剪切刺激因子-2通过PTB-HBV PRE协同跨核转运途径抑制HBV复制的机制研究

The innate and adaptive immune response is thought to be responsible for viral clearance and disease pathogenesis during hepatitis B virus (HBV) infection. The mechanism of host factors regulating chronic HBV replication remains unknown. From our previous studies, the intrahepatic CSTF2 levels presented negative correlation with serum HBV DNA titers from 83 HBV-infected patients. In vitro experiments showed that silence of CSTF2 by small interfering RNA could enhance HBV replication, and over expression of CSTF2 could suppress HBV replication significantly. In addition, our previous study revealed a significant inhibition on HBV PRE by CSTF2 over expression. PRE is reported to be vitally responsible for the nuclear export of HBV mRNA. Moreover, CSTF2 exhibited a potential interaction with polypyrimidine tract-binding protein (PTB), which is an export factor for PRE-containing HBV mRNAs. Thus we hypothesize that CSTF2 may inhibit HBV replication by an impact on PTB-PRE nuclear export function via the interaction with PTB. This project is supposed to clarify the above hypothesis using in vitro HBV infection cell model and high-pressure tail vein injection mouse model, with knocking down or over expression of genes, to verify the inhibitory effect of CSTF2 on HBV replication. Furthermore, novel far-red fluorescence complementation systems will be used to observe protein-protein and protein-RNA interaction, in order to analyze the interaction of CSTF2 and PRE or PTB and the impact on PTB-PRE nuclear export by CSTF2. This study will help to identify the novel mechanism of host genes on the regulation of HBV replication and throw light on new targets for anti-HBV therapy.

肝细胞调控HBV复制的机制尚未阐明。我们的前期研究发现，肝细胞内剪切刺激因子-2 （CSTF2）基因表达水平与病毒载量呈负相关；在体外过表达或敲除CSTF2，可分别显著下调和上调HBV复制；而CSTF2可显著抑制转录后调节元件（PRE），并与PRE的协同作用蛋白-多嘧啶区结合蛋白（PTB）存在相互作用。我们推测，CSTF2通过PTB-HBV PRE协同跨核转运途径抑制HBV复制。我们拟在前期研究基础上，利用HBV感染细胞和小鼠模型，采用基因敲除或过表达的方法，进一步验证CSTF2对HBV复制的抑制作用；采用远红mNeptone荧光互补技术，观察CSTF2是否与HBV RNA发生结合，以及是否通过与PTB蛋白相互作用，影响PTB-PRE对HBV mRNA协同跨核转运，以阐明CSTF2抑制HBV复制的机制。本研究有助于阐明肝细胞调控 HBV 复制的机制，为寻找抗 HBV新靶位提供理论依据。

肝细胞调控HBV复制的机制尚未阐明。我们的前期研究发现，肝细胞内剪切刺激因子-2 ( CSTF2)基因表达水平与病毒载量呈负相关;在体外过表达或敲除CSTF2，可分别显著下调和上调HBV复制;而CSTF2可显著抑制转录后调节元件(PRE)，并与PRE的协同作用蛋白-多嘧啶区结合蛋白(PTB)存在相互作用。体外实验进一步证实，CSTF2可明显降低HBV S蛋白的表达，但并未与之发生结合，而CSTF2可与HBV core蛋白结合，却并不降低其表达水平。我们进一步利用HBV感染的小鼠模型，发现将不同浓度梯度的CSTF2基因表达质粒通过尾静脉注射方法注入小鼠，实验组和对照组血清HBsAg和HBeAg并没有显著差异。我们进一步观察到CSTF2与HBV RNA发生了结合，并通过与PTB蛋白相互作用，影响PTB-PRE对HBV mRNA协同跨核转运。本研究有助于阐明肝细胞调控 HBV 复制的机制，为寻找抗 HBV新靶位提供理论依据。