Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1997 Nov;71(11):8448–8455. doi: 10.1128/jvi.71.11.8448-8455.1997

Effects of mutations within and adjacent to the terminal repeats of hepatitis B virus pregenomic RNA on viral DNA synthesis.

S Perri 1, D Ganem 1
PMCID: PMC192307  PMID: 9343201

Abstract

The viral polymerase and several cis-acting sequences are essential for hepadnaviral DNA replication, but additional host factors are likely to be involved in this process. We previously identified two sequences, UBS and DBS (upstream and downstream binding sites), present in multiple copies in and adjacent to the pregenomic RNA (pgRNA) terminal redundancy, that were specifically recognized by a 65-kDa host factor, p65. The possible roles of these two sequences in hepatitis B virus (HBV) replication were investigated in the context of the intact viral genome. UBS is contained within the terminal redundancy of pgRNA, and the 5' copy of this sequence is essential for viral replication. Mutations within the central core of UBS ablate p65 binding and selectively block synthesis of plus-strand DNA, without affecting RNA packaging or minus-strand synthesis. The DBS sequence, which is located downstream of the pgRNA polyadenylation site, overlaps the core (C) protein coding region. All mutations introduced into this site severely affected viral replication. However, these effects were shown to result from dominant negative effects of mutant core polypeptides rather than from cis-acting effects on RNA recognition. Thus, the 5' UBS but not DBS sites play important cis-acting roles in HBV DNA replication; however, the involvement of p65 in these roles remains a matter for investigation.

Full Text

The Full Text of this article is available as a PDF (746.1 KB).

Selected References

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

  1. Beames B., Lanford R. E. Insertions within the hepatitis B virus capsid protein influence capsid formation and RNA encapsidation. J Virol. 1995 Nov;69(11):6833–6838. doi: 10.1128/jvi.69.11.6833-6838.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bosch V., Bartenschlager R., Radziwill G., Schaller H. The duck hepatitis B virus P-gene codes for protein strongly associated with the 5'-end of the viral DNA minus strand. Virology. 1988 Oct;166(2):475–485. doi: 10.1016/0042-6822(88)90518-1. [DOI] [PubMed] [Google Scholar]
  3. Bruss V., Ganem D. The role of envelope proteins in hepatitis B virus assembly. Proc Natl Acad Sci U S A. 1991 Feb 1;88(3):1059–1063. doi: 10.1073/pnas.88.3.1059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Böttcher B., Wynne S. A., Crowther R. A. Determination of the fold of the core protein of hepatitis B virus by electron cryomicroscopy. Nature. 1997 Mar 6;386(6620):88–91. doi: 10.1038/386088a0. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Chang C., Hirsch R. C., Ganem D. Sequences in the preC region of duck hepatitis B virus affect pregenomic RNA accumulation. Virology. 1995 Mar 10;207(2):549–554. doi: 10.1006/viro.1995.1115. [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. Chang L. J., Pryciak P., Ganem D., Varmus H. E. Biosynthesis of the reverse transcriptase of hepatitis B viruses involves de novo translational initiation not ribosomal frameshifting. Nature. 1989 Jan 26;337(6205):364–368. doi: 10.1038/337364a0. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Cherrington J., Russnak R., Ganem D. Upstream sequences and cap proximity in the regulation of polyadenylation in ground squirrel hepatitis virus. J Virol. 1992 Dec;66(12):7589–7596. doi: 10.1128/jvi.66.12.7589-7596.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cohen B. J., Richmond J. E. Electron microscopy of hepatitis B core antigen synthesized in E. coli. Nature. 1982 Apr 15;296(5858):677–679. doi: 10.1038/296677a0. [DOI] [PubMed] [Google Scholar]
  12. Conway J. F., Cheng N., Zlotnick A., Wingfield P. T., Stahl S. J., Steven A. C. Visualization of a 4-helix bundle in the hepatitis B virus capsid by cryo-electron microscopy. Nature. 1997 Mar 6;386(6620):91–94. doi: 10.1038/386091a0. [DOI] [PubMed] [Google Scholar]
  13. Enders G. H., Ganem D., Varmus H. E. 5'-terminal sequences influence the segregation of ground squirrel hepatitis virus RNAs into polyribosomes and viral core particles. J Virol. 1987 Jan;61(1):35–41. doi: 10.1128/jvi.61.1.35-41.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. 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]
  17. 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]
  18. Knaus T., Nassal M. The encapsidation signal on the hepatitis B virus RNA pregenome forms a stem-loop structure that is critical for its function. Nucleic Acids Res. 1993 Aug 25;21(17):3967–3975. doi: 10.1093/nar/21.17.3967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lanford R. E., Notvall L. Expression of hepatitis B virus core and precore antigens in insect cells and characterization of a core-associated kinase activity. Virology. 1990 May;176(1):222–233. doi: 10.1016/0042-6822(90)90247-o. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Miller R. H., Marion P. L., Robinson W. S. Hepatitis B viral DNA-RNA hybrid molecules in particles from infected liver are converted to viral DNA molecules during an endogenous DNA polymerase reaction. Virology. 1984 Nov;139(1):64–72. doi: 10.1016/0042-6822(84)90330-1. [DOI] [PubMed] [Google Scholar]
  22. Miyanohara A., Imamura T., Araki M., Sugawara K., Ohtomo N., Matsubara K. Expression of hepatitis B virus core antigen gene in Saccharomyces cerevisiae: synthesis of two polypeptides translated from different initiation codons. J Virol. 1986 Jul;59(1):176–180. doi: 10.1128/jvi.59.1.176-180.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Nassal M. The arginine-rich domain of the hepatitis B virus core protein is required for pregenome encapsidation and productive viral positive-strand DNA synthesis but not for virus assembly. J Virol. 1992 Jul;66(7):4107–4116. doi: 10.1128/jvi.66.7.4107-4116.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Perri S., Ganem D. A host factor that binds near the termini of hepatitis B virus pregenomic RNA. J Virol. 1996 Oct;70(10):6803–6809. doi: 10.1128/jvi.70.10.6803-6809.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Pollack J. R., Ganem D. An RNA stem-loop structure directs hepatitis B virus genomic RNA encapsidation. J Virol. 1993 Jun;67(6):3254–3263. doi: 10.1128/jvi.67.6.3254-3263.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. Russnak R., Ganem D. Sequences 5' to the polyadenylation signal mediate differential poly(A) site use in hepatitis B viruses. Genes Dev. 1990 May;4(5):764–776. doi: 10.1101/gad.4.5.764. [DOI] [PubMed] [Google Scholar]
  29. Schlicht H. J., Radziwill G., Schaller H. Synthesis and encapsidation of duck hepatitis B virus reverse transcriptase do not require formation of core-polymerase fusion proteins. Cell. 1989 Jan 13;56(1):85–92. doi: 10.1016/0092-8674(89)90986-0. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Simonsen C. C., Levinson A. D. Analysis of processing and polyadenylation signals of the hepatitis B virus surface antigen gene by using simian virus 40-hepatitis B virus chimeric plasmids. Mol Cell Biol. 1983 Dec;3(12):2250–2258. doi: 10.1128/mcb.3.12.2250. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. 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]
  34. 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]
  35. Upender M., Raj L., Weir M. Megaprimer method for in vitro mutagenesis using parallel templates. Biotechniques. 1995 Jan;18(1):29-30, 32. [PubMed] [Google Scholar]
  36. 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]
  37. Wei Y., Tavis J. E., Ganem D. Relationship between viral DNA synthesis and virion envelopment in hepatitis B viruses. J Virol. 1996 Sep;70(9):6455–6458. doi: 10.1128/jvi.70.9.6455-6458.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Yu M., Summers J. A domain of the hepadnavirus capsid protein is specifically required for DNA maturation and virus assembly. J Virol. 1991 May;65(5):2511–2517. doi: 10.1128/jvi.65.5.2511-2517.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Zhou S. L., Standring D. N. Production of hepatitis B virus nucleocapsidlike core particles in Xenopus oocytes: assembly occurs mainly in the cytoplasm and does not require the nucleus. J Virol. 1991 Oct;65(10):5457–5464. doi: 10.1128/jvi.65.10.5457-5464.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Zhou S., Standring D. N. Hepatitis B virus capsid particles are assembled from core-protein dimer precursors. Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10046–10050. doi: 10.1073/pnas.89.21.10046. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Ziermann R., Ganem D. Homologous and heterologous complementation of HBV and WHV capsid and polymerase functions in RNA encapsidation. Virology. 1996 May 15;219(2):350–356. doi: 10.1006/viro.1996.0260. [DOI] [PubMed] [Google Scholar]

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

RESOURCES