Replication of hepatitis B virus in differentiated adult rat hepatocytes transfected with cloned viral DNA.
The possibility of obtaining expression of human hepatitis B virus (HBV) genes and production of virus particles in normal liver cells from heterologous species like normal adult rat hepatocytes, by transfecting the complete HBV genome, was investigated. Various techniques for hepatocyte transfection were assayed including the usual calcium-phosphate coprecipitation technique, the Pasco and Fagan modified calcium-phosphate procedure, and the lipofection technique. Transfection efficiency was determined by measuring the production of HBV surface antigen under various culture conditions. Transfection was the most efficient when assayed 1 or 2 days after hepatocyte plating at low density. Few variations in the efficiency were observed between the different transfection procedures. We show that under these culture conditions, replication of HBV can be achieved in differentiated adult rat hepatocytes. Synthesis of relaxed circular and single-stranded DNA forms and of viral transcripts including pregenome RNA occurred in the cells whereas viral antigens and mature and immature viral particles were released into the culture medium. The production of viral proteins was always higher in hepatocytes cocultivated with rat liver epithelial cells and maintained at a low density. In contrast, viral replication was not obtained by transfecting undifferentiated rat liver epithelial cells. These results demonstrate that replication of HBV can occur in hepatocytes from mammalian species non-closely related to primates and strongly support the idea that attachment of the virus and its penetration into the cells are critical steps in the host-specificity of the infection process and that hepatic-specific regulating factors could be essential for viral replication.(ABSTRACT TRUNCATED AT 250 WORDS)
Diot
C
C
INSERM-U49, Hôpital Pontchaillou, Rennes, France.
Gripon
P
P
Rissel
M
M
Guguen-Guillouzo
C
C
D000818:Animals; D002454:Cell Differentiation; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000235:genetics*; D015870:Gene Expression; D005814:Genes, Viral; D006514:Hepatitis B Surface Antigens / Q000096:biosynthesis / Q000235:genetics; D006513:Hepatitis B e Antigens / Q000096:biosynthesis / Q000235:genetics; D006515:Hepatitis B virus / Q000235:genetics* / Q000276:immunology / Q000502:physiology*; D008099:Liver / Q000166:cytology / Q000382:microbiology*; D051381:Rats; D014158:Transcription, Genetic; D014162:Transfection; D014779:Virus Replication
D004279:DNA, Viral; D006514:Hepatitis B Surface Antigens; D006513:Hepatitis B e Antigens