Recombinant covalently closed circular DNA of hepatitis B virus induces long‐term viral persistence with chronic hepatitis in a mouse model

Abstract

rcccDNA was intrinsically stable in vivo, enabling long-term persistence in the context of chronic hepatitis, and viral persistence, in turn, may promote progression of chronic liver disease; our study also presented a surrogate model of HBV cccDNA persistence in mice that could advance our understanding of the pathogenesis of chronic hepatitis B. (Hepatology 2018;67:56-70).

Nuclear Sensor Interferon‐Inducible Protein 16 Inhibits the Function of Hepatitis B Virus Covalently Closed Circular DNA by Integrating Innate Immune Activation and Epigenetic Suppression

“…To investigate the potential sensor for HBV cccDNA, we established a recombinant cccDNA (rcccDNA) cell model by transfecting prcccDNA plus pCMV‐Cre (prcccDNA/Cre) into Huh7 cells as described . This model is credited with producing high levels of rcccDNA in the hepatic nuclei, which has been proven to be a surrogate for the natural cccDNA minichromosome . As PRRs often possess two properties (physical interaction with DNA and being transcriptionally inducible by cytokines as part of a positive feedback loop to sustain innate immune responses), we treated this rcccDNA cell model with or without IFN‐α and then tested the possible sensors for cccDNA by ChIP assays.…”

“…Precursor plasmid rcccDNA (prcccDNA) and Crerecombinase expression plasmid (pCMV-Cre) were cotransfected into Huh7 cells to produce recombinant cccDNA (rcccDNA) as reported by Qi et al (15) Nuclear rcccDNA has been demonstrated to be epigenetically organized as a minichromosome and can be used as a cccDNA surrogate for HBV studies. (15)(16)(17) To investigate the effect of IFI16 overexpression on cccDNA, prcccDNA/pCMV-Cre together with FLAGtagged plasmid IFI16 (pIFI16-FLAG; Addgene, #35064) was transfected into Huh7 cells using lipofectamine 2000 (Invitrogen, Carlsbad, CA). Small interfering RNA (siRNA) specific for IFI16 (sense: 5′-UGCUGAACGCAACAGAAUCAU-3′ and antisense: 5′-AUGAUUCUGUUGCGUUCAGCA-3′) or control siRNA was transfected into Huh7 cells by HiPerFect Transfection Reagent (Qiagen, Hilden, Germany).…”

“…(15) This model is credited with producing high levels of rcccDNA in the hepatic nuclei, which has been proven to be a surrogate for the natural cccDNA minichromosome. (15)(16)(17) As PRRs often possess two properties (physical interaction with DNA and being transcriptionally inducible by cytokines as part of a positive feedback loop to sustain innate immune responses (24) ), we treated this rcccDNA cell model with or without IFN-α and then tested the possible sensors for cccDNA by ChIP assays. Results showed that IFI16, but not other possible nuclear-located innate DNA sensors (including cGAS, AIM2, and myeloid cell nuclear differentiation antigen), bound to the rcccDNA, especially after IFN-α treatment (Fig.…”

Restriction of exogenous DNA expression by SAMHD1

“…The Tet-Off and Tet-On gene expression systems were from Clontech (Mountain View, USA). prcccDNA and pCMV-Cre plasmids were constructed previously [20,21]. prcccDNA contains a precursor of recombinant HBV cccDNA which can be formed by Cre-mediated recombination in cells and mice.…”

“…S12i (online), no obvious binding of SAMHD1 to HBV DNA was detected. For in vivo study, a mouse model of HBV cccDNA was employed [20,21]. In such a system, a recombinant cccDNA (rcccDNA) precursor plasmid (prcccDNA) and pCMV-Cre were hydrodynamically injected through tail vein to transfect mouse liver.…”