IGF2BP1竞争性结合病毒RNA的A1907位点促进HBV复制

IGF2BP1 competitively binds to the A1907 site of viral RNA to promote HBV replication

摘要

目的 探究HBV RNA上m6A修饰位点A1907对病毒复制的影响,揭示m6A阅读蛋白IGF2BP1与YTHDF2通过竞争结合该位点调控HBV复制的机制。方法 构建A1907C单突变及A1907C&T1851G双突变的HBV表达质粒;在HepG2细胞中转染上述质粒后,采用qPCR、化学发光免疫分析及Western blot实验检测HBV DNA、HBsAg、HBeAg及衣壳(core)蛋白等病毒复制指标;通过siRNA敲减细胞中m6A甲基转移酶METTL3&14,利用RNA免疫沉淀(RNA immunoprecipitation,RIP)实验检测IGF2BP1和YTHDF2与HBV RNA的结合;采用抑制剂或siRNA敲减方法,探究IGF2BP1和YTHDF2对HBV复制的拮抗作用。结果 A1907位点突变可抑制HBV复制,且单突变与双突变抑制效应相当(P<0.01);IGF2BP1和YTHDF2与HBV RNA结合均依赖A1907位点的m6A修饰,且二者存在竞争结合关系(P<0.01);IGF2BP1通过拮抗YTHDF2的抑制作用促进HBV复制(P<0.05)。结论 A1907是维持HBV有效复制的重要m6A修饰位点;IGF2BP1与YTHDF2通过竞争性结合该位点来调控HBV的复制水平。因此,靶向A1907位点的m6A修饰及其相关阅读蛋白,有望成为抗HBV治疗的新方向。

Abstract

Objective To investigate the impact of A1907 site in HBV RNA on viral replication and to elucidate the mechunism by which the m6A reader proteins IGF2BP1 and YTHDF2 regulate HBV replication through competitive binding to this site. Methods HBV expression plasmids containing the A1907C single mutation and the A1907C&T1851G double mutation were constructed. After HepG2 cells were transfected with wild-type or mutant HBV plasmids, viral replication indicators, including supernatant HBV DNA, HBsAg, HBeAg, and intracellular core protein, were detected using qPCR, CLIA, and Western blot. siRNA was employed to knock down the m6A methyltransferases METTL3&14, and RNA immunoprecipitation (RIP) assays were used to assess the binding of IGF2BP1 and YTHDF2 to HBV RNA. The antagonistic effects of IGF2BP1 and YTHDF2 on HBV replication were further explored using activation inhibitors or siRNA-mediated knockdown approaches. Results Mutation at the A1907 site inhibits HBV replication, and the inhibitory effect of the single mutation and the double mutation are comparable (P<0.01). The binding of both IGF2BP1 and YTHDF2 to HBV RNA is dependent on the m6A modification at the A1907 site, and a competitive binding relationship is observed between the two proteins (P<0.01). IGF2BP1 is found to promote HBV replication by antagonizing the inhibitory effect of YTHDF2 (P<0.05). Conclusion A1907 is a critical m6A modification site for maintaining efficient HBV replication. IGF2BP1 and YTHDF2 regulate HBV replication through competitively binding to the m6A-modified A1907 site. This m6A modification site and its associated reader proteins represent promising potential targets for anti-HBV treatment.

关键词

HBV / m6A修饰 / A1907位点 / m6A阅读蛋白 / IGF2BP1 / YTHDF2

Key words

HBV / m6A modification / A1907 / m6A readers proteins / IGF2BP1 / YTHDF2

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基金资助

国家自然科学基金面上项目(82572552)