乙肝病毒BCP区突变(A1762T/G1764A)对乙肝病毒复制的影响研究

The Effects of Hepatitis B Virus BCP Region Mutation(A1762T/G1764A)on HBV Replication

通过构建乙型肝炎病毒(Hepatitis B virus, HBV)单倍体分子,阐明HBV基本核心启动子突变(A1762T/+G1764A,BCP)对HBV复制的影响。本研究收集了HBeAg阴性的慢乙肝患者,将其中两例BCP突变的序列通过重叠PCR克隆到HBV1.3倍体的复制质粒;构建环状的HBV单倍体分子,转染Huh7.0细胞后,通过ELISA、实时荧光定量PCR等分析BCP突变对病毒复制的影响;通过荧光素酶实验分析BCP突变对HBV preC mRNA转录的影响。55例HBeAg阴性的慢乙肝患者中有30例带有A1762T/G1764A的突变;克隆带有BCP突变的临床序列到HBV 1.3倍体转染细胞后,上清中HBeAg为阴性,表面抗原降低10%(P>0.05);构建的HBV单倍体环化后形成闭合环状的cccDNA分子,转染细胞后,结果表明BCP突变后,HBeAg下降55.48%(P<0.05),表面抗原无显著性差异(P>0.05),细胞内病毒颗粒中DNA水平为野生型的3.18倍,上清中HBV DNA为野生型的1.36倍;BCP突变后preC mRNA水平降低81.05%,而3.5kb RNA水平无显著性差异,核心抗原(HBcAg)表达增多。本实验结果表明基本核心启动子突变(A1762T/G1764A)减少了前核心RNA(preC mRNA)的转录,导致HBeAg分泌减少,而病毒核心蛋白表达增加,导致细胞内病毒DNA复制能力增强。

The effects of Hepatitis B virus(HBV)basic core promoter mutation(A1762T/+G1764A,BCP)on HBV replication was elucidated by constructing HBV monomers.In this study,HBeAg-negative chronic hepatitis B patients were collected,and the sequences of BCP mutations in two cases were cloned into HBV1.3 replication plasmids by overlapping PCR;The circular HBV monomers were constructed,and transfected into Huh7.0 cells.The effects of BCP mutation on virus replication were analyzed by ELISA or real-time quantitative PCR;the effects of BCP mutation on HBV preC mRNA transcription were analyzed by luciferase assay.Thirty of the 55 chronic hepatitis B(CHB)patients had A1762T/G1764A mutations in the BCP region.The constructed HBV monomers were circularized to form a closed-circular cccDNA molecule.After transfection into the cells,the HBeAg levels decreased by 55.48%(P<0.05),and there was no significant difference in surface antigen(P>0.05)for the BCP mutants compared with wide type.The viral DNA level in the intracellular virus particles was 3.18 times than that of the wild type,and HBV DNA in the supernatant increased 1.36 times;PreC mRNA level decreased by 81.05%after BCP mutation,while the 3.5kb RNA level had no significant difference.The results of this study showed that the mutation of the basic core promoter(A1762T/G1764A)reduced the transcription of precore RNA(preC mRNA),leading to the decrease of HBeAg secretion,while the increase of viral core protein expression led to the enhancement of viral DNA replication in the cells.