乙型肝炎病毒RNA上La蛋白结合位点缺失对S基因mRNA稳定性的影响

The deletion of La protein binding site in HBV RNA leads to instability of S gene mRNA

目的研究乙型肝炎病毒(HBV):RNA上La蛋白结合位点的缺失对S基因mRNA在细胞内稳定性的影响,评价HBV RNA上这个位点对HBV生命周期的重要性,从而寻找新的抗HBV靶点。方法构建HBV突变载体, 使其转录出来的HBV RNA缺失La蛋白结合位点;计算机预测突变后这个位点所在的HBV RNA片段的二级结构;将未突变的HBV载体和HBV突变载体分别瞬时转染HepG2细胞;半定量逆转录聚合酶链反应法检测HBV S基因的 mRNA,酶联免疫吸附法检测乙型肝炎表面抗原。结果酶切鉴定和测序证明,成功构建了La蛋白结合相关位点部分碱基缺失的HBV突变载体——pHBV—mLa;突变后的HBV RNA二级结构同突变前的比较,完全改变;转染细胞后半定量逆转录聚合酶链反应法检测发现,突变后HBV S基因的mRNA水平显著降低(t'=12．703,P<0．05);酶联免疫吸附法检测发现突变引起乙型肝炎表面抗原表达明显下调(f=4_4．648,P<0．01)。结论HBV RNA上La蛋白的结合位点以及局部特殊二级结构的形成,影响着自身转录后的稳定性,对于HBV的生命周期至关重要。

Objective To investigate the effect of deletion of the La protein binding site of HBV RNA, caused by its mutation, on the FIBV S-mRNA stability ofS gene, to study the role of the site in hepatits B virus life cycle, and to try to find a new anti-HBV target in the future. Methods A HBV vector with mutation related to the La protein binding site was constructed using molecular cloning and PCR based site directed mutagenensis, and the vector was named pHBV-mLa. The HBV RNA secondary structure of the site was calculated using a computer. Normal HBV vectors and mutant vectors were respectively transfected into HepG2 cells by Lipofectamine2000. HBV S-mRNA levels in the two groups were analyzed using semi-quantitative RT-PCR, and HBsAg secretion into the culture media was tested using ELISA.Results A HBV vector with mutation related to the La protein binding site was successfully constructed, and it was identified and confirmed using restriction analysis and sequencing. The HBV RNA secondary structure of the mutant vector was completely different to the stem-loop structure of the normal HBV vector. Semi-quantitative RT-PCR and ELISA analyses showed that the level of HBV S-mRNA in the mutant vector group was significantly lower than that in the normal HBV vector group （t′= 12.703, P 〈 0.05）, and the expression efficacy of HBsAg was reduced in the mutant vector group （t = 44.648, P 〈 0.01）. Conclusions The change of La protein binding site in the HBV RNA caused by the mutation in HBV DNA disorganizes the stem-loop structure in the HBV RNA site. With the structural change, the La protein cannot bind the site and stabilize the HBV RNA （HBV S-mRNA）, as the cleavage site in the upstream of the stem-loop structure is exposed to endoribonuclease. This results in HBV S-mRNA decay and affects the expression of the S gene. This study shows that only the sequence of this site in the HBV DNA is reserved, then the stem-loop structure in the La protein binding site will remain intact, and the disorganization of the stem-loop structure affects the stability of the transcripted HBV RNA. The La protein binding site in HBV RNA and the special secondary structure of the site are crucial to the life cycle of the hepatitis B virus.