一种新的反式调控乙型肝炎病毒复制的转录因子NF-YA的鉴定及其作用机制的研究

The number of patients with hepatitis b virus (HBV) infection in China is the largest worldwide. It’s of great significance for the research and treatment of HBV infection. HBV core promoter (Cp) activity is a key factor for viral replication. The molecular mechanism of viral replication could be further understood based on the identification of transcription factor(s) and cis-acting element(s) involved in regulation of Cp activity. This study will help develop new insights for antiviral therapy of HBV. Our previous studies revealed that cis-acting element “ATTGG” is highly conserved in HBV Cp, however, the biological sense is unknown. Recently, we found transcription factor NF-YA could tans-promote Cp activity depending on this element; NF-YA over-expression and knockdown could respectively up- and down-regulate HBV transcription/replication; Knockdown transcription factor Sp1, a potential binding protein of NF-YA, could suppress HBV. Previous studies indicated that NF-YA promotes or inhibits the transcription of target genes via recruitement to cis-acting element “ATTGG” and interaction with various transcription factor(s). Therefore, we hypothesized that NF-YA promotes HBV transcription and replication relying on recruitement to Cp and interaction with Sp1. We would clarify how NF-YA regulates HBV by means of various hepatoma cell lines, HBV infection cell model and mouse models simulating HBV chronic infection.

中国是乙型肝炎病毒（HBV）感染人数最多的国家，对HBV感染的研究和治疗具有重大的现实意义。核心启动子（Cp）的转录活性是影响病毒复制的关键因素，鉴定出调控Cp活性的顺式作用元件和转录因子将有助于阐明宿主调控病毒复制的分子机制，为抗病毒治疗提供新靶点。我们前期报道，顺式元件“ATTGG”在Cp内高度保守，但生物学意义尚不明确。近期研究发现，转录因子NF-YA依赖于该元件反式激活Cp；NF-YA的过表达和敲除则分别促进和抑制HBV的转录和复制；敲除NF-YA的潜在结合因子Sp1的表达则抑制HBV。既往报道，NF-YA能识别并结合该元件，同时招募不同的转录因子激活或抑制靶基因的表达。因此，我们推测：NF-YA通过募集于Cp上并招募Sp1形成转录激活物，进而促进HBV的转录和复制。我们拟用肝癌细胞模型、HBV感染的细胞模型和模拟HBV慢性感染的小鼠模型，阐明NF-YA调控HBV的分子机制。

乙型肝炎病毒（HBV）感染是一个全球公共卫生问题，全世界约有2.6亿人慢性感染HBV，每年约60万人死于HBV引起的相关肝脏性疾病。在病毒感染的肝细胞内HBV共价闭合环状DNA（cccDNA）一直处在转录活化状态，因此从病毒和宿主两方面开展对HBV转录调控机制的研究将有助于慢乙肝治疗药物的开发。在病毒方面，本研究中我们发现HBV核心启动子（Cp）存在一个新的顺式调控元件“ATTGG”，该元件突变将显著抑制不同基因型HBV Cp的转录活性；同时由于Cp同增强子II（EnII）存在重叠，故该元件突变后对HBV启动子Sp1，Sp2和Xp的活化水平也显著降低。在宿主方面，通过体外转染系统，发现过表达宿主基因NF-YA则显著促进HBV的基因表达和复制，反之，则抑制HBV；同时，在HBV感染系统中，NF-YA也能显著促进HBV的基因表达和复制；在慢乙肝小鼠体内，我们发现敲低小鼠内源NF-YA的表达，则显著抑制HBV，综上结果提示NF-YA可以作为抗HBV治疗的靶点。既往研究表面NF-YA发挥作用需要招募宿主基因SP1，体外研究证明敲低SP1的表达则显著抑制HBV的基因表达和复制，提示NF-YA和SP1可能通过形成转录复合物后调控HBV cccDNA的转录活性。综上，本研究将为抗HBV治疗提供一定的思路。