Efficient genome replication of hepatitis B virus using adenovirus vector: a compact pregenomic RNA-expression unit.
Efficient genome replication of hepatitis B virus using adenovirus vector: a compact pregenomic RNA-expression unit.
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DOI：
10.1038/srep41851
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发表时间：
2017-02-03
影响因子：
4.6
通讯作者：
Kanegae Y
中科院分区：
文献类型：
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作者：
Suzuki M;Kondo S;Yamasaki M;Matsuda N;Nomoto A;Suzuki T;Saito I;Kanegae Y
B型肝炎病毒（HBV）复杂的复制机制阻碍了HBV研究和抗HBV治疗的发展。在此，我们报告了应用腺病毒载体（AdVs）的HBV的有效基因组复制，其显示出高转导效率。即使在来自人源化小鼠的原代肝细胞中，使用AdV的转导效率也比使用质粒的转导效率高450倍。通过使用由CMV启动子、1.03拷贝HBV基因组和外源poly（A）信号组成的表达单元，我们成功地产生了改进的AdV（HBV 103-AdV），其有效地提供了比先前报道的AdV多58倍的前基因组RNA。HBV 103-AdV介导的HBV复制在原代肝细胞以及HepG 2细胞中使用定量实时PCR容易且精确地检测。值得注意的是，当转导含有1.14拷贝的复制缺陷型HBV基因组的AdV时，我们观察到产生了含有HBV DNA的环状分子（假ccc DNA），其可能通过同源重组产生。而复制缺陷型HBV 103-AdV几乎不产生假ccc，可能是因为重复序列非常短。此外，恩替卡韦和拉米夫定的疗效进行了定量评价，使用该系统在感染HBV 103-AdV后仅4天。因此，该系统提供了高产量的HBV基因组复制，因此可以广泛使用。
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.