Goff S. P. Domain structure of the Moloney murine leukemia virus reverse transcriptase: mutational analysis and separate expression of the DNA polymerase and RNase H activities. Proc Natl Acad Sci U S A. 1988 Mar;85(6):1777–1781. doi: 10.1073/pnas.85.6.1777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Temin H. M., Mizutani S. RNA-dependent DNA polymerase in virions of Rous sarcoma virus. Nature. 1970 Jun 27;226(5252):1211–1213. doi: 10.1038/2261211a0. [DOI] [PubMed] [Google Scholar]
  40. Toh H., Hayashida H., Miyata T. Sequence homology between retroviral reverse transcriptase and putative polymerases of hepatitis B virus and cauliflower mosaic virus. 1983 Oct 27-Nov 2Nature. 305(5937):827–829. doi: 10.1038/305827a0. [DOI] [PubMed] [Google Scholar]
  41. Voliva C. F., Martin S. L., Hutchison C. A., 3rd, Edgell M. H. Dispersal process associated with the L1 family of interspersed repetitive DNA sequences. J Mol Biol. 1984 Oct 5;178(4):795–813. doi: 10.1016/0022-2836(84)90312-7. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES