title: HBVcircle: A novel tool to investigate hepatitis B virus covalently closed circular DNA.
abstract: Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) persists as a stable episome in infected hepatocytes and serves as a template for the transcription of all viral genes. Due to the narrow host range of HBV, the development of a robust mouse model that supports cccDNA-dependent viral replication is a key hurdle in the development of novel HBV therapeutics. This study aimed to develop a novel tool to investigate HBV cccDNA.
  
  
  
  Through minicircle technology, HBVcircle, a recombinant cccDNA, was easily generated and extracted from a genetically engineered E. coli strain. We characterized the performance of HBVcircle in cell culture by transfection and in immunocompetent mice by hydrodynamic injection (HDI).
  
  
  
  We demonstrated that HBVcircle formed authentic cccDNA-like molecules in vitro in transiently transfected hepatic cells and in vivo in mouse liver after HDI. HBVcircle supported high levels and persistent HBV replication. In addition, we investigated different factors affecting HBV in vivo replication and persistence, including the host genetic background, vector design and dosage, viral genes and genotypes, and immune activation status. Furthermore, different classes of anti-HBV drugs were also assessed with the HBVcircle system.
  
  
  
  Compared with previous reported HBV mouse models which employ other viral vectors to introduce overlength HBV genomes, viral gene expression and associated phenotypes are entirely driven by cccDNA-like viral genomes in the HBVcircle mouse model. Therefore, the HBVcircle is a close mimic of cccDNA, and it represents a novel tool for addressing HBV cccDNA related biological questions and for anti-HBV drug discovery.
  
  
  
  To establish a mouse model that supports cccDNA-dependent transcription, a novel tool named HBVcircle, was developed with minicircle technology. HBVcircle formed authentic cccDNA-like molecules in hepatocytes, and supported high levels and persistent HBV replication in vivo. The HBVcircle is a close mimic of cccDNA, and it represents a novel tool for addressing HBV cccDNA related biological questions and for anti-HBV drug discovery.
authors.affiliation: Roche Innovation Center Shanghai, Shanghai 201203, China.
authors.forename: Zhipeng
authors.initials: Z
authors.lastname: Yan
authors.affiliation: Roche Innovation Center Shanghai, Shanghai 201203, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Zeng
authors.affiliation: Roche Innovation Center Shanghai, Shanghai 201203, China.
authors.forename: Youjun
authors.initials: Y
authors.lastname: Yu
authors.affiliation: Roche Innovation Center Shanghai, Shanghai 201203, China.
authors.forename: Kunlun
authors.initials: K
authors.lastname: Xiang
authors.affiliation: Roche Innovation Center Shanghai, Shanghai 201203, China.
authors.forename: Hui
authors.initials: H
authors.lastname: Hu
authors.affiliation: Roche Innovation Center Shanghai, Shanghai 201203, China.
authors.forename: Xue
authors.initials: X
authors.lastname: Zhou
authors.affiliation: Roche Innovation Center Shanghai, Shanghai 201203, China.
authors.forename: Lili
authors.initials: L
authors.lastname: Gu
authors.affiliation: Roche Innovation Center Shanghai, Shanghai 201203, China.
authors.forename: Li
authors.initials: L
authors.lastname: Wang
authors.affiliation: Roche Innovation Center Shanghai, Shanghai 201203, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Zhao
authors.affiliation: Roche Innovation Center Basel, 4070 Basel, Switzerland.
authors.forename: John A T
authors.initials: JAT
authors.lastname: Young
authors.affiliation: Roche Innovation Center Shanghai, Shanghai 201203, China. Electronic address: lu.gao@roche.com.
authors.forename: Lu
authors.initials: L
authors.lastname: Gao
mesh_terms: D056704:Adaptive Immunity; D000818:Animals; D002460:Cell Line; D004270:DNA, Circular / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D004279:DNA, Viral / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D055808:Drug Discovery; D004353:Drug Evaluation, Preclinical; D005814:Genes, Viral; D005818:Genetic Engineering; D005821:Genetic Techniques; D056945:Hep G2 Cells; D006509:Hepatitis B / Q000188:drug therapy / Q000821:virology; D006515:Hepatitis B virus / Q000235:genetics* / Q000276:immunology / Q000502:physiology; D022781:Hepatocytes / Q000821:virology; D006801:Humans; D008297:Male; D051379:Mice; D008809:Mice, Inbred C3H; D008957:Models, Genetic; D014158:Transcription, Genetic; D014162:Transfection; D014779:Virus Replication / Q000235:genetics
chemical_list: D004270:DNA, Circular; D004279:DNA, Viral
keywords: Anti-HBV drug discovery; HBV mouse model; Hydrodynamic injection; Minicircle; Recombinant cccDNA