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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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Serum, saliva, and semen from fifteen homosexual men who were chronic surface antigen (HBsAg) carriers were quantitatively assayed for hepatitis B virus (HBV) DNA by Southern analysis. HBV DNA (2 pg/ml-42,000 pg/ml) was detected in 11 carriers and correlated with endogenous DNA polymerase activity and e antigen (HBeAg) titer greater than or equal to 1:640. Viral DNA was detected in the saliva, at concentrations indicating a virus titer of 10(5)-10(7)/ml, of eight carriers and in semen from three patients (10(6)-10(7) virions/ml). HBV DNA in saliva and semen had electrophoretic mobilities similar to those in serum HBV DNA, but HBV DNA was present at much lower concentrations (10(-2)-10(-4) that of serum DNA). Results of isopycnic centrifugation suggested that DNA in saliva and semen was virion associated. These data provide an indication of the quantity of virus present in the semen and saliva of HBV carriers and the relative frequency with which shedding of high titers of virus (greater than 10(5) virions/ml) occurs in these secretions.