The recombined cccDNA produced using minicircle technology mimicked HBV genome in structure and function closely.

Xiaoyan Guo,Ping Chen,Dan Wang,Boping Zhou,Zhi-Liang Gao,Wenjuan Xu,Liping Zhang,Gang Zheng,Cheng-Yi He,Zhi-Ying Chen,Xiaohu Hou,Tian-Yan Wang

doi:10.1038/srep25552

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.

Highlights

Nucleotide Position the residual closed circular DNA (cccDNA) is the key for curing Hepatitis B virus (HBV) infection[7,13]
Like HBV, duck hepatitis B virus (DHBV) belongs to the hepadnavirus family so that is widely used as a surrogate to study HBV infection, replication, and cccDNA formation[15,19,20,21]
Production of HBV recombined cccDNA (rcccDNA) using minicircle technology. rcccDNA was generated from a minicircle-producing plasmid harboring a 3.2 kb HBV monomeric genome DNA using a ΦC 31-mediated DNA recombination for the minicircle production[22,23]

Summary

Introduction

Nucleotide Position the residual cccDNA is the key for curing HBV infection[7,13]. Like HBV, DHBV belongs to the hepadnavirus family so that is widely used as a surrogate to study HBV infection, replication, and cccDNA formation[15,19,20,21]. We described a protocol based on minicircle technique[22,23] to produce milligrams of recombined cccDNA (rcccDNA) in a common laboratory setting. The rcccDNA making technique will contribute greatly to the study of cccDNA, such asin searching for the treatment leading to its elimination and the cure of HBV infection

Methods

Results

Conclusion