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DNA Polymerase alpha is essential for intracellular amplification of hepatitis B virus covalently closed circular DNA.

ABSTRACT: Persistent hepatitis B virus (HBV) infection relies on the establishment and maintenance of covalently closed circular (ccc) DNA, a 3.2 kb episome that serves as a viral transcription template, in the nucleus of an infected hepatocyte. Although evidence suggests that cccDNA is the repair product of nucleocapsid associated relaxed circular (rc) DNA, the cellular DNA polymerases involving in repairing the discontinuity in both strands of rcDNA as well as the underlying mechanism remain to be fully understood. Taking a chemical genetics approach, we found that DNA polymerase alpha (Pol α) is essential for cccDNA intracellular amplification, a genome recycling pathway that maintains a stable cccDNA pool in infected hepatocytes. Specifically, inhibition of Pol α by small molecule inhibitors aphidicolin or CD437 as well as silencing of Pol α expression by siRNA led to suppression of cccDNA amplification in human hepatoma cells. CRISPR-Cas9 knock-in of a CD437-resistant mutation into Pol α genes completely abolished the effect of CD437 on cccDNA formation, indicating that CD437 directly targets Pol α to disrupt cccDNA biosynthesis. Mechanistically, Pol α is recruited to HBV rcDNA and required for the generation of minus strand covalently closed circular rcDNA, suggesting that Pol α is involved in the repair of the minus strand DNA nick in cccDNA synthesis. Our study thus reveals that the distinct host DNA polymerases are hijacked by HBV to support the biosynthesis of cccDNA from intracellular amplification pathway compared to that from de novo viral infection, which requires Pol κ and Pol λ.

SUBMITTER: Tang L

PROVIDER: S-EPMC6505960 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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:Covalently closed circular DNA (cccDNA) of hepadnaviruses exists as an episomal minichromosome in the nucleus of an infected hepatocyte and serves as the template for the transcription of viral mRNAs. It had been demonstrated by others and us that interferon alpha (IFN-α) treatment of hepatocytes induced a prolonged suppression of human and duck hepatitis B virus cccDNA transcription, which is associated with the reduction of cccDNA-associated histone modifications specifying active transcription (H3K9ac or H3K27ac), but not the histone modifications marking constitutive (H3K9me3) or facultative (H3K27me3) heterochromatin formation. In our efforts to identify IFN-induced cellular proteins that mediate the suppression of cccDNA transcription by the cytokine, we found that downregulating the expression of signal transducer and activator of transcription 1 (STAT1), structural maintenance of chromosomes flexible hinge domain containing 1 (SMCHD1), or promyelocytic leukemia (PML) protein increased basal level of cccDNA transcription activity and partially attenuated IFN-α suppression of cccDNA transcription. In contrast, ectopic expression of STAT1, SMCHD1, or PML significantly reduced cccDNA transcription activity. SMCHD1 is a noncanonical SMC family protein and implicated in epigenetic silencing of gene expression. PML is a component of nuclear domain 10 (ND10) and is involved in suppressing the replication of many DNA viruses. Mechanistic analyses demonstrated that STAT1, SMCHD1, and PML were recruited to cccDNA minichromosomes and phenocopied the IFN-α-induced posttranslational modifications of cccDNA-associated histones. We thus conclude that STAT1, SMCHD1, and PML may partly mediate the suppressive effect of IFN-α on hepadnaviral cccDNA transcription.IMPORTANCE Pegylated IFN-α is the only therapeutic regimen that can induce a functional cure of chronic hepatitis B in a small, but significant, fraction of treated patients. Understanding the mechanisms underlying the antiviral functions of IFN-α in hepadnaviral infection may reveal molecular targets for development of novel antiviral agents to improve the therapeutic efficacy of IFN-α. By a loss-of-function genetic screening of individual IFN-stimulated genes (ISGs) on hepadnaviral mRNAs transcribed from cccDNA, we found that downregulating the expression of STAT1, SMCHD1, or PML significantly increased the level of viral RNAs without altering the level of cccDNA. Mechanistic analyses indicated that those cellular proteins are recruited to cccDNA minichromosomes and induce the posttranslational modifications of cccDNA-associated histones similar to those induced by IFN-α treatment. We have thus identified three IFN-α-induced cellular proteins that suppress cccDNA transcription and may partly mediate IFN-α silencing of hepadnaviral cccDNA transcription.

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DNA Polymerase alpha is essential for intracellular amplification of hepatitis B virus covalently closed circular DNA.

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