A simple and efficient strategy for the de novo construction of greater-than-genome-length hepatitis B virus replicons

J Virol Methods. 2014 Oct;207:158-62. doi: 10.1016/j.jviromet.2014.07.009. Epub 2014 Jul 12.

Abstract

Hepatitis B virus (HBV) uses small covalently closed circular DNA for transcription and replication; linearization at any site would destruct at least one HBV open reading frame and interrupt the virus life cycle. Therefore, greater-than-genome-length (GGL) HBV replicons have been widely used for HBV replication studies. However, the existing strategies for the de novo construction of GGL HBV replicons are too complex to implement, especially when multiple replicons need to be cloned. In this study, the pHBV-basic plasmid was constructed for efficient cloning of GGL HBV replicons; changing the orientation of the site for type IIs restriction enzyme SapI in this plasmid reduced the de novo construction of various GGL HBV replicons to only one to three steps. Furthermore, Q5 high-fidelity DNA polymerase was found to be ideal for HBV genome amplification. In vitro experiments showed that the HBV replicon containing 1.31 genome copies replicated with better efficiency as evidenced by the titers of HBV DNA and HBsAg and HBeAg markers. The vector described in this study could serve as a powerful vehicle for in vitro and in vivo investigation of HBV replication.

Keywords: Construction strategy; Greater-than-genome-length; Hepatitis B virus; Q5 high-fidelity DNA polymerase; Replicon.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA, Viral / chemistry
  • DNA, Viral / genetics
  • Genetic Engineering / methods
  • Genetic Vectors
  • Genome, Viral*
  • Hepatitis B Surface Antigens / biosynthesis
  • Hepatitis B virus / genetics*
  • Hepatitis B virus / physiology
  • Humans
  • Molecular Biology / methods*
  • Molecular Sequence Data
  • Plasmids
  • Replicon*
  • Sequence Analysis, DNA
  • Virology / methods*
  • Virus Replication

Substances

  • DNA, Viral
  • Hepatitis B Surface Antigens

Associated data

  • GENBANK/KP027477