Efficient genome replication of hepatitis B virus using adenovirus vector: a compact pregenomic RNA-expression unit.
The complicated replication mechanisms of hepatitis B virus (HBV) have impeded HBV studies and anti-HBV therapy development as well. Herein we report efficient genome replication of HBV applying adenovirus vectors (AdVs) showing high transduction efficiency. Even in primary hepatocytes derived from humanized mice the transduction efficiencies using AdVs were 450-fold higher compared than those using plasmids. By using an expression unit consisting of the CMV promoter, 1.03-copy HBV genome and foreign poly(A) signal, we successfully generated an improved AdV (HBV103-AdV) that efficiently provided 58 times more pregenomic RNA than previously reported AdVs. The HBV103-AdV-mediated HBV replication was easily and precisely detected using quantitative real-time PCR in primary hepatocytes as well as in HepG2 cells. Notably, when the AdV containing replication-defective HBV genome of 1.14 copy was transduced, we observed that HBV DNA-containing circular molecules (pseudo-ccc DNA) were produced, which were probably generated through homologous recombination. However, the replication-defective HBV103-AdV hardly yielded the pseudo-ccc, probably because the repeated sequences are vey short. Additionally, the efficacies of entecavir and lamivudine were quantitatively evaluated using this system at only 4 days postinfection with HBV103-AdVs. Therefore, this system offers high production of HBV genome replication and thus could become used widely.
Laboratory of Molecular Genetics, The Institute of Medical Science, The University of Tokyo, Tokyo, 108-8639, Japan.
Mariko
M
Suzuki
Laboratory of Molecular Genetics, The Institute of Medical Science, The University of Tokyo, Tokyo, 108-8639, Japan.
Saki
S
Kondo
Laboratory of Virology, Institute of Microbial Chemistry (BIKAKEN), Microbial Chemistry Research Foundation, Tokyo 141-0021, Japan.
Manabu
M
Yamasaki
Laboratory of Virology, Institute of Microbial Chemistry (BIKAKEN), Microbial Chemistry Research Foundation, Tokyo 141-0021, Japan.
Norie
N
Matsuda
Laboratory of Virology, Institute of Microbial Chemistry (BIKAKEN), Microbial Chemistry Research Foundation, Tokyo 141-0021, Japan.
Akio
A
Nomoto
Department of Virology and Parasitology, Hamamatsu University School of Medicine, Shizuoka 431-3192, Japan.
Tetsuro
T
Suzuki
Laboratory of Molecular Genetics, The Institute of Medical Science, The University of Tokyo, Tokyo, 108-8639, Japan.
Izumu
I
Saito
Laboratory of Molecular Genetics, The Institute of Medical Science, The University of Tokyo, Tokyo, 108-8639, Japan.|Core Research Facilities of Basic Science (Molecular Genetics), Research Center for Medical Science, Jikei University School of Medicine, Tokyo 105-8461, Japan.
Yumi
Y
Kanegae
D000256:Adenoviridae / Q000235:genetics; D003587:Cytomegalovirus / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D016679:Genome, Viral; D057809:HEK293 Cells; D056945:Hep G2 Cells; D006515:Hepatitis B virus / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D014162:Transfection / Q000379:methods*; D014779:Virus Replication
D012333:RNA, Messenger; D012367:RNA, Viral