TRAF2-Neddylation通路调控HBV转录复制的作用机制研究

The ultimate goals of treatment for chronic hepatitis B virus (HBV) infection are entirely clearance of Hepatitis B virus. Current treatments including Interferon (IFN)-α and nucleoside analogues (NUC) only achieves viral suppression. Therefore, there is an unmet medical need for an efficient HBV cure. During acute infection of HBV, the early host immune response inhibits HBV gene expression and DNA replication by a noncytopathic mechanism of activation of cytotoxic T lymphocytes (CTLs) through the induction of interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α). However, the molecular mechanism and the downstream effector molecules remain poorly understood. Our preliminary data showed that tumor necrosis factor receptor-associated factor 2 (TRAF2), an important downstream effector of TNFα, is capable of the binding to NAE1 protein and subsequently inhibiting its ability of Neddylation activation which is an ubiquitin-like protein modification process. Meanwhile, we found that the inhibition of neddylation by knockdown of NAE1 significantly decreased HBV DNA level and HBsAg, HBeAg expressions in the supernatant of HepG2.2.15 cells, indicating that overexpression of TRAF2 may inhibit HBV replication through Neddyaltion pathway, and hypothesizing that TRAF2 is a key molecule in the TNFα-mediated HBV inhibition. In this project, we plan to further investigate the effects of TRAF2 on HBV transcripts, viral replication intermediates, viral proteins in HepG2.2.15 cells using methods of Southern blot, qPCR and ELISA for HBV DNA and viral proteins. The key functional domain of TRAF2 and HBV-related transcription factors on the inhibition of HBV replication by TRAF2 will be demonstrated using Co-IP，Western blot，CRISPR/Cas9 system methods, as well as the mechanism of HBV fighting against TRAF2. In addition, the function of TRAF2 on TNFα-mediated HBV inhibition will also be explored. Finally, the effect of TRAF2 on HBV replication will be confirmed on HBV transgenic mice by lentivirus technology.Our study will demonstrate a novel function of TRAF2 as a natural inhibitor of HBV infection and provide a new mechanism by which TNF-α suppresses HBV replication noncytolytically. These findings may open up a new option for the control of the persistent HBV infection.

病毒清除是乙肝治疗的最终目标，核苷类药物和干扰素是目前治疗的主流选择,但仍面对核苷类耐药以及干扰素对人体的毒副作用对疗效的影响，因此新的抗HBV靶点还是亟需寻找。研究报道γ干扰素/TNF-α可激活细胞毒性T淋巴细胞，以非溶细胞性的方式来清除肝细胞内HBV，同时TNF-α还能直接抑制HBV复制，但机理尚不明确。最近研究表明类泛素化(Neddylation)在HBV转录复制中有重要的调控作用，我们的前期研究发现TNF-α下游重要蛋白TRAF2能直接结合Neddylation关键蛋白NAE1并调控该通路，敲低NAE1能显著抑制HBV转录复制。项目拟采用Co-IP、CRISPR/Cas9、重组慢病毒等技术，在细胞和动物模型中深入研究TRAF2如何通过Neddylation通路调控HBV转录复制；阐明TRAF2及其下游关键分子在TNF-α抗HBV复制中新的分子机制，并探讨其作为药物靶点潜能。

慢乙肝的病毒清除一直是临床治疗面临的严重挑战，虽然核苷类药物和干扰素是目前治疗的主流选择，但是由于核苷类耐药以及干扰素对人体的毒副作用影响了临床治疗的疗效。新型抗HBV靶点的发现，并研发相应抗病毒策略是近年来乙肝研究领域的热点和焦点问题。基于前期研究所发现的TNF-α下游重要蛋白TRAF2与HBV转录复制相关，以及TRAF2能直接结合Neddylation关键分子并影响其蛋白水平的重要基础，本项目采用Co-IP、泛素化调控分析、重组慢病毒等技术，在细胞和动物模型中深入研究了TRAF2如何通过Neddylation通路调控HBV转录复制，并提出Neddylation可作为乙肝抗病毒治疗的新靶点。研究发现，HBV感染会上调肝细胞内Neddylation修饰水平，而Neddylation修饰又通过Nedd8与cullin蛋白的保守赖氨酸残基的结合来激活CRL4 HBx，进而影响HBV复制。而敲除TRAF2则能抑制Neddylation，从而通过影响cullin蛋白下游通路来抑制HBV的复制，并且还能有效降低HBV cccDNA的水平。另外，通过对HBV启动子的研究，发现Neddylation还会影响HNF1α，HNF4α，CEB/Pα等HBV重要转录因子的水平，进而影响HBV的转录。进一步的机制研究发现，TRAF2可以与Cullin家族中Cullin4的重要底物DCAF7相结合，进而影响Neddylation通路介导的HBV复制转录及病毒蛋白的表达。我们还提出，MLN4924作为经典的Neddylation抑制剂有望作为抗HBV的新策略。我们在细胞水平和小鼠模型中分析了MLN4924的抗HBV作用，研究结果显示MLN4924可以有效降低HBV DNA以及病毒抗原HBsAg、HBeAg和HBcAg的表达，并有效抑制HBV cccDNA水平。本项目初步阐明了TRAF2-Neddylation介导的HBV转录复制调控新机制，并证实了Neddylation作为新型抗HBV药靶的可行性。