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J Virol. 1995 June; 69(6): 3350–3357.
PMCID: PMC189047

The covalently closed duplex form of the hepadnavirus genome exists in situ as a heterogeneous population of viral minichromosomes.


Replication of hepadnaviruses requires a persistent population of covalently closed circular (CCC) DNA molecules in the nucleus of the infected cell. It is widely accepted that the vital role of this molecule is to be the sole DNA template for the synthesis by RNA polymerase II of all viral transcripts throughout the infection process. Since the transcriptional activity of eukaryotic nuclear DNA is considered to be determined in part by its specific organization as chromatin, the nucleoprotein disposition of the hepadnavirus CCC DNA was investigated. These studies were undertaken on the duck hepatitis B virus (DHBV) CCC DNA present in the liver cell nuclei of DHBV-infected ducks. The organization and protein associations of the DHBV CCC DNA in situ were inferred from sedimentation, micrococcal nuclease digestion, and DNA superhelicity analyses. These three lines of investigation demonstrate that the DHBV CCC DNA is stably associated with proteins in the nuclei of infected liver cells. Moreover, they provide compelling evidence that the viral nucleoprotein complex is indeed a minichromosome composed of classical nucleosomes but in arrays that are atypical for chromatin. When the DHBV chromatin is digested with micrococcal nuclease, a ladder of viral DNA fragments that exhibits a 150-bp repeat is produced. This profile for the viral chromatin is obtained from the same nuclei in which the duck chromatin shows the standard 200-bp ladder. The superhelicity of the DHBV CCC DNA ranges from 0 to 20 negative supertwists per molecule, with all possible 21 topoisomers present in each DNA preparation. The 21 topoisomers of DHBV CCC DNA are inferred to derive from an identically diverse array of viral minichromosomes. In the DHBV minichromosomes composed of 20 nucleosomes, 96.7% of the viral DNA is calculated to be compacted into these chromatin subunits spaced on average by 5 bp of linker DNA; other minichromosomes contain fewer nucleosomes and proportionately more linker DNA. Two major subpopulations of DHBV minichromosomes are detected with comparable prevalence. The two groups correspond to minichromosomes which contain essentially a full or half complement of nucleosomes. The functional significance of this minichromosome diversity is unknown but is suggestive of transcriptional regulation of the viral DNA template.

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Selected References

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  • Bock CT, Schranz P, Schröder CH, Zentgraf H. Hepatitis B virus genome is organized into nucleosomes in the nucleus of the infected cell. Virus Genes. 1994 Jul;8(3):215–229. [PubMed]
  • Civitico GM, Locarnini SA. The half-life of duck hepatitis B virus supercoiled DNA in congenitally infected primary hepatocyte cultures. Virology. 1994 Aug 15;203(1):81–89. [PubMed]
  • Deshmane SL, Fraser NW. During latency, herpes simplex virus type 1 DNA is associated with nucleosomes in a chromatin structure. J Virol. 1989 Feb;63(2):943–947. [PMC free article] [PubMed]
  • Freiman JS, Cossart YE. Natural duck hepatitis B virus infection in Australia. Aust J Exp Biol Med Sci. 1986 Oct;64(Pt 5):477–484. [PubMed]
  • Germond JE, Hirt B, Oudet P, Gross-Bellark M, Chambon P. Folding of the DNA double helix in chromatin-like structures from simian virus 40. Proc Natl Acad Sci U S A. 1975 May;72(5):1843–1847. [PubMed]
  • Green MH, Miller HI, Hendler S. Isolation of a polyoma-nucleoprotein complex from infected mouse-cell cultures. Proc Natl Acad Sci U S A. 1971 May;68(5):1032–1036. [PubMed]
  • Griffith JD. Chromatin structure: deduced from a minichromosome. Science. 1975 Mar 28;187(4182):1202–1203. [PubMed]
  • Griffith JD, Christiansen G. The multifunctional role of histone H1, probed with the SV40 minichromosome. Cold Spring Harb Symp Quant Biol. 1978;42(Pt 1):215–226. [PubMed]
  • Hewish DR, Burgoyne LA. Chromatin sub-structure. The digestion of chromatin DNA at regularly spaced sites by a nuclear deoxyribonuclease. Biochem Biophys Res Commun. 1973 May 15;52(2):504–510. [PubMed]
  • Hirt B. Selective extraction of polyoma DNA from infected mouse cell cultures. J Mol Biol. 1967 Jun 14;26(2):365–369. [PubMed]
  • Kajino K, Jilbert AR, Saputelli J, Aldrich CE, Cullen J, Mason WS. Woodchuck hepatitis virus infections: very rapid recovery after a prolonged viremia and infection of virtually every hepatocyte. J Virol. 1994 Sep;68(9):5792–5803. [PMC free article] [PubMed]
  • Kornberg RD. Chromatin structure: a repeating unit of histones and DNA. Science. 1974 May 24;184(4139):868–871. [PubMed]
  • Lentine AF, Bachenheimer SL. Intracellular organization of herpes simplex virus type 1 DNA assayed by staphylococcal nuclease sensitivity. Virus Res. 1990 Jul;16(3):275–292. [PubMed]
  • 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. [PMC free article] [PubMed]
  • Mason WS, Aldrich C, Summers J, Taylor JM. Asymmetric replication of duck hepatitis B virus DNA in liver cells: Free minus-strand DNA. Proc Natl Acad Sci U S A. 1982 Jul;79(13):3997–4001. [PubMed]
  • Miller RH, Robinson WS. Hepatitis B virus DNA forms in nuclear and cytoplasmic fractions of infected human liver. Virology. 1984 Sep;137(2):390–399. [PubMed]
  • Noll M. Subunit structure of chromatin. Nature. 1974 Sep 20;251(5472):249–251. [PubMed]
  • Oberhaus SM, Newbold JE. Detection of DNA polymerase activities associated with purified duck hepatitis B virus core particles by using an activity gel assay. J Virol. 1993 Nov;67(11):6558–6566. [PMC free article] [PubMed]
  • Olszewski N, Hagen G, Guilfoyle TJ. A transcriptionally active, covalently closed minichromosome of cauliflower mosaic virus DNA isolated from infected turnip leaves. Cell. 1982 Jun;29(2):395–402. [PubMed]
  • Peck LJ, Wang JC. Energetics of B-to-Z transition in DNA. Proc Natl Acad Sci U S A. 1983 Oct;80(20):6206–6210. [PubMed]
  • Ruiz-Opazo N, Chakraborty PR, Shafritz DA. Evidence for supercoiled hepatitis B virus DNA in chimpanzee liver and serum Dane particles: possible implications in persistent HBV infection. Cell. 1982 May;29(1):129–136. [PubMed]
  • Sells MA, Chen ML, Acs G. Production of hepatitis B virus particles in Hep G2 cells transfected with cloned hepatitis B virus DNA. Proc Natl Acad Sci U S A. 1987 Feb;84(4):1005–1009. [PubMed]
  • Shaw JE, Levinger LF, Carter CW., Jr Nucleosomal structure of Epstein-Barr virus DNA in transformed cell lines. J Virol. 1979 Feb;29(2):657–665. [PMC free article] [PubMed]
  • Shure M, Pulleyblank DE, Vinograd J. The problems of eukaryotic and prokaryotic DNA packaging and in vivo conformation posed by superhelix density heterogeneity. Nucleic Acids Res. 1977;4(5):1183–1205. [PMC free article] [PubMed]
  • Sprengel R, Kuhn C, Manso C, Will H. Cloned duck hepatitis B virus DNA is infectious in Pekin ducks. J Virol. 1984 Dec;52(3):932–937. [PMC free article] [PubMed]
  • Sprengel R, Schneider R, Marion PL, Fernholz D, Wildner G, Will H. Comparative sequence analysis of defective and infectious avian hepadnaviruses. Nucleic Acids Res. 1991 Aug 11;19(15):4289–4289. [PMC free article] [PubMed]
  • Summers J, Mason WS. Replication of the genome of a hepatitis B--like virus by reverse transcription of an RNA intermediate. Cell. 1982 Jun;29(2):403–415. [PubMed]
  • Summers J, Smith PM, Huang MJ, Yu MS. Morphogenetic and regulatory effects of mutations in the envelope proteins of an avian hepadnavirus. J Virol. 1991 Mar;65(3):1310–1317. [PMC free article] [PubMed]
  • Tate VE, Philipson L. Parental adenovirus DNA accumulates in nucleosome-like structures in infected cells. Nucleic Acids Res. 1979 Jun 25;6(8):2769–2785. [PMC free article] [PubMed]
  • Tuttleman JS, Pourcel C, Summers J. Formation of the pool of covalently closed circular viral DNA in hepadnavirus-infected cells. Cell. 1986 Nov 7;47(3):451–460. [PubMed]
  • Vinograd J, Lebowitz J, Watson R. Early and late helix-coil transitions in closed circular DNA. The number of superhelical turns in polyoma DNA. J Mol Biol. 1968 Apr 14;33(1):173–197. [PubMed]
  • Wang JC. Interaction between DNA and an Escherichia coli protein omega. J Mol Biol. 1971 Feb 14;55(3):523–533. [PubMed]
  • Waring M. Variation of the supercoils in closed circular DNA by binding of antibiotics and drugs: evidence for molecular models involving intercalation. J Mol Biol. 1970 Dec 14;54(2):247–279. [PubMed]
  • Weiser B, Ganem D, Seeger C, Varmus HE. Closed circular viral DNA and asymmetrical heterogeneous forms in livers from animals infected with ground squirrel hepatitis virus. J Virol. 1983 Oct;48(1):1–9. [PMC free article] [PubMed]
  • 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. [PMC free article] [PubMed]

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