Mechanism of Hepatitis B Virus cccDNA Formation

Abstract

Hepatitis B virus (HBV) remains a major medical problem affecting at least 257 million chronically infected patients who are at risk of developing serious, frequently fatal liver diseases. HBV is a small, partially double-stranded DNA virus that goes through an intricate replication cycle in its native cellular environment: human hepatocytes. A critical step in the viral life-cycle is the conversion of relaxed circular DNA (rcDNA) into covalently closed circular DNA (cccDNA), the latter being the major template for HBV gene transcription. For this conversion, HBV relies on multiple host factors, as enzymes capable of catalyzing the relevant reactions are not encoded in the viral genome. Combinations of genetic and biochemical approaches have produced findings that provide a more holistic picture of the complex mechanism of HBV cccDNA formation. Here, we review some of these studies that have helped to provide a comprehensive picture of rcDNA to cccDNA conversion. Mechanistic insights into this critical step for HBV persistence hold the key for devising new therapies that will lead not only to viral suppression but to a cure.

Keywords: DNA repair; HBV; cccDNA biogenesis; hepatitis B virus; rcDNA; viral replication.

Figure 1

The HBV life cycle and cccDNA biogenesis. HBV life cycle is a multi-step process composed of viral entry, cccDNA biogenesis, progeny nucleocapsid production, virion formation, and egress. Viral entry is mediated by NTCP, heparin sulfate proteoglycans (HSPGs, such as glypican 5), and EGFR on the surface of hepatocytes. cccDNA biogenesis is an essential process to establish infection, which consists of three distinct steps: nuclear transport of rcDNA and uncoating, repair of rcDNA to form cccDNA, and cccDNA chromatinization. cccDNA can serve as the template for pre-genomic and precore mRNAs and multiple sub-genomic mRNAs, which can be translated to several viral proteins, including HBV POL, core antigen (capsid), surface antigens, HBx, and core-antigen related proteins (not shown). Progeny nucleocapsid production is initiated by the binding of HBV POL to pre-genomic mRNA, which triggers the packaging (encapsidation) and synthesis of rcDNA. The resultant nucleocapsids can either be re-imported into the nucleus, and the rcDNA repaired to form cccDNA and maintain cccDNA pool through the intracellular amplification pathway, or it can be enveloped in the multivesicular body (MVB) to complete virion assembly. Subsequent virion egress completes the HBV life cycle.

Figure 2

The repair factors and steps involved in conversion of HBV rcDNA to cccDNA. (a) Removal of HBV polymerase (POL) adduct from rcDNA can be achieved by: (1) tyrosylphosphodiesterases (TDPs), such as TDP2; (2) nucleases, such as FEN-1; (3) proteases; and (4) other mechanism such as self-release of POL, or TOP1 mediated release. The first three mechanisms will lead to formation of types A−C of deproteinated rcDNA (dp-rcDNA). Type A and C dp-rcDNAs contain the terminal redundancy DNA flap, whereas type B dp-rcDNA does not. The repair intermediates of POL self-release or TOP1-mediated release are not clear and are thus denoted by a question mark. (b) After the removal of POL, the minus strand is further repaired by removal of the terminal redundancy DNA flap via FEN-1 or other nucleases and ligation of the nick by LIG1 or LIG3. The steps involved in repair of the plus strand are: (1) completion of DNA synthesis by various host DNA polymerases; (2) removal of the displaced RNA primer via FEN-1; and (3) ligation of the nick by LIG1 and LIG3. Additional factors POLα, TOP1, and TOP2 are shown to be involved in cccDNA formation; however, it is not clear which steps they are involved in. ATR has been shown to be involved in preventing the degradation of the minus stand. Red wavy line, RNA primer; gray wavy line, remnant peptide post protease digestion.