Skip to main content
Journal of Virology logoLink to Journal of Virology
. 1994 Jan;68(1):6–13. doi: 10.1128/jvi.68.1.6-13.1994

Reverse transcription in hepatitis B viruses is primed by a tyrosine residue of the polymerase.

F Zoulim 1, C Seeger 1
PMCID: PMC236258  PMID: 7504742

Abstract

All known DNA polymerases require primers for the initiation of DNA synthesis. While cellular polymerases and reverse transcriptases use free hydroxyl groups of RNA or DNA, the DNA polymerases of certain animal viruses and bacteriophages depend upon hydroxyl groups of amino acid residues within proteins as primers for DNA synthesis. Recently, the reverse transcriptase of a hepadnavirus has been shown to prime RNA-directed DNA synthesis from an internal site of the polypeptide (G.H. Wang and C. Seeger, Cell 71:663-670, 1992). In this report we demonstrate that a tyrosine residue of the polymerase polypeptide is the site of a phosphodiester linkage with the first nucleotide of minus-strand DNA. This tyrosine residue is located within an amino-terminal domain of the polymerase polypeptide and is indispensable for the priming of reverse transcription. Our results demonstrate that the hepatitis B virus reverse transcriptase can initiate DNA synthesis without the requirement for tRNA as a primer.

Full text

PDF
10

Images in this article

Selected References

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

  1. Ajiro K., Borun T. W., Shulman S. D., McFadden G. M., Cohen L. H. Comparison of the structures of human histone 1A and 1B and their intramolecular phosphorylation sites during the HeLa S-3 cell cycle. Biochemistry. 1981 Mar 17;20(6):1454–1464. doi: 10.1021/bi00509a008. [DOI] [PubMed] [Google Scholar]
  2. Bamford D. H., Mindich L. Characterization of the DNA-protein complex at the termini of the bacteriophage PRD1 genome. J Virol. 1984 May;50(2):309–315. doi: 10.1128/jvi.50.2.309-315.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bartenschlager R., Kuhn C., Schaller H. Expression of the P-protein of the human hepatitis B virus in a vaccinia virus system and detection of the nucleocapsid-associated P-gene product by radiolabelling at newly introduced phosphorylation sites. Nucleic Acids Res. 1992 Jan 25;20(2):195–202. doi: 10.1093/nar/20.2.195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Bellacosa A., Testa J. R., Staal S. P., Tsichlis P. N. A retroviral oncogene, akt, encoding a serine-threonine kinase containing an SH2-like region. Science. 1991 Oct 11;254(5029):274–277. doi: 10.1126/science.254.5029.274. [DOI] [PubMed] [Google Scholar]
  6. Boyle W. J., van der Geer P., Hunter T. Phosphopeptide mapping and phosphoamino acid analysis by two-dimensional separation on thin-layer cellulose plates. Methods Enzymol. 1991;201:110–149. doi: 10.1016/0076-6879(91)01013-r. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Cooper J. A., Hunter T. Changes in protein phosphorylation in Rous sarcoma virus-transformed chicken embryo cells. Mol Cell Biol. 1981 Feb;1(2):165–178. doi: 10.1128/mcb.1.2.165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. García P., Hermoso J. M., García J. A., García E., López R., Salas M. Formation of a covalent complex between the terminal protein of pneumococcal bacteriophage Cp-1 and 5'-dAMP. J Virol. 1986 Apr;58(1):31–35. doi: 10.1128/jvi.58.1.31-35.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Garmendia C., Salas M., Hermoso J. M. Site-directed mutagenesis in the DNA linking site of bacteriophage phi 29 terminal protein: isolation and characterization of a Ser232----Thr mutant. Nucleic Acids Res. 1988 Jul 11;16(13):5727–5740. doi: 10.1093/nar/16.13.5727. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gerlich W. H., Robinson W. S. Hepatitis B virus contains protein attached to the 5' terminus of its complete DNA strand. Cell. 1980 Oct;21(3):801–809. doi: 10.1016/0092-8674(80)90443-2. [DOI] [PubMed] [Google Scholar]
  12. Guan K. L., Dixon J. E. Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase. Anal Biochem. 1991 Feb 1;192(2):262–267. doi: 10.1016/0003-2697(91)90534-z. [DOI] [PubMed] [Google Scholar]
  13. Hermoso J. M., Salas M. Protein p3 is linked to the DNA of phage phi 29 through a phosphoester bond between serine and 5'-dAMP. Proc Natl Acad Sci U S A. 1980 Nov;77(11):6425–6428. doi: 10.1073/pnas.77.11.6425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hochuli E., Döbeli H., Schacher A. New metal chelate adsorbent selective for proteins and peptides containing neighbouring histidine residues. J Chromatogr. 1987 Dec 18;411:177–184. doi: 10.1016/s0021-9673(00)93969-4. [DOI] [PubMed] [Google Scholar]
  15. Hostomsky Z., Hostomska Z., Fu T. B., Taylor J. Reverse transcriptase of human immunodeficiency virus type 1: functionality of subunits of the heterodimer in DNA synthesis. J Virol. 1992 May;66(5):3179–3182. doi: 10.1128/jvi.66.5.3179-3182.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Khudyakov YuE, Makhov A. M. Prediction of terminal protein and ribonuclease H domains in the gene P product of hepadnaviruses. FEBS Lett. 1989 Jan 30;243(2):115–118. doi: 10.1016/0014-5793(89)80110-3. [DOI] [PubMed] [Google Scholar]
  17. Kohlstaedt L. A., Wang J., Friedman J. M., Rice P. A., Steitz T. A. Crystal structure at 3.5 A resolution of HIV-1 reverse transcriptase complexed with an inhibitor. Science. 1992 Jun 26;256(5065):1783–1790. doi: 10.1126/science.1377403. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  20. Lichy J. H., Horwitz M. S., Hurwitz J. Formation of a covalent complex between the 80,000-dalton adenovirus terminal protein and 5'-dCMP in vitro. Proc Natl Acad Sci U S A. 1981 May;78(5):2678–2682. doi: 10.1073/pnas.78.5.2678. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Linn S. How many pols does it take to replicate nuclear DNA? Cell. 1991 Jul 26;66(2):185–187. doi: 10.1016/0092-8674(91)90608-2. [DOI] [PubMed] [Google Scholar]
  23. 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]
  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. Molnar-Kimber K. L., Summers J., Taylor J. M., Mason W. S. Protein covalently bound to minus-strand DNA intermediates of duck hepatitis B virus. J Virol. 1983 Jan;45(1):165–172. doi: 10.1128/jvi.45.1.165-172.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Pettit S. C., Horwitz M. S., Engler J. A. Mutations of the precursor to the terminal protein of adenovirus serotypes 2 and 5. J Virol. 1989 Dec;63(12):5244–5250. doi: 10.1128/jvi.63.12.5244-5250.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Pugh J. C., Yaginuma K., Koike K., Summers J. Duck hepatitis B virus (DHBV) particles produced by transient expression of DHBV DNA in a human hepatoma cell line are infectious in vitro. J Virol. 1988 Sep;62(9):3513–3516. doi: 10.1128/jvi.62.9.3513-3516.1988. [DOI] [PMC free article] [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. Roth M. J., Brown D. R., Hurwitz J. Analysis of bacteriophage phi X174 gene A protein-mediated termination and reinitiation of phi X DNA synthesis. II. Structural characterization of the covalent phi X A protein-DNA complex. J Biol Chem. 1984 Aug 25;259(16):10556–10568. [PubMed] [Google Scholar]
  30. Salas M. Protein-priming of DNA replication. Annu Rev Biochem. 1991;60:39–71. doi: 10.1146/annurev.bi.60.070191.000351. [DOI] [PubMed] [Google Scholar]
  31. Stillman B. W., Lewis J. B., Chow L. T., Mathews M. B., Smart J. E. Identification of the gene and mRNA for the adenovirus terminal protein precursor. Cell. 1981 Feb;23(2):497–508. doi: 10.1016/0092-8674(81)90145-8. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. Tobin G. J., Young D. C., Flanegan J. B. Self-catalyzed linkage of poliovirus terminal protein VPg to poliovirus RNA. Cell. 1989 Nov 3;59(3):511–519. doi: 10.1016/0092-8674(89)90034-2. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. Vartapetian A. B., Bogdanov A. A. Proteins covalently linked to viral genomes. Prog Nucleic Acid Res Mol Biol. 1987;34:209–251. doi: 10.1016/s0079-6603(08)60497-3. [DOI] [PubMed] [Google Scholar]

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

RESOURCES