Uracil DNA glycosylase counteracts APOBEC3G-induced hypermutation of hepatitis B viral genomes: excision repair of covalently closed circular DNA.
Uracil DNA glycosylase counteracts APOBEC3G-induced hypermutation of hepatitis B viral genomes: excision repair of covalently closed circular DNA.
复制标题
DOI：
10.1371/journal.ppat.1003361
复制
发表时间：
2013
期刊：
影响因子：
6.7
通讯作者：
Muramatsu M
中科院分区：
文献类型：
--
作者：
Kitamura K;Wang Z;Chowdhury S;Simadu M;Koura M;Muramatsu M
B型肝炎病毒（HBV）的共价闭合环状DNA（cccDNA）在慢性肝炎中起重要作用。提出细胞修复系统将细胞质核衣壳（NC）DNA（部分双链DNA）转化为细胞核中的cccDNA。最近，抗病毒胞苷脱氨酶，AID/APOBEC蛋白，显示通过脱氨在NC-DNA中产生尿嘧啶残基，导致病毒基因组的胞苷到尿嘧啶（C-to-U）超突变。我们研究了嗜肝DNA病毒DNA中的尿嘧啶残基是否被尿嘧啶-DNA糖基化酶（UNG）切除，尿嘧啶-DNA糖基化酶是一种用于碱基切除修复（BER）的宿主因子。当UNG活性被UNG抑制蛋白（UGI）的表达抑制时，APOBEC 3G或干扰素处理诱导的NC-DNA超突变在人肝细胞系中增强。为了评估UNG对cccDNA病毒中间体的影响，我们使用鸭HBV（DHBV）复制模型。DHBV DNA的序列分析表明，cccDNA在表达APOBEC 3G的细胞中积累了G到A或C到T的突变，并且这被UNG抑制广泛增强。cccDNA超突变在P基因中产生了许多过早的终止密码子。UNG抑制还增强了APOBEC 3G介导的病毒复制抑制，包括在长期培养中NC-DNA、前C mRNA和分泌的病毒颗粒相关DNA的减少。当APOBEC 3G的催化位点突变时，未观察到UNG抑制增强APOBEC 3G介导的抑制。再克隆的cccDNA的转染实验显示，UNG抑制和APOBEC 3G表达的组合降低了cccDNA的复制能力。总之，这些数据表明，UNG在细胞核中形成cccDNA期间或之后从病毒基因组中切除尿嘧啶残基，并暗示BER途径活性降低了APOBEC 3介导的超突变的抗病毒作用。人胞苷脱氨酶（AID/APOBECs）是针对各种类型病毒的限制性因子。这些蛋白质具有在嗜肝DNA病毒肝炎B病毒（HBV）和鸭HBV（DHBV）模型的病毒DNA中引入胞苷至尿苷（C-to-U）超突变的能力。众所周知，人基因组DNA中的尿嘧啶残基被尿嘧啶-DNA糖基化酶（UNG）去除，导致产生由下游修复因子修复的脱碱基位点。然而，从病毒基因组DNA中去除尿嘧啶的结果仍然存在争议，因为脱碱基位点可能引发DNA降解并伴有链断裂。我们使用体外细胞培养系统研究了UNG在病毒超突变和嗜肝DNA病毒复制中的作用。我们发现UNG抑制增强了APOBEC 3G诱导的嗜肝DNA病毒DNA的超突变，特别是DHBV cccDNA，一种用于病毒在细胞核中复制的模板。我们测量了纯化的cccDNA的复制能力，发现来自由APOBEC 3G和UNG抑制剂蛋白表达的细胞的再克隆的cccDNA由于较高的超突变率而复制效率较低。这些结果表明嗜肝DNA病毒篡夺宿主细胞的修复系统与AID/APOBEC突变体竞争。
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. Human cytidine deaminases, AID/APOBECs, are restriction factors against various types of viruses. These proteins have the ability to introduce a cytidine-to-uridine (C-to-U) hypermutation in the viral DNAs of the hepadnaviruses hepatitis B virus (HBV) and duck HBV (DHBV) models. It is well known that uracil residues in human genomic DNA are removed by uracil-DNA glycosylase (UNG), resulting in the creation of abasic sites that are repaired by downstream repair factors. However, the consequence of uracil removal from the viral genomic DNA remains controversial, given that it may be possible for abasic sites to trigger DNA degradation with strand breakage. We investigated the role of UNG in viral hypermutation and hepadnaviruses replication using in vitro cell culture systems. We found that UNG inhibition enhanced APOBEC3G-induced hypermutation of hepadnaviral DNAs, especially DHBV cccDNA, a template used for viral replication in the nucleus. We measured the replication ability of purified cccDNA and found that recloned cccDNA from cells expressed by both APOBEC3G and UNG inhibitor protein replicated less efficiently due to higher hypermutation rates. These results suggest that hepadnaviruses usurp the repair system of host cells to compete with AID/APOBEC mutators.
登录
查看更多内容
影响因子：
3.7
作者：
Huovinen T;Brockmann EC;Akter S;Perez-Gamarra S;Ylä-Pelto J;Liu Y;Lamminmäki U
通讯作者：
Lamminmäki U
影响因子：
5.4
作者：
Jost, Stephanie;Turelli, Priscilla;Trono, Didier
通讯作者：
Trono, Didier
影响因子：
8
作者：
Endo, Y.;Marusawa, H.;Chiba, T.
通讯作者：
Chiba, T.
影响因子：
4.8
作者：
Chen, RX;Le Rouzic, E;Benichou, S
通讯作者：
Benichou, S
影响因子：
3.7
作者：
Henry M;Guétard D;Suspène R;Rusniok C;Wain-Hobson S;Vartanian JP
通讯作者：
Vartanian JP