title: The recombined cccDNA produced using minicircle technology mimicked HBV genome in structure and function closely.
abstract: HBV covalently closed circular DNA (cccDNA) is drug-resistant and responsible for viral persistence. To facilitate the development of anti-cccDNA drugs, we developed a minicircle DNA vector (MC)-based technology to produce large quantity of recombined cccDNA (rcccDNA) resembling closely to its wild-type counterpart both in structure and function. The rcccDNA differed to the wild-type cccDNA (wtcccDNA) only in that it carried an extra 36-bp DNA recombinant product attR upstream of the preC/C gene. Using a procedure similar to standard plasmid production, milligrams of rcccDNA can be generated in common laboratories conveniently. The rcccDNA demonstrated many essential biological features of wtcccDNA, including: (1) undergoing nucleation upon nucleus entry; (2) serving as template for production of all HBV RNAs and proteins; (3) deriving virions capable of infecting tree shrew, and subsequently producing viral mRNAs, proteins, rcccDNA and infectious virions. As an example to develop anti-cccDNA drugs, we used the Crispr/Cas9 system to provide clear-cut evidence that rcccDNA was cleaved by this DNA editing tool in vitro. In summary, we have developed a convenient technology to produce large quantity of rcccDNA as a surrogate of wtcccDNA for investigating HBV biology and developing treatment to eradicate this most wide-spreading virus.
authors.affiliation: The Laboratory for Gene and Cell Engineering, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, 518055, China.|Department of Infectious Diseases, The Third Affiliated Hospital of Sun Yat-Sen University, 600 Tian He Road, Guangzhou, 510630, China.
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Guo
authors.affiliation: The Laboratory for Gene and Cell Engineering, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, 518055, China.
authors.forename: Ping
authors.initials: P
authors.lastname: Chen
authors.affiliation: The Laboratory for Gene and Cell Engineering, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, 518055, China.
authors.forename: Xiaohu
authors.initials: X
authors.lastname: Hou
authors.affiliation: The Laboratory for Gene and Cell Engineering, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, 518055, China.
authors.forename: Wenjuan
authors.initials: W
authors.lastname: Xu
authors.affiliation: The Laboratory for Gene and Cell Engineering, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, 518055, China.
authors.forename: Dan
authors.initials: D
authors.lastname: Wang
authors.affiliation: The Laboratory for Gene and Cell Engineering, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, 518055, China.
authors.forename: Tian-Yan
authors.initials: TY
authors.lastname: Wang
authors.affiliation: The Laboratory for Gene and Cell Engineering, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, 518055, China.
authors.forename: Liping
authors.initials: L
authors.lastname: Zhang
authors.affiliation: The Laboratory for Gene and Cell Engineering, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, 518055, China.
authors.forename: Gang
authors.initials: G
authors.lastname: Zheng
authors.affiliation: Department of Infectious Diseases, The Third Affiliated Hospital of Sun Yat-Sen University, 600 Tian He Road, Guangzhou, 510630, China.
authors.forename: Zhi-Liang
authors.initials: ZL
authors.lastname: Gao
authors.affiliation: The Laboratory for Gene and Cell Engineering, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, 518055, China.
authors.forename: Cheng-Yi
authors.initials: CY
authors.lastname: He
authors.affiliation: Institute of Hepatology, Shenzhen Third People's Hospital, Shenzhen, 518112, China.
authors.forename: Boping
authors.initials: B
authors.lastname: Zhou
authors.affiliation: The Laboratory for Gene and Cell Engineering, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, 518055, China.
authors.forename: Zhi-Ying
authors.initials: ZY
authors.lastname: Chen
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems; D045744:Cell Line, Tumor; D004270:DNA, Circular / Q000235:genetics*; D004274:DNA, Recombinant / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics*; D006509:Hepatitis B / Q000821:virology; D006515:Hepatitis B virus / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D054884:Host-Pathogen Interactions; D006801:Humans; D012367:RNA, Viral / Q000235:genetics; D014418:Tupaiidae / Q000821:virology; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism; D014771:Virion / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication / Q000235:genetics
chemical_list: D004270:DNA, Circular; D004274:DNA, Recombinant; D004279:DNA, Viral; D012367:RNA, Viral; D014764:Viral Proteins