Role of hepatitis B core protein in HBV transcription and recruitment of histone acetyltransferases to cccDNA minichromosome
Introduction
Currently approved therapeutic agents against chronic hepatitis B, despite very effective in the suppression of hepatitis B virus (HBV) replication, fail to completely eradicate HBV. This is partly due to the persistence of HBV covalently closed circular DNA (cccDNA) inside the infected hepatocytes. cccDNA is the template for the transcription of all HBV RNAs, and is not a direct target of current therapeutic agents. A better understanding of the HBV replication cycle, especially in HBV transcriptional regulation, is desirable for the development of novel effective therapy.
The hepatitis B core protein (HBc) plays multiple roles in HBV replication. HBc is a 21.5 kDa protein and is the structural component of the viral nucleocapsids. It contains a globular N-terminal domain and a C-terminal domain (CTD). The HBc N-terminal domain is essential for nucleocapsid formation. The HBc-CTD is arginine-rich, and the arginine residues can be grouped into four clusters named clusters I - IV, with each clusters being composed of 3 or 4 arginine residues.
During HBV replication, the HBV pregenomic RNA (pgRNA) is transcribed from cccDNA. The pgRNA is then encapsidated by HBc in the cytoplasm. Inside the viral capsids, HBc facilitates reverse transcription of the pgRNA to synthesize the HBV DNA minus and plus strands. This is attributed to the nucleic acid-binding properties of the HBc CTD (Lewellyn and Loeb, 2011, Nassal, 1992, Seeger and Mason, 2000). In addition to its action inside the nucleocapsids, HBc may also play a role in HBV replication inside the hepatocyte nucleus. Electron-microscopic and chromatin immunoprecipitation (ChIP) studies demonstrate that HBc is a component of the cccDNA minichromosome and involved in the epigenetic regulation of HBV transcription (Bock et al., 2001, Pollicino et al., 2006). HBc binds to cccDNA and reduces the nucleosome spacing of the cccDNA-histones complex, which may regulate HBV transcription by altering the nucleosomal arrangement of the HBV genome (Bock et al., 2001, Pollicino et al., 2006). The role of HBc in the regulation of HBV transcription is further reinforced by a study demonstrating that HBc preferentially binds to the CpG islands in cccDNA (Guo et al., 2011). The binding of HBc to cccDNA is associated with hypomethylation in CpG island 2 in cccDNA, increased binding of cyclic AMP-responsive enhancer binding protein (CREB) binding protein (CBP), and increased histone acetylation status, all of which are suggested to increase HBV transcription (Guo et al., 2011, Pollicino et al., 2006). It can be envisaged that blocking HBc-to-cccDNA binding can be a potential novel therapeutic approach. However, due to the lack of a convenient in vitro system that supports cccDNA-initiated HBV transcription, studies on the effect of HBc mutations on HBV transcription are limited.
The association between HBc and cccDNA is likely to be mediated by HBc arginine residues, which confer its nucleic acid binding property. In this study, we used a transient transfection system in which transcription of HBV RNA was originated from cccDNA (Pollicino et al., 2006) to assess the effects of HBc-CTD mutations on HBV replication. We aimed to identify the HBc arginine region(s) or residue(s) that are crucial to HBV transcription and replication. In addition, we aimed to study the interaction between mutant HBc and cccDNA using ChIP experiments. The relationship between HBc-cccDNA binding and HBV transcription and replication was also studied.
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Cell lines and plasmids
HepG2 cell expressing the sodium taurocholate cotransporting polypeptide (NTCP) was a kind gift from Professor DY Jin. Full-length HBV DNA was amplified from a 31 years old, hepatitis B e antigen positive, male chronic hepatitis B patient using primers and methods described previously (Gunther et al., 1995). The amplified HBV DNA was cloned into pUC19. Sequence analysis of the resulting clone, named pHBV3, revealed that it was of HBV genotype C, without any mutations in the basal core promoter
Hepatitis B virus replication in the HBc-negative mutant
We first verified the absence of HBc protein production in the early truncated HBc mutant HBcStop38 by Western blot analysis. As shown in Fig. 2A, an HBc protein band of approximately 22 kDa was detected in the HBc-null strain complement with the wild-type HBc plasmid, while HBc was not detectable in HBcStop38.
The effect of HBc-deletion on HBV replication was evaluated by real-time PCR measurement of intracellular encapsidated HBV DNA. As shown in Fig. 2B, the level of intracellular
Discussion
In the present study, we used a cccDNA-dependent transfection system to study the effect of HBc mutations on HBV transcription. In this system, circularized HBV DNA were transfected into hepatoma cells and served as the “ccc” transcriptional template for HBV. Following transfection, cccDNA was detectable in the nucleus, and its downstream replication products, including HBV RNA, encapsidated rcDNA, and HBsAg, were also detectable. This indicated that the transient transfection system was
Conflict of interest
All authors have no conflict of interest pertinent to this study.
Funding
This study was supported by the Health and Medical Research Fund, Food and Health Bureau, Hong Kong SAR Government (project number 12111282).
Acknowledgement
The authors would like to thank Professor DY Jin, The University of Hong Kong, for providing the hepatoma cells for this study.
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