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

Sci Rep. 2016 May 13;6:25552. 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CRISPR-Cas Systems
  • Cell Line, Tumor
  • DNA, Circular / genetics*
  • DNA, Recombinant / genetics*
  • DNA, Viral / genetics*
  • Hepatitis B / virology
  • Hepatitis B virus / genetics*
  • Hepatitis B virus / metabolism
  • Hepatitis B virus / physiology
  • Host-Pathogen Interactions
  • Humans
  • RNA, Viral / genetics
  • Tupaiidae / virology
  • Viral Proteins / genetics
  • Viral Proteins / metabolism
  • Virion / genetics
  • Virion / metabolism
  • Virus Replication / genetics

Substances

  • DNA, Circular
  • DNA, Recombinant
  • DNA, Viral
  • RNA, Viral
  • Viral Proteins