胞嘧啶脱氨酶APOBEC3G可能通过G→A突变抑制乙型肝炎病毒的复制

Cytidine deaminase APOBEC3G my suppress HBV replication through G to A mutation of HBV X gene region

目的 研究胞嘧啶脱氨酶APOBEC3G（A3G）对HBV复制的影响及作用机制。方法 利用脂质体介导A3G和HBV的真核表达质粒瞬时共转染人肝癌细胞株HepG2，以空载体pcDNA3．1与HBV真核表达质粒共转染为对照，同时转染含增强型绿色荧光蛋白（EGFP）基因的质粒载体以判定转染效率。共转染两天后用荧光定量PCR方法定量细胞内核壳体（cofe）相关HBV DNA水平，用Western blot检测细胞内A3G和HBcAg的表达，并对细胞内cofe相关HBVDNAX基因进行PCR扩增、T．A克隆和测序，分析X基因中的碱基突变。结果 经EGFP判定的转染效率平均为29％。共转染0．5PgA3G和0．5μg HBV的HepG2细胞内core相关HBVDNA量平均下降为对照的8．9％，共转染2μgA3G和0．5μgHBV的平均下降为对照的0．6％。共转染A3G和HBV表达质粒后，HepG2细胞内HBV的X基因中发生G→A突变的数目明显增多，33个克隆中有9个克隆检测到了16-37个G→A突变，突变总数达254个，而对照的33个克隆中仅有2个克隆各检测到2个G→A突变。进一步的分析发现，共转染A3G后发生的G→A突变大多集中于几个热点区域，突变靶点常在GG二核苷酸中的第一个G上。结论 胞嘧啶脱氨酶APOBEC3G可能通过诱导HBVDNA的X基因发生G→A超突变，抑制HBV在人肝癌细胞HepG2中的复制。

Objective To investigate whether APOBEC3G（A3G） can mediate cytosine deamination of HBV DNA and suppress HBV replication. Methods An expressing vector encoding A3G or a control vector pcD- NA3.1 was co-transfected with a HBV-producing plasmid into human hepatoma cell line HepG2. Meanwhile, an expressing vector encoding green fluorescence protein was also transfected into HepG2 in order to determine the transfection efficiency. Cells were harvested two days after transfection, and introcellular core-associated HBV DNA was extracted. Purified HBV DNA was quantified by real-tlme PCR. Wostem blot analysis was performed on cytoplasmic extracts from these cells, with the antibodies against HBV core protein and HA. X gene region of coreassociated HBV DNA was amplified, and the PCR products were cloned into pGEM-T Easy and individual recombinant clones were sequenced. GeneDoc software was used to analyze the sequences. Results The transfection effi- ciency was about 29%. Compared to the empty vector control, 0.5 p-g A3G reduced the formation of intracellular core-associated HBV DNA to 8.9%, and 2μg A3G to 0.6%. Low level of G to A mutation in the X gene region of HBV DNA was detected from samples collected from HepG2 cells transfected with a control vector. A3G increased G to A mutations in the X region of HBV DNA. About 2 of 33 recombinant bacterial clones from control HepG2 cells mmsfected with vector contained only two G to A mutations. On the other hand, 9 of 33 clones conrained sixteen or more G to A mutations in the presence of A3G, and the total number of G to A mutations was up to 254. Further analysis indicated that the G to A mutation showed a preference for the first G of a dinucleotide sequence of GG. Conclusion Cytidine deaminase A3G may suppress HBV replication in HepG2 cells through G to A hypermutation of the X region of HBV DNA.