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目的:研究APOBEC3A抑制HBV复制的分子机制。方法:首先在肝癌细胞HuH7中过表达APOBEC3A,通过MTT法检测了APOBEC3A对细胞毒性的影响;通过免疫荧光检测了APOBEC3A在细胞中的定位,通过IP试验进一步证实了APOBEC3A与病毒颗粒的相互作用;并通过ELISA,特异性荧光定量PCR检测了HBV复制的参数包括上清中HBsAg,病毒核心颗粒中HBV DNA以及核内的cccDNA的表达水平;最后通过3D PCR方法分析了核心颗粒中HBV DNA脱氨基作用。结果:过表达APOBEC3A对HuH7细胞毒性没有显著性差异;APOBEC3A主要位于细胞核,但APOBEC3A可以与病毒颗粒结合,在逆转录环节对HBV复制发生抑制作用;共转染HBV复制质粒和APOBEC3A表达质粒后,细胞上清中HBsAg,核心颗粒中的HBV DNA以及核内的cccDNA均显著下降;最后通过3D PCR和克隆测序表明核心颗粒中的HBV DNA负链发生了大量的G-A突变,同时正链也发生了较多的C-T突变。结论:APOBEC3A可与病毒颗粒结合,在HBV复制的逆转录环节可作用于HBV单链,发生脱氨基作用,从而抑制HBV的复制。
Objective: To study the molecular mechanism of APOBEC3A inhibiting HBV replication. Methods: APOBEC3A was overexpressed in HuH7 cells and the effect of APOBEC3A on cytotoxicity was detected by MTT assay. The localization of APOBEC3A in cells was detected by immunofluorescence. The interaction between APOBEC3A and virus particles was further confirmed by IP assay. HBV replication parameters such as HBsAg in the supernatant, HBV DNA in the virus core particle and cccDNA in the nucleus were detected by ELISA and specific fluorescence quantitative PCR. Finally, the deamination of HBV DNA in the core particle was analyzed by 3D PCR effect. Results: APOBEC3A overexpression had no significant difference on the cytotoxicity of HuH7 cells. APOBEC3A mainly located in the nucleus, but APOBEC3A could bind to the virus particles and inhibit the replication of HBV in the reverse transcription section. After transfected with HBV replication plasmid and APOBEC3A expression plasmid, The HBsAg in the cell supernatant, the HBV DNA in the core particle and the cccDNA in the nucleus all decreased significantly. Finally, by 3D PCR and cloning sequencing, a large number of GA mutations occurred in the negative strand of the HBV DNA in the core particle and the positive strand also occurred More CT mutations. CONCLUSION: APOBEC3A can bind to virus particles and act on the single strand of HBV during the process of reverse transcription of HBV replication, resulting in deamination, thereby inhibiting HBV replication.