TY - JOUR
T1 - Establishment of Cre-mediated HBV recombinant cccDNA (rcccDNA) cell line for cccDNA biology and antiviral screening assays
AU - Wu, Min
AU - Li, Jin
AU - Yue, Lei
AU - Bai, Lu
AU - Li, Yaming
AU - Chen, Jieliang
AU - Zhang, Xiaonan
AU - Yuan, Zhenghong
N1 - Funding Information:
We thank Prof. Qiang Deng for his generous gift of prcccDNA plasmid and Prof. Ulrike Protzer for plenti6.3-A3A plasmid; we thank Zhuying Chen for her excellent technical assistance. This study is supported by the Innovation Program of Shanghai Municipal Education Commission (2017-01-07-00-07-E00057), the National Key Research and Development Program (2016YFC0100604), the National Natural Science Foundation (81671998, 91542207), the Shanghai Science and Technology Commission (16411960100) and the Sino-GermanTransregional Collaborative Research Centre (81461130019).
Funding Information:
We thank Prof. Qiang Deng for his generous gift of prcccDNA plasmid and Prof. Ulrike Protzer for plenti6.3-A3A plasmid; we thank Zhuying Chen for her excellent technical assistance. This study is supported by the Innovation Program of Shanghai Municipal Education Commission ( 2017-01-07-00-07-E00057 ), the National Key Research and Development Program ( 2016YFC0100604 ), the National Natural Science Foundation ( 81671998 , 91542207 ), the Shanghai Science and Technology Commission ( 16411960100 ) and the Sino-GermanTransregional Collaborative Research Centre ( 81461130019 ). Appendix A
Publisher Copyright:
© 2018 The Authors
PY - 2018/4
Y1 - 2018/4
N2 - Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA), existing in hepatocyte nuclei as a stable minichromosome, plays a central role in the life cycle of the virus and permits the persistence of infection. Despite being essential for HBV infection, little is known about the molecular mechanisms of cccDNA formation, regulation and degradation, and there is no therapeutic agents directly targeting cccDNA, fore mostly due to the lack of robust, reliable and quantifiable HBV cccDNA models. In this study, combined the Cre/loxP and sleeping beauty transposons system, we established HepG2-derived cell lines integrated with 2-60 copies of monomeric HBV genome flanked by loxP sites (HepG2-HBV/loxP). After Cre expression via adenoviral transduction, 3.3-kb recombinant cccDNA (rcccDNA) bearing a chimeric intron can be produced in the nuclei of these HepG2-HBV/loxP cells. The rcccDNA could be accurately quantified by quantitative PCR using specific primers and cccDNA pool generated in this model could be easily detected by Southern blotting using the digoxigenin probe system. We demonstrated that the rcccDNA was epigenetically organized as the natural minichromosome and served as the template supporting pgRNA transcription and viral replication. As the expression of HBV S antigen (HBsAg) is dependent on the newly generated cccDNA, HBsAg is the surrogate marker of cccDNA. Additionally, the efficacies of 3 classes of anti-HBV agents were evaluated in HepG2-HBV/loxP cells and antiviral activities with different mechanisms were confirmed. These data collectively suggested that HepG2-HBV/loxP cell system will be powerful platform for studying cccDNA related biological mechanisms and developing novel cccDNA targeting drugs.
AB - Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA), existing in hepatocyte nuclei as a stable minichromosome, plays a central role in the life cycle of the virus and permits the persistence of infection. Despite being essential for HBV infection, little is known about the molecular mechanisms of cccDNA formation, regulation and degradation, and there is no therapeutic agents directly targeting cccDNA, fore mostly due to the lack of robust, reliable and quantifiable HBV cccDNA models. In this study, combined the Cre/loxP and sleeping beauty transposons system, we established HepG2-derived cell lines integrated with 2-60 copies of monomeric HBV genome flanked by loxP sites (HepG2-HBV/loxP). After Cre expression via adenoviral transduction, 3.3-kb recombinant cccDNA (rcccDNA) bearing a chimeric intron can be produced in the nuclei of these HepG2-HBV/loxP cells. The rcccDNA could be accurately quantified by quantitative PCR using specific primers and cccDNA pool generated in this model could be easily detected by Southern blotting using the digoxigenin probe system. We demonstrated that the rcccDNA was epigenetically organized as the natural minichromosome and served as the template supporting pgRNA transcription and viral replication. As the expression of HBV S antigen (HBsAg) is dependent on the newly generated cccDNA, HBsAg is the surrogate marker of cccDNA. Additionally, the efficacies of 3 classes of anti-HBV agents were evaluated in HepG2-HBV/loxP cells and antiviral activities with different mechanisms were confirmed. These data collectively suggested that HepG2-HBV/loxP cell system will be powerful platform for studying cccDNA related biological mechanisms and developing novel cccDNA targeting drugs.
KW - Cre/loxP
KW - Hepatitis B virus (HBV)
KW - Recombinant covalently closed circular DNA (rcccDNA)
KW - Stable cell line
UR - http://www.scopus.com/inward/record.url?scp=85042061342&partnerID=8YFLogxK
U2 - 10.1016/j.antiviral.2018.02.007
DO - 10.1016/j.antiviral.2018.02.007
M3 - Article
C2 - 29432776
SN - 0166-3542
VL - 152
SP - 45
EP - 52
JO - Antiviral Research
JF - Antiviral Research
ER -
