The enhancer I core region contributes to the replication level of hepatitis B virus in vivo and in vitro.
Chronic hepatitis B virus (HBV) infection can lead to liver cirrhosis and hepatocellular carcinoma. Long-term interaction of the immune system with the virus results in the selection of escape mutants and viral persistence. In this work we characterize mutations in the enhancer I region isolated prior to liver transplantation from the HBV genomes of 10 patients with chronic HBV infection. The HBV-genomes were sequenced, and the enhancer I region was cloned into luciferase reporter constructs to determine the transcriptional activity. Functional studies were performed by transfecting HBV replication-competent plasmids into hepatoma cells. Analyses of the replication fitness of the mutant strains were conducted by biochemical analysis. In all HBV genomes the enhancer I region was mutated. Most of these mutations resulted in decreased transcriptional activity. The strongest effects were detectable in strains with mutations in the hepatocyte nuclear factor 3 and 4 (HNF3 and HNF4) binding sites of the enhancer I core domain. Replication-competent HBV constructs containing these mutations demonstrated up to 10-fold-reduced levels of virus replication. Before liver transplantation, when the mutant strains were detected in the patients' sera, low HBV DNA levels were found. After transplantation and reinfection with a wild-type virus, the levels of replication were up to 240-fold higher. Our results show that mutations in the enhancer I region of HBV have a major impact on HBV replication. These mutations may also determine the switch from high to low levels of viral replication which is frequently observed during chronic HBV infection.
Bock
C T
CT
Department of Gastroenterology and Hepatology, Medizinische Hochschule Hannover, D-30625 Hannover, Germany.
Malek
N P
NP
Tillmann
H L
HL
Manns
M P
MP
Trautwein
C
C
D001665:Binding Sites; D002460:Cell Line; D004279:DNA, Viral / Q000032:analysis; D004268:DNA-Binding Proteins / Q000235:genetics; D015536:Down-Regulation; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D016679:Genome, Viral; D006515:Hepatitis B virus / Q000235:genetics*; D019694:Hepatitis B, Chronic / Q000601:surgery / Q000821:virology*; D006801:Humans; D016031:Liver Transplantation; D009154:Mutation; D009363:Neoplasm Proteins; D014157:Transcription Factors / Q000235:genetics; D014162:Transfection; D014779:Virus Replication / Q000235:genetics*
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D009363:Neoplasm Proteins; D014157:Transcription Factors