肝细胞核因子3结合位点的突变及对抑制乙型肝炎病毒复制的影响

The effect of hepatocyte nuclear factor 3 binding site mutation on the inhibition of hepatitis B virus transcription and replication

目的研究肝细胞核因子（HNF）3结合位点HNF3β对HBV转录和复制调节作用的机制。方法通过分别位于HBV基因组的多聚酶编码区Pp、PS1P和Sp区上的HNF3结合位点，构建HBV基因组2个或3个位点的复合突变，获得4个复合突变型重组质粒；分别转染非肝源性细胞小鼠成纤维细胞株（NIH3T3），Northern印迹法检测3．5kb、2．4kb／2．1kb、0．7kbHBVRNA的转录水平，Southern印迹法检测HBVDNA复制中间体水平．观察上述突变是否会影响HNF313对HBV转录和复制的抑制作用。结果在被转染的非肝源性细胞3T3中，HNF313仍能降低4个复合突变型HBV重组质粒3．5kbHBVRNA的转录水平，抑制HBVDNA的复制；与未共转染HNF3B相比，转录水平下降50％～75％、复制水平下降80％～96％。结论在位于HBV基因组的多聚酶编码区Pp、PS1p和Sp区上的3个HNF3结合位点中，即使其中2个或3个结合位点同时联合突变，HNF313仍能抑制HBV的转录和复制。Pp、PS1p和Sp区HNF3结合位点的突变不能阻断HNF313对HBV复制的抑制作用。

Objective To investigate the effect of hepatocyte nuclear factor（HNF） 3 binding site mutations on the inhibition potency of HNF 3 on hepatitis B virus（HBV） transcription and replication. Methods With three one-mutation pHBV4. 1HNF3mut plasmids（with mutation in P open reading frame（ORF）, PSlp and Sp promoter, respectively）, four multi-mutation pHBV4.1 HNF3mut plasmids were constructed, and then were co-transfected into nonhepatic cell line（NIH3T3）. The transcription levels of 3.5 kb, 2.4 kb/2.1 kb and 0.7 kb HBV RNA were analyzed by Northern blot hybridization, and the levels of HBV DNA replication intermediates were detected by Southern blot hybridization analysis. Results It was shown that, in nonhepatic cell line 3T3, HNF3β reduced nuclear hormone receptor-mediated transcription of 3.5 kb HBV RNA and HBV replication. The level of 3.5 kb HBV RNA was reduced 50% to 75% and HBV DNA replication level was reduced approximately 80 % to 96 %. Conclusions Even with combined mutations in two or three HNF3 binding sites of HBV polymerase coding regions, Pp, PSlp and Sp, HNF3β can still inhibit the transcription and replication of HBV. The mutations in Pp, PSlp and Sp regions can not reduce the inhibition potency of HNF3β on HBV replication.