Characterization of Nucleosome Positioning in Hepadnaviral Covalently Closed Circular DNA Minichromosomes

Abstract

<jats:p>Hepadnaviral covalently closed circular DNA (cccDNA) exists as an episomal minichromosome in the nucleus of virus-infected hepatocytes, and serves as the transcriptional template for the synthesis of viral mRNAs. To obtain insight on the structure of hepadnaviral cccDNA minichromosomes, we utilized ducks infected with the duck hepatitis B virus (DHBV) as a model and determined the<jats:italic>in vivo</jats:italic>nucleosome distribution pattern on viral cccDNA by the micrococcal nuclease (MNase) mapping and genome-wide PCR amplification of isolated mononucleosomal DHBV DNA. Several nucleosome-protected sites in a region of the DHBV genome [nucleotides (nt) 2000 to 2700], known to harbor various<jats:italic>cis</jats:italic>transcription regulatory elements, were consistently identified in all DHBV-positive liver samples. In addition, we observed other nucleosome protection sites in DHBV minichromosomes that may vary among individual ducks, but the pattern of MNase mapping in those regions is transmittable from the adult ducks to the newly infected ducklings. These results imply that the nucleosomes along viral cccDNA in the minichromosomes are not random but sequence-specifically positioned. Furthermore, we showed in ducklings that a significant portion of cccDNA possesses a few negative superhelical turns, suggesting the presence of intermediates of viral minichromosomes assembled in the liver, where dynamic hepatocyte growth and cccDNA formation occur. This study supplies the initial framework for the understanding of the overall complete structure of hepadnaviral cccDNA minichromosomes.</jats:p>

Two Populations of Viral Minichromosomes Are Present in a Geminivirus-Infected Plant Showing Symptom Remission (Recovery)

“…This activity has been associated with a structure of a relaxed minichromosome containing only a few nucleosomes, referred as "nucleosome unstable," whereas in latency, virus transcriptional activity is very low and the chromatin is condensed (48)(49)(50). A similar trend is found in hepatitis B virus (HBV) (51). In this case, it has been observed that cccDNA methylation in regulatory regions can regulate the expression of their genes (52-54), but if methylation by RNA silencing (TGS) is induced, it can suppress viral transcription and adversely affect its replication mechanism (55,56).…”

“…Similarly, different minichromosome populations were also observed in duck hepatitis B virus infection. Although 21 topoisomers were reported, the two most abundant populations are the ones with 8 to 10 nucleosomes for active minichromosomes and a second group with 18 to 20 nucleosomes in less active or inactive minichromosomes (51,57).…”

Core protein: A pleiotropic keystone in the HBV lifecycle

“…Cp was identified on cccDNA isolated from stably transfected AD38 cells and found to correlate with changes in the number of nucleosomes (Bock et al, 2001). Indeed, nucleosome spacing, at least on duck HBV cccDNA appears non-random (Shi et al, 2012). Chromatin Immuno-Precipitation (ChIP) has shown that Cp is preferentially associated with CpG islands (Guo et al, 2011).…”

An Efficient Antiviral Strategy for Targeting Hepatitis B Virus Genome Using Transcription Activator-Like Effector Nucleases

“…Since it was reported that there is a nucleosome distribution pattern on DHBV cccDNA and the nucleosomes along viral cccDNA in the minichromosomes are not random but sequence-specifically positioned (20), it is possible that the target sites of the TALENs-R3 were overlapped with the nucleosome binding to the cccDNA and thus the binding of R3 proteins to the cccDNA is blocked. These results suggest that the efficacy of a given pair of TALENs highly depends on its target sequences on the HBV DNA and it requires further design and test to optimize the most efficient TALENs against HBV.…”