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

Abstract

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.

Initial Amplification of Duck Hepatitis B Virus Covalently Closed Circular Dna After In Vitro Infection of Embryonic Duck Hepatocytes Is Increased by Cell Cycle Progression

“…22 It is likely that growth factors such as EGF, HGF, TGF-␣, and TGF-␤, which activate or repress many important cellular genes, may modulate viral replication at least at the transcriptional level, independently of their effects on cell cycle. Sodium butyrate, which induces hyperacetylation of histones, hypophosphorylation of histone H1, 23 and cell differentiation, may also act directly on the viral minichromosome 15,16 and not through its action on the cell cycle. However, aphidicolin, an inhibitor of cellular DNA polymerase ␣, 24 butyrate, and TGF-␤ all act in very different ways on the cellular machinery but have the same effect on viral DNA synthesis.…”

“…8, lane 4), indicating that high amounts of open circular DNA may lead in some cases to nonspecific detection of CCC DNA. To make sure of the specificity of the CCC PCR, DNA samples were heat denatured using the conditions described by Newbold et al 16 in which closed DNA rapidly renatures while open circular forms were stably denatured. After this treatment, the band correspond-ing to the gap region could no longer be detected in PCR with serum-derived RC DNA (Fig.…”

“…However, butyrate, which is an inhibitor of histone deacetylase, may act on CCC DNA amplification either through an indirect action on the cell cycle or through a direct action on CCC DNA, which persists in the nucleus as a mini chromosome associated with histone proteins. 15,16 To establish the potential relationship between the cell cycle and the regulation of CCC DNA synthesis, we have used primary duck fetal (eggs) hepatocytes. These cells have the capacity to proliferate in culture with at least one complete cellular cycle.…”

Impact of postoperative hepatitis B virus reactivation in hepatocellular carcinoma patients who formerly had naturally suppressed virus

“…The amount (copies/cell) of total DNA and HBV cccDNA were quantified and normalized with the β-globin housekeeping gene as suggested on the previous studies. [21][22][23] Statistical analysis. Categorical variables were compared using two-sided χ 2 -test or Fisher's exact test when appropriate, and continuous variables were compared using the independent sample t-test.…”

Intrahepatic response markers in chronic hepatitis B patients treated with peginterferon alpha‐2a and adefovir

“…2 Most of the newly formed nucleocapsids recycle back into the nucleus to ensure accumulation and maintenance of a pool of cccDNA. [2][3][4][5] Eradication of chronic HBV infection requires the elimination of this cccDNA from infected hepatocytes. 3 However, cccDNA can be present in the liver at very low levels several years after successful clearance of HBV DNA and hepatitis B surface antigen (HBsAg).…”