Development of cell cultures that express hepatitis B virus to high levels and accumulate cccDNA.
Establishment of an infection with hepatitis B virus (HBV) requires synthesis and maintenance of a covalently closed circular DNA (cccDNA) form of the viral genome in the nucleus of host cells. To facilitate the investigation of the synthesis of cccDNA, cell cultures were developed that express HBV to high levels. Cell lines derived from hepatoma cells Huh7 and HepG2 were created that express Epstein-Barr virus (EBV) nuclear antigen-1 and a fusion protein of the Tet repressor and Kox1 transcriptional repression domain stably. Transfection of these cell lines with an expression plasmid for HBV that contains the origin of plasmid replication of EBV (oriP) led to increases in the intracellular levels of HBV core protein ( approximately 8- to 51-fold) and encapsidated HBV DNA ( approximately 3- to 12-fold) in comparison to Huh7 and HepG2 cells. Virion production was also increased ( approximately 3- to 12-fold) in these cell cultures and an increase in the level of cccDNA ( approximately 3-fold) was observed in the Huh7-derived cell lines. In addition, these cell lines maintained the HBV expression plasmid upon selection and expressed HBV conditionally. Thus, these cell cultures exhibit several features that facilitate study of the synthesis of cccDNA and other aspects of replication of HBV.
Lentz
Thomas B
TB
McArdle Laboratory for Cancer Research, University of Wisconsin School of Medicine and Public Health, Madison, WI 53706, USA.
Loeb
Daniel D
DD
D002460:Cell Line; D004279:DNA, Viral / Q000096:biosynthesis*; D019309:Epstein-Barr Virus Nuclear Antigens / Q000096:biosynthesis / Q000235:genetics; D006512:Hepatitis B Core Antigens / Q000096:biosynthesis / Q000235:genetics; D006515:Hepatitis B virus / Q000502:physiology*; D022781:Hepatocytes / Q000821:virology; D006801:Humans; D051741:Kruppel-Like Transcription Factors / Q000096:biosynthesis / Q000235:genetics; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D012097:Repressor Proteins / Q000096:biosynthesis / Q000235:genetics; D019065:Virus Assembly; D014779:Virus Replication
D004279:DNA, Viral; D019309:Epstein-Barr Virus Nuclear Antigens; D006512:Hepatitis B Core Antigens; D051741:Kruppel-Like Transcription Factors; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C109484:ZNF10 protein, human; C034811:tetracycline resistance-encoding transposon repressor protein; C417029:EBV-encoded nuclear antigen 1