KRAB锌指蛋白调控tRNA引物结合位点抑制逆转录病毒元件的分子机制研究

外源性和内源性逆转录病毒与宿主长期共进化，对人类健康或基因组稳定性带来巨大威胁。逆转录病毒利用基因组中特有的引物结合位点（PBS）结合宿主tRNA，并以之为引物逆转录成双链DNA插入基因组完成入侵和复制。因此PBS是逆转录病毒的必备元件。申请人之前研究发现，哺乳动物最大的转录因子家族锌指蛋白可通过锚定特定种类的PBS DNA，建立表观修饰以抑制相应逆转录病毒的活化。本项目拟在系统梳理所有PBS信息的基础上，结合锌指蛋白-逆转录病毒数据库，筛选鉴定潜在具有PBS DNA结合活性的候选锌指蛋白；并结合CRISPR/Cas9技术及多种测序手段，阐明上述锌指蛋白的生物学功能和其抑制性调控逆转录病毒的分子机理。本项目将完善PBS与锌指蛋白的数据库信息，深刻解析锌指蛋白点对点锚定PBS从而抑制逆转录病毒活化的作用机理，系统性地绘制锌指蛋白与囊括所有PBS种类的逆转录病毒元件之间的对应关系。

Retroviruses co-evolve with the host cells, posing huge threats to human health or genome stability. As RNA viruses, they can replicate and invade into the host genome by reverse-transcribing RNA into double-stranded DNA, which starts from utilizing their primer binding site (PBS) to bind the specific primer tRNA of the host cells. Therefore, PBS is an essential element for the replication of retroviruses including both exogenous and endogenous retroviruses. Our previous study has found that zinc finger proteins (ZFPs), the largest family of transcriptional factors in vertebrates, are capable to anchor specific types of PBS DNA to establish epigenetic modifications and inhibit the corresponding retroviruses. However, the detailed molecular mechanisms have not been systematically studied so far. Based on our established ZFP-retrotransposon database, this project will screen and identify the ZFP candidates with potential PBS DNA binding activity. Combined with CRISPR/Cas9 technique and multiple sequencing methods, this project will also demonstrate the biological functions of the above ZFPs and their molecular mechanisms that specifically recognize the PBS DNA to regulate endogenous and exogenous retroviruses. This study will greatly improve our understanding of ZFPs and retroviruses, clarify the point-to-point anchoring mechanisms between ZFPs and PBS, and systematically depict the regulatory network between ZFPs and retroviruses with all types of PBS.