Recombinant Covalently Closed Circular Hepatitis B Virus DNA Induces Prolonged Viral Persistence in Immunocompetent Mice

Abstract

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/loxPmediated 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 g 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. (cccDNA) is an essential component of the HBV replication cycle and is regarded as a primary molecular mechanism for HBV persistence. The amount of cccDNA in cells is low, with around 5 to 50 copies in the nucleus. Nevertheless, cccDNA is stable, with a loss rate that correlates with the mitosis or death of infected hepatocytes (1, 2). Current antiviral treatments fail to eliminate the preexisting cccDNA pool that is responsible for viral rebound after therapy cessation. On the other hand, there is still lack of convenient techniques with high sensitivity and specificity to measure the HBV cccDNA pool in the hepatocyte nucleus (3)(4)(5).A laboratory animal model will be crucial for studying chronic HBV infection and disease. Mice are not susceptible to HBV infection because they lack a receptor(s) for viral entry. Even in HBV transgenic (Tg) mice, cccDNA is not formed, for unknown reasons (6, 7). These barriers can be experimentally overcome by hydrodynamic injection of naked plasmid DNA encoding an overlength HBV replicon, which enables intracellular replication of HBV in murine hepatocytes (8)(9)(10)(11)(12). However, the HBV replicon-based hydrodynamic injection induces only transient HBV viremia resembling an acute infection in immunocompetent mice. In this regard, a recent model of HBV persistence generated by inject...

An HBV-tolerant immunocompetent model that effectively simulates chronic hepatitis B virus infection in mice

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A non-viral CRISPR/Cas9 delivery system for therapeutically targeting HBV DNA and pcsk9 in vivo

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Two-way molecular ligation for efficient conversion of monomeric hepatitis B virus DNA constructs into tandem dimers

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