Initiation of hepatitis B virus genome replication and production of infectious virus following delivery in HepG2 cells by novel recombinant baculovirus vector.
One of the major problems in gaining further insight into hepatitis B virus (HBV)/host-cell interactions is to improve the existing cellular models for the study of HBV replication. The first objective of this study was to improve the system based on transduction of HepG2 cells with a recombinant baculovirus to study HBV replication. A new HBV recombinant baculovirus, Bac-HBV-1.1, in which the synthesis of pre-genomic RNA is driven by a strong mammalian promoter, was generated. Transduction with this new recombinant baculovirus led to higher levels of HBV replication in HepG2 cells compared with levels obtained with previously described baculovirus vectors. The initiation of a complete HBV DNA replication cycle in Bac-HBV-1.1-transduced HepG2 cells was shown by the presence of HBV replicative intermediates, including covalently closed circular DNA (cccDNA). Only low levels of cccDNA were detected in the nucleus of infected cells. Data showed that cccDNA resulted from the recycling of newly synthesized nucleocapsids and was bound to acetylated histones in a chromatin-like structure. HBV particles released into the supernatant of transduced HepG2 cells were infectious in differentiated HepaRG cells. Several Bac-HBV-1.1 baculoviruses containing HBV strains carrying mutations conferring resistance to lamivudine and/or adefovir were constructed. Phenotypic analysis of these mutants confirmed the results obtained with the transfection procedures. In conclusion, an improved cell-culture system was established for the transduction of replication-competent HBV genomes. This will be useful for future studies of the fitness of HBV mutants.
Lucifora
J
J
Université Lyon 1, IFR62 Lyon Est, 69008 Lyon, France.|INSERM, U871, 151 Cours Albert Thomas, 69003 Lyon, France.
Durantel
D
D
Hospices Civils de Lyon, Hôtel Dieu Hospital, 69002 Lyon, France.|Université Lyon 1, IFR62 Lyon Est, 69008 Lyon, France.|INSERM, U871, 151 Cours Albert Thomas, 69003 Lyon, France.
Belloni
L
L
Laboratoire Associé INSERM, U785, Villejuif, France.|Department of Internal Medicine and Laboratory of Gene Expression, Fondazione A. Cesalpino, University of Rome La Sapienza, Rome, Italy.
Barraud
L
L
Department of Internal Medicine and Laboratory of Gene Expression, Fondazione A. Cesalpino, University of Rome La Sapienza, Rome, Italy.
Villet
S
S
Université Lyon 1, IFR62 Lyon Est, 69008 Lyon, France.|INSERM, U871, 151 Cours Albert Thomas, 69003 Lyon, France.
Vincent
I E
IE
Université Lyon 1, IFR62 Lyon Est, 69008 Lyon, France.|INSERM, U871, 151 Cours Albert Thomas, 69003 Lyon, France.
Margeridon-Thermet
S
S
Université Lyon 1, IFR62 Lyon Est, 69008 Lyon, France.|INSERM, U871, 151 Cours Albert Thomas, 69003 Lyon, France.
Hantz
O
O
Université Lyon 1, IFR62 Lyon Est, 69008 Lyon, France.|INSERM, U871, 151 Cours Albert Thomas, 69003 Lyon, France.
Kay
A
A
Université Lyon 1, IFR62 Lyon Est, 69008 Lyon, France.|INSERM, U871, 151 Cours Albert Thomas, 69003 Lyon, France.
Levrero
M
M
Eurofins-Viralliance, BioAlliance Pharma SA, Paris, France.|Laboratoire Associé INSERM, U785, Villejuif, France.
Zoulim
F
F
Hospices Civils de Lyon, Hôtel Dieu Hospital, 69002 Lyon, France.|Université Lyon 1, IFR62 Lyon Est, 69008 Lyon, France.|INSERM, U871, 151 Cours Albert Thomas, 69003 Lyon, France.
D016367:Baculoviridae / Q000235:genetics*; D045744:Cell Line, Tumor; D004261:DNA Replication; D005822:Genetic Vectors; D016679:Genome, Viral; D006515:Hepatitis B virus / Q000235:genetics / Q000472:pathogenicity / Q000502:physiology*; D022781:Hepatocytes / Q000821:virology*; D006801:Humans; D011995:Recombination, Genetic; D014161:Transduction, Genetic; D014771:Virion / Q000378:metabolism / Q000472:pathogenicity; D014773:Virology / Q000379:methods; D014779:Virus Replication