载脂蛋白质B mRNA编辑酶催化多肽3G抑制乙型肝炎病毒和鸭乙型肝炎病毒复制

目的了解载脂蛋白质BmRNA编辑酶催化多肽3G（APOBEC3G）对乙型肝炎病毒（HBV）和鸭乙型肝炎炎病毒（DHBV）复制的抑制作用。方法从健康人外周血单个核细胞提取RNA，逆转录聚合酶链反应扩增APOBEC3G，将产物克隆到pXF3H载体的EcoRⅠ和Hind Ⅲ酶切位点以构建真核表达质粒；以ayw亚型HBV全长质粒构建具有复制能力的1．3倍HBV质粒（pHBV1．3）。不同剂量的APOBEC3G真核表达质粒与pHBV1．3共转染HepG2细胞；酶联免疫吸附法检测细胞培养上清液的乙型肝炎表面抗原和e抗原水平，Southernblot和Northernblot分析HBV核衣壳相关DNA和RNA的水平变化。不同剂量APOBEC3G真核表达质粒与头尾相接的2倍DHBV质粒共转染LMH鸡肝癌细胞，Southernblot分析DHBV核衣壳相关DNA水平变化。结果成功构建APOBEC3G真核表达质粒和具有复制能力的1．3倍HBV质粒。APOBEC3G抑制乙型肝炎表面抗原和e抗原的分泌，转染细胞内HBV核衣壳相关RNA表达水平下降，而对核心蛋白质的表达没有影响；APOBEC3G对转染细胞内HBV和DHBV核衣壳相关DNA水平具有剂量依赖的抑制效应。结论APOBEC3G对HBV和DHBV复制具有抑制作用。

Inhibition of hepatitis B and duck hepatitis B virus replication by APOBEC3G

OBJECTIVE: To investigate the effect of apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G) mediated antiviral activity against hepatitis B virus (HBV) and duck hepatitis B virus (DHBV). METHODS: Total RNA was extracted from peripheral blood mononuclear cells (PBMCs), RT-PCR product was cloned into the EcoR I/Hind III restriction sites of the CMV-driven expression vector fused with a hemagglutinin fusion epitope tag at its carboxyl terminal. Replication competent 1.3 fold over-length HBV was constructed with full-length HBV of ayw subtype. The mammalian hepatoma cell HepG2 was cotransfected with the replication competent 1.3 fold over-length HBV and various amounts of CMV-driven expression vector encoding APOBEC3G-HA. Levels of HBsAg and HBeAg in the media of the transfected cells were determined by ELISA, HBV DNA. RNA from intracellular core particles was examined using Northern and Southern blot analyses. Chicken hepatoma cell LMH was cotransfected with head-to-tail dimer of an EcoR I monomer of DHBV and various amounts of CMV-driven expression vector encoding APOBEC3G-HA. DHBV DNA from intracellular core particles was examined using Southern blot analysis. RESULTS: CMV-driven expression vector encoding APOBEC3G-HA and replication competent 1.3 fold over-length HBV were constructed. There was a dose dependent decrease in the levels of intracellular core-associated viral (HBV and DHBV) DNA and extracellular production of HBsAg and HBeAg. Levels of intracellular core-associated viral RNA were also decreased, but the expression of HBcAg remained almost unchanged. CONCLUSION: APOBEC3G suppresses HBV and DHBV replication and also suppresses HBsAg and HBeAg expression.