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Hepatitis B Virus Covalently Closed Circular DNA Predicts Postoperative Liver Cancer Metastasis Independent of Virological Suppression.

ABSTRACT: New antiviral therapies against hepatitis B virus (HBV) focus on the elimination of covalently closed circular DNA (cccDNA). However, traditional cccDNA-specific quantitative PCR (qPCR) has a narrow effective range, hindering a reliable comparison between the levels of biopsy-derived cccDNAs. Collaterally, the prognostic role of cccDNA in HBV-related hepatocellular carcinoma (HCC) cannot be clearly defined. Here, we developed a peptide nucleic acid (PNA)-clamping qPCR method to provide a wider range of specific cccDNA quantification (up to 5 logs of effective range). Extrachromosomal DNA was extracted from para-neoplastic tissues for cccDNA quantification. In total, 350 HBV-related HCC patients were included for an outcome analysis. Without differential pre-dilution, cccDNA levels in para-neoplastic liver tissues were determined, ranging from < 2 × 10³ to 123.0 × 10⁶ copies/gram. The multivariate linear regression analysis showed that cccDNA was independently correlated with the HBV e antigen (p < 0.001) and serum HBV-DNA levels (p = 0.012). The Cox proportional hazard model analysis showed that cccDNA independently predicted overall survival (p = 0.003) and extrahepatic metastasis-free survival (p = 0.001). In virologically suppressed HCC patients, cccDNA still effectively predicted intrahepatic recurrence-free (p = 0.003) and extrahepatic metastasis-free (p = 0.009) survivals. In conclusion, cccDNA independently predicted postoperative extrahepatic metastasis-free survival.

SUBMITTER: Hsu CW

PROVIDER: S-EPMC7867012 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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Hepatitis B Virus Covalently Closed Circular DNA Predicts Postoperative Liver Cancer Metastasis Independent of Virological Suppression.

Hsu Chao-Wei CW Chu Yu-De YD Lai Ming-Wei MW Lin Chih-Lang CL Liang Kung-Hao KH Lin Yang-Hsiang YH Yeh Chau-Ting CT

Cancers 20210131 3

New antiviral therapies against hepatitis B virus (HBV) focus on the elimination of covalently closed circular DNA (cccDNA). However, traditional cccDNA-specific quantitative PCR (qPCR) has a narrow effective range, hindering a reliable comparison between the levels of biopsy-derived cccDNAs. Collaterally, the prognostic role of cccDNA in HBV-related hepatocellular carcinoma (HCC) cannot be clearly defined. Here, we developed a peptide nucleic acid (PNA)-clamping qPCR method to provide a wider r ...[more]

PMID: 33572617

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Project description:The covalently closed circular (CCC) DNA of hepatitis B virus (HBV) functions as the only viral transcriptional template capable of producing all viral RNA species and is essential to initiate and sustain viral replication. CCC DNA is converted from a relaxed circular (RC) DNA, in which neither of the two DNA strands is covalently closed. As RC DNA mimics damaged cellular DNA, the host cell DNA damage repair (DDR) system is thought to be responsible for HBV CCC DNA formation. The potential role of two major cellular DDR pathways, the ataxia telangiectasia mutated (ATM) pathway and the ATM and Rad3-related (ATR) pathway, in HBV CCC DNA formation was thus investigated. Inhibition, or expression knockdown, of ATR and its downstream signaling factor CHK1, but not of ATM, decreased CCC DNA formation during de novo HBV infection, as well as intracellular CCC DNA amplification, when RC DNA from extracellular virions and intracellular nucleocapsids, respectively, is converted to CCC DNA. Furthermore, a novel RC DNA processing product with 5' truncated minus strands was detected when the ATR-CHK1 pathway was inhibited, further indicating that this pathway controls RC DNA processing during its conversion to CCC DNA. These results provide new insights into how host cells recognize and process HBV RC DNA in order to produce CCC DNA and have implications for potential means to block CCC DNA production.IMPORTANCE Hepatitis B virus (HBV) chronically infects hundreds of millions of people and remains a major cause of viral hepatitis, cirrhosis, and liver cancer. HBV persistence is sustained by a viral nuclear episome that directs all viral gene expression needed to support viral replication. The episome is converted from an incomplete DNA precursor in viral particles in an ill-understood process. We report here that the incomplete DNA precursor is recognized by the host cell in a way similar to the sensing of damaged cellular DNA for subsequent repair to form the nuclear episome. Intense efforts are ongoing to develop novel antiviral strategies to eliminate CCC DNA so as to cure chronic HBV infection. Our results here provide novel insights into, and suggest novel ways of perturbing, the process of episome formation. Furthermore, our results inform mechanisms of cellular DNA damage recognition and repair, processes essential for normal cell growth.

Project description:It remains crucial to develop a laboratory model for studying hepatitis B virus (HBV) chronic infection. We hereby produced a recombinant covalently closed circular DNA (rcccDNA) in view of the key role of cccDNA in HBV persistence. A loxP-chimeric intron was engineered into a monomeric HBV genome in a precursor plasmid (prcccDNA), which was excised using Cre/loxP-mediated DNA recombination into a 3.3-kb rcccDNA in the nuclei of hepatocytes. The chimeric intron was spliced from RNA transcripts without interrupting the HBV life cycle. In cultured hepatoma cells, cotransfection of prcccDNA and pCMV-Cre (encoding Cre recombinase) resulted in accumulation of nuclear rcccDNA that was heat stable and epigenetically organized as a minichromosome. A mouse model of HBV infection was developed by hydrodynamic injection of prcccDNA. In the presence of Cre recombinase, rcccDNA was induced in the mouse liver with effective viral replication and expression, triggering a compromised T-cell response against HBV. Significant T-cell hyporesponsiveness occurred in mice receiving 4 μg prcccDNA, resulting in prolonged HBV antigenemia for up to 9 weeks. Persistent liver injury was observed as elevated alanine transaminase activity in serum and sustained inflammatory infiltration in the liver. Although a T-cell dysfunction was induced similarly, mice injected with a plasmid containing a linear HBV replicon showed rapid viral clearance within 2 weeks. Collectively, our study provides an innovative approach for producing a cccDNA surrogate that established HBV persistence in immunocompetent mice. It also represents a useful model system in vitro and in vivo for evaluating antiviral treatments against HBV cccDNA. Importance: (i) Unlike plasmids that contain a linear HBV replicon, rcccDNA established HBV persistence with sustained liver injury in immunocompetent mice. This method could be a prototype for developing a mouse model of chronic HBV infection. (ii) An exogenous intron was engineered into the HBV genome for functionally seamless DNA recombination. This original approach could be also extended to other viral studies. (iii) rcccDNA was substantially induced in the nuclei of hepatocytes and could be easily distinguished by its exogenous intron using PCR. This convenient model system affords the opportunity to test antivirals directly targeting HBV cccDNA.

Project description:Hepatitis B virus (HBV) delivers a partially double-stranded, relaxed circular (RC) DNA genome in complete virions to the host cell nucleus for conversion to the covalently closed circular (CCC) DNA, which establishes and sustains viral infection. An overlength pregenomic RNA (pgRNA) is then transcribed from CCC DNA and packaged into immature nucleocapsids (NCs) by the viral core (HBc) protein. pgRNA is reverse transcribed to produce RC DNA in mature NCs, which are then enveloped and secreted as complete virions, or delivered to the nucleus to replenish the nuclear CCC DNA pool. RC DNA, whether originating from extracellular virions or intracellular mature NCs, must be released upon NC disassembly (uncoating) for CCC DNA formation. HBc is known to undergo dynamic phosphorylation and dephosphorylation at its C-terminal domain (CTD) to facilitate pgRNA packaging and reverse transcription. Here, two putative phosphorylation sites in the HBc N-terminal domain (NTD), S44 and S49, were targeted for genetic and biochemical analysis to assess their potential roles in viral replication. The NTD mutant that mimics the non-phosphorylated state (N2A) was competent in all steps of viral replication tested from capsid assembly, pgRNA packaging, reverse transcription, to virion secretion, except for a decrease in CCC DNA formation. On the other hand, the phosphor-mimetic mutant N2E showed a defect in the early step of pgRNA packaging but enhanced the late step of mature NC uncoating and consequently, increased CCC DNA formation. N2E also enhanced phosphorylation in CTD and possibly elsewhere in HBc. Furthermore, inhibition of the cyclin-dependent kinase 2 (CDK2), which is packaged into viral capsids, could block CCC DNA formation. These results prompted us to propose a model whereby rephosphorylation of HBc at both NTD and CTD by the packaged CDK2, following CTD dephosphorylation during NC maturation, facilitates uncoating and CCC DNA formation by destabilizing mature NCs.

Dataset Information

Hepatitis B Virus Covalently Closed Circular DNA Predicts Postoperative Liver Cancer Metastasis Independent of Virological Suppression.

Publications

Hepatitis B Virus Covalently Closed Circular DNA Predicts Postoperative Liver Cancer Metastasis Independent of Virological Suppression.

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