新型HBV抑制剂干扰病毒分泌并下调PreS和PreC/C转录水平的分子机制

The development of non-nucleoside inhibitors of HBV has attracted considerable attention due to the emergence of drug-resistant viruses after long-term use of nucleoside drugs targeting HBV DNA polymerase. ADN-9, an anti-HBV agent targeted secretion of HBV, synthesized by our group previously, obviously decreased the extracellular levels of HBV antigens and HBV DNA in vitro. Additionally, the quantity of intracellular HBsAg and MHBs were increased, while the mRNA levels of PreS and PreC/C were reduced. Based on these data, we hypothesized that the HBV surface (HBs) proteins, which were blocked by ADN-9, probably would be the key reason for interfering the virus secretion and down-regulating the transcriptional levels of PreS and PreC/C, as well as inhibiting the replication of HBV DNA. In this study, the methods of Western Blot and co-immunoprecipitation will be used to investigate the mechanism that ADN-9 interferes with glycosylation and folding of membrane proteins. We will adopt the methods of Transcription Factor activation profiling plate array, RNA interference, co-transfection, EMSA, etc, to probe the mechanism that down-regulates the transcriptional levels of PreS and PreC/C. By using BALB/c mice transfected with recombinant adenovirus (Ad-HBV), we will further study, then clarify the anti-HBV mechanism, and verify the ADN-9-targeted gene at the whole animal level. Our studies will provide theoretical basis for the development of novel anti-HBV drugs targeting secretion of the virus particles.

由于靶向HBV DNA聚合酶的核苷类药物长期使用易产生耐药性，开发非核苷类HBV抑制剂备受关注。课题组前期自主研发的HBV分泌抑制剂ADN-9，大幅降低胞外HBV DNA和抗原水平；胞内HBsAg和MHBs蛋白水平升高，而PreS和PreC/C mRNA水平降低。据此，我们提出“HBV膜蛋白受阻滞是ADN-9干扰病毒分泌，下调PreS和PreC/C转录水平，抑制病毒复制的主要分子机制”这一科学假说。本课题将通过Western Blot、免疫共沉淀等实验研究ADN-9干扰膜蛋白糖基化、阻碍蛋白折叠的机制；通过转录因子活性微孔板阵列检测技术，基因干扰、转染和EMSA等方法，探索ADN-9下调PreS和PreC/C转录的分子机制。利用重组腺病毒（Ad-HBV）转染BALB/c小鼠，从动物整体水平研究并阐明AND-9抗HBV机制，验证靶基因。本研究将为基于HBV分泌抑制剂的新药开发提供理论依据。

乙型肝炎病毒(HBV)持续感染是导致原发性肝细胞癌的主要因素之一。目前，临床上治疗HBV感染的主要药物包括干扰素和核苷类似物。干扰素应答率低和耐受性差，核苷类药物需终身服药，且易产生耐药性。因此，研发新型HBV药物意义重大。穿心莲内酯衍生物ADN-9显著降低HepG2.2.15细胞培养上清中HBV主要标志物及鸭乙肝病毒（DHBV）感染雏鸭血清和肝组织中DHBV DNA水平。本项目研究通过Western Blot和免疫荧光技术动态分析药物处理后HBV相关结构蛋白变化，发现ADN-9优先下调LHBs，随后抑制LHBs、MHBs及HBc的表达。qPCR结果表明ADN-9作用24h显著下调2.4+3.5kb mRNA、2.1+2.4+3.5kb mRNA水平，其中2.4+3.5kb mRNA幅度最大；作用48h HBV mRNA全面下调，同时胞内衣壳相关HBV DNA拷贝数、LHBs相关病毒颗粒、裸衣壳颗粒显著减少，但HBc相关病毒颗粒无明显改变。Northern Blot结果进一步证明了ADN-9作用24h仅使2.1/2.4kb mRNA下调，3.5kb mRNA未受显著影响。由于未观察到cccDNA降低，进一步通过构建双荧光素酶报告基因系统，证明了ADN-9特异性抑制HBV启动子SPI活性，进而下调preS1 mRNA（2.4kb mRNA）和LHBs的表达。采用数字表达谱分析和RT-qPCR发现ADN-9可抑制SP1、AP-1和IRF1等与HBV转录相关的转录因子表达，通过过表达SP1验证了SP1蛋白对PreS1启动子活性有正调节作用，并可部分减弱ADN-9对启动子SPI的抑制。进一步采用高压水动力法C57BL/6J 小鼠尾静脉注射pAAV-HBV1.2模型验证了ADN-9抑制HBV表达的作用与机制。本研究结果对以LHBs为靶点的抗HBV研究与药物开发提供新的思路和契机。