title: A new strategy for constructing in vitro replication-competent 1.3 copies of hepatitis B virus genome.
abstract: In the absence of a robust infectable cell culture system, assays related to replication of clinical HBV isolates are based on the transfection of replication-competent HBV DNA into hepatoma cell lines that are able to replicate and secrete HBV virions. Current methods for constructing HBV 1.1 genomes work well for drug susceptibility assays, but are not very suitable for research on HBV replication capacity or regulation since a heterogeneous promoter is required to drive pgRNA transcription. A new strategy for constructing HBV 1.3 genomes that contain HBV intrinsic promoter necessary for pgRNA transcription is reported in this paper. Using this strategy, three HBV 1.3 genomes from isolates of three patients were constructed. When the three HBV 1.3 genomes were transfected into the HepG2 cell line, replicative intermediates were detectable by Southern blotting with digoxigenin-labeled DNA probe in two of the three constructs. Using overlap extension PCR and avoiding as much as possible the digestion-and-ligation process, this strategy could be applied to constructing longer-than-genome units for most genotypes of HBV strains.
authors.affiliation: Institute for Viral Hepatitis, Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, Chongqing University of Medical Sciences, Chongqing, China.
authors.forename: Jie-li
authors.initials: JL
authors.lastname: Hu
authors.forename: Jing
authors.initials: J
authors.lastname: Cui
authors.forename: Xiao-yan
authors.initials: XY
authors.lastname: Deng
authors.forename: Wen-lu
authors.initials: WL
authors.lastname: Zhang
authors.forename: Qing-ling
authors.initials: QL
authors.lastname: Li
authors.forename: Jin-jun
authors.initials: JJ
authors.lastname: Guo
authors.forename: Ai-zhong
authors.initials: AZ
authors.lastname: Zeng
authors.forename: Ai-long
authors.initials: AL
authors.lastname: Huang
mesh_terms: D015139:Blotting, Southern / Q000379:methods; D045744:Cell Line, Tumor; D004279:DNA, Viral / Q000235:genetics; D016679:Genome, Viral; D006515:Hepatitis B virus / Q000235:genetics / Q000254:growth & development* / Q000302:isolation & purification; D022781:Hepatocytes / Q000821:virology; D006801:Humans; D014779:Virus Replication
chemical_list: D004279:DNA, Viral