Uracil DNA Glycosylase Counteracts APOBEC3G-Induced Hypermutation of Hepatitis B Viral Genomes: Excision Repair of Covalently Closed Circular DNA

The covalently closed circular DNA (cccDNA) of the hepatitis B virus (HBV) plays an essential role in chronic hepatitis. The cellular repair system is proposed to convert cytoplasmic nucleocapsid (NC) DNA (partially double-stranded DNA) into cccDNA in the nucleus. Recently, antiviral cytidine deaminases, AID/APOBEC proteins, were shown to generate uracil residues in the NC-DNA through deamination, resulting in cytidine-to-uracil (C-to-U) hypermutation of the viral genome. We investigated whether uracil residues in hepadnavirus DNA were excised by uracil-DNA glycosylase (UNG), a host factor for base excision repair (BER). When UNG activity was inhibited by the expression of the UNG inhibitory protein (UGI), hypermutation of NC-DNA induced by either APOBEC3G or interferon treatment was enhanced in a human hepatocyte cell line. To assess the effect of UNG on the cccDNA viral intermediate, we used the duck HBV (DHBV) replication model. Sequence analyses of DHBV DNAs showed that cccDNA accumulated G-to-A or C-to-T mutations in APOBEC3G-expressing cells, and this was extensively enhanced by UNG inhibition. The cccDNA hypermutation generated many premature stop codons in the P gene. UNG inhibition also enhanced the APOBEC3G-mediated suppression of viral replication, including reduction of NC-DNA, pre-C mRNA, and secreted viral particle-associated DNA in prolonged culture. Enhancement of APOBEC3G-mediated suppression by UNG inhibition was not observed when the catalytic site of APOBEC3G was mutated. Transfection experiments of recloned cccDNAs revealed that the combination of UNG inhibition and APOBEC3G expression reduced the replication ability of cccDNA. Taken together, these data indicate that UNG excises uracil residues from the viral genome during or after cccDNA formation in the nucleus and imply that BER pathway activities decrease the antiviral effect of APOBEC3-mediated hypermutation.

乙型肝炎病毒（HBV）的共价闭合环状DNA（cccDNA）在慢性肝炎的发生发展中发挥关键作用。研究表明，宿主细胞修复系统可将细胞质核衣壳（NC）DNA（部分双链DNA）转运至细胞核内，并转化为cccDNA。近期研究证实，抗病毒胞嘧啶脱氨酶AID/APOBEC家族蛋白可通过脱氨基作用在NC-DNA中引入尿嘧啶残基，进而引发病毒基因组发生胞嘧啶向尿嘧啶（C-to-U）的超突变。本研究针对肝DNA病毒DNA中的尿嘧啶残基是否会被碱基切除修复（BER）的宿主因子尿嘧啶DNA糖苷酶（UNG）切除展开了探究。在人类肝细胞系中，当通过表达UNG抑制蛋白（UGI）抑制UNG活性后，APOBEC3G或干扰素处理诱导的NC-DNA超突变水平显著增强。为评估UNG对cccDNA病毒中间产物的影响，我们采用了鸭乙型肝炎病毒（DHBV）复制模型。对DHBV DNA的序列分析结果显示，在表达APOBEC3G的细胞中，cccDNA积累了G到A（G-to-A）或C到T（C-to-T）突变，且UNG抑制可进一步大幅强化该突变程度。cccDNA的超突变可在P基因中产生大量提前终止密码子。UNG抑制还可增强APOBEC3G介导的病毒复制抑制效果，包括长期培养条件下NC-DNA、前C（pre-C）mRNA以及分泌型病毒颗粒相关DNA的水平下降。当APOBEC3G的催化结构域发生突变后，UNG抑制便不再能增强APOBEC3G介导的病毒复制抑制效应。对重新克隆的cccDNA进行转染实验证实，联合UNG抑制与APOBEC3G表达可降低cccDNA的复制能力。综上，本研究数据表明，UNG可在细胞核内cccDNA形成过程中或形成后从病毒基因组中切除尿嘧啶残基，这也提示碱基切除修复通路的活性会削弱APOBEC3介导的超突变所带来的抗病毒效应。