HBc-A3A/UNG2靶向融合蛋白构建及其清除HBV cccDNA作用和机制研究

Inability to clear hepatitis B virus covalently closed circular DNA (cccDNA) from the nuclei of infected cells is primarily responsible for the failure to cure chronic HBV infection. It was recently reported that IFN-α can upregulate hepatic nuclear APOBEC3A (A3A) expression, which deaminates dC base of the cccDNA through binding to HBV core protein and converts it to dU. The resulting dU becomes a target for the excision repair by uracil DNA glycosylase (UNG2) and it ultimately leads to degradation of cccDNA. The mechanisms underlying the degradation of cccDNA by the base excision repair process remain unknown. To illustrate the mechanisms for cccDNA degradation mediated by both A3A and UNG2 and to establish a strong and targeted antiviral strategy for clearing cccDNA, we propose to modify HBc and to construct and express a HBc-A3A / UNG2 antiviral fusion protein that directly targets cccDNA, on the platform of our previous study of HBV core protein. We will investigate specific functions of this fusion protein in degrading cccDNA, explore a new strategy through studying both factors targeting cccDNA clearance, and dissect the specific mechanisms for cccDNA degradation. We will use HBV infected cell and mouse model as well as molecular and cell biology techniques of qPCR, 3D-PCR, ChIP and DNA sequencing for accomplishing proposed experiments. The results by completing this proposal are expected to provide theoretical and experimental basis for a new antiviral therapy, which systematically targets cccDNA clearance.

细胞核HBV cccDNA难以清除是慢性乙型肝炎难治愈的主要原因。近年发现细胞核APOBEC3A（A3A）可能通过与HBc结合，使cccDNA 的dC脱氨基为dU，进而尿嘧啶DNA糖基化酶2（UNG2）启动对dU切除修复，最终引起cccDNA特异性降解。而碱基切除修复如何引起cccDNA特异性降解的机制还不十分清楚。为了利用此效应建立清除cccDNA效应更强的抗病毒方法并阐明特异性cccDNA降解机制，我们拟在前期HBc研究基础上，重构HBc并构建核cccDNA靶向的HBc-A3A/UNG2抗病毒融合蛋白。用细胞和小鼠模型、qPCR、3D-PCR、ChIP和基因测序等技术，研究靶向蛋白引发特异性cccDNA降解的作用和机制，探索建立靶向双因子清除cccDNA的新策略，进一步阐明A3A、UNG2引发特异性cccDNA降解的机制，为研究建立系统的靶向清除乙肝病毒的治疗方案奠定理论和实验基础。

为探索靶向清除细胞核内HBV cccDNA的方法，构建了由APOBEC3A(A3A)与HBc C端区域（CTD）组成的A3A-CTD融合蛋白的表达pcDNA3.0载体，从中选择出表达量高、胞嘧啶脱氨基酶活性高、能够定位细胞核的4个A3A-CTD融合蛋白（A3A-144S、A3A-144E、A3A-144AAA、A3A-144A）。抗HBV初筛实验采用融合蛋白表达载体分别与HBV复制载体（pCH9-HBV）共转染Huh7细胞，培养3天后，用ELISA和定量PCR检测细胞上清HBsAg和HBeAg以及细胞内HBV DNA。结果显示，4种A3A-CTDs均有抑制HBV复制的作用，其中，与A3A相比，作用显著的融合蛋白为A3A-144S、A3A-144E和A3A-144AAA(P<0.05)。进一步的抗HBV实验采用融合蛋白慢病毒感染HBV阳性的HepAD38细胞。结果显示，慢病毒瞬时表达或稳转表达的A3A-144S、A3A-144E、A3A-144AAA以及A3A对细胞上清HBsAg和HBeAg以及细胞内HBV DNA水平均无显著降低。研究发现A3A-CTDs相比A3A有增强HBV cccDNA损伤的作用。这种损伤对双链DNA复制抑制明显，但对双链DNA的转录和RNA逆转录无明显抑制，因此，在HepAD38细胞中，A3A-CTDs对以整合HBV基因组转录为复制基础的HBV DNA复制无明显抑制，但能有效抑制Huh7细胞中以质粒DNA复制（pCH9-HBV）为基础的HBV DNA复制。此外，A3A-CTDs可非特异结合细胞基因组DNA，增加细胞基因组DNA损伤和突变，引起细胞G1、S期阻滞以细胞生长抑制。总之，（1）A3A-CTDs可介导A3A对cccDNA脱氨基损伤，但是不能特异清除细胞核cccDNA；（2）A3A-CTDs引起的DNA损伤可抑制双链DNA复制，而对双链DNA的转录以及RNA逆转录影响不显著；（3）A3A-CTDs可非特异结合细胞基因组DNA，增加对细胞基因组DNA的损伤和突变，引起细胞周期阻滞和生长抑制。该研究阐明了A3A-CTDs对HBV DNA的作用及机制，拓展了对A3A抑制HBV DNA复制的局限性的认识。鉴于显著抑制癌细胞生长，该融合蛋白在抗肿瘤研究中具有应用前景。

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