人源APOBEC3G蛋白CD2结构域催化DNA脱氨基化抗HIV感染分子机制研究

APOBEC3G (A3G) is a member of the family of deoxycytidine deaminases APOBEC, which can convert dC residues in single-stranded DNA (ssDNA) to dU, and act as DNA mutators. APOBEC3G contains two active domains, A3G-CD1 at its N-terminus and A3G-CD2 at its C-terminus. A3G-CD1 mediates the interactions of A3G with Vif protein of the virus. A3G-CD2 catalyzes cytidine deamination, induces G-to-A hypermutation in double-stranded DNA, impairs biological function of HIV-1 virus genes, and suppresses virus replication. The deamination site of A3G-CD2 locates at the cytidine at 3’-terminus of the hot spot 5’-CCC-3’ in the antisense ssDNA of virus gene, and is performed by jumping or sliding through 3’->5’ direction. The NMR or X-ray structures of A3G-CD2 in its free state have been reported, however, due to the fast deamination, it’s difficult to capture the stable complex of A3G-CD2 with ssDNA, thus how A3G-CD2 specifically recognizes the hot spot 5’-CCC-3’ in ssDNA and performs deamination reactions on cytidine is still unclear. In the project, we plan to do chemical modification on cytidine by inducing a electron donor group at 5-position of its aromatic ring, to hinder the deamination reaction, and to get the stable complex of A3G-CD2 with ssDNA containing hot motif. Then we will try to determine the NMR solution structure of A3G-CD2 in complex with the ssDNA, to explain the molecular basis of how A3G-CD2 performs its deamination reaction on virus ssDNA, to present the structural basis of A3G’s anti-virus activities.

APOBEC3G（A3G）是胞嘧啶脱氨基化酶家族APOBEC3成员，含N-端A3G-CD1结构域和C-端A3G-CD2结构域。A3G-CD1介导HIV病毒壳体化。A3G-CD2负责催化病毒单链DNA胞嘧啶脱氨基化，诱导G→A超突变，使HIV基因组不能执行其功能，抑制病毒复制。A3G-CD2催化胞嘧啶脱氨基化一般发生在病毒DNA负链含5’-CCC-3’序列的3’端胞嘧啶上，通过由3’→5’跳跃或滑动来持续发挥该功能。自由态A3G-CD2结构已有报道，但催化脱氨基化反应较快，难以捕获A3G与DNA瞬态复合体；故A3G识别5’-CCC-3’并进行脱氨基化反应的机制没有文献报道。本项目计划在活性位点碱基胞嘧啶芳环5-位引入供电子基团，阻碍脱氨基化发生，得到A3G-CD2与DNA稳定复合物，通过NMR技术解析其结构，阐述A3G-CD2催化胞嘧啶脱氨基化分子机制, 开展A3G抗HIV活性机制研究。

APOBEC3G（A3G）是胞嘧啶脱氨基化酶家族APOBEC3成员，含N-端A3G-CD1结构域和C-端A3G-CD2结构域。A3G-CD1介导HIV病毒壳体化。A3G-CD2负责催化病毒单链DNA胞嘧啶脱氨基化，诱导G→A超突变，使HIV基因组不能执行其功能，抑制病毒复制。A3G-CD2催化胞嘧啶脱氨基化一般发生在病毒DNA负链含5’-CCC-3’序列的3’端胞嘧啶上，通过由3’→5’跳跃或滑动来持续发挥该功能。自由态A3G-CD2结构已有报道，但催化脱氨基化反应较快，难以捕获A3G与DNA瞬态复合体；在本项目启动前A3G识别5’-CCC-3’并进行脱氨基化反应机制没有文献报道。项目团队主要开展了如下工作：（1）在活性位点碱基胞嘧啶芳环5-位引入供电子基团碘原子（即：5’-ATTC4C5C6IA7ATT-3’ ，简称TCCC6I)，减慢了脱氨基化发生，但碘原子将A3G-CD2脱氨基序列偏好从CCC切换到TCC。（2）通过NMR技术解析A3G-CD2与产物DNA TCUC6I和TCUU6I的复合物溶液结构，结果表明底物DNA以TCC和CCC模式结合A3G-CD2。dC6脱氨基与第四种碱基类型有关。CCC模式有利于dC6脱氨基，而TCC模式有利于dC5脱氨基。这些结果很好地阐述A3G-CD2催化胞嘧啶脱氨基化分子机制。（3）开展了靶向A3G与Vif相互作用的抗HIV的小分子抑制剂筛选研究，筛选了两组含有氨甲酰磺胺键或二硫键的小分子作为两种不同芳香环的桥接物。最终鉴定出5种不对称的二硫化合物，IC50值接近或小于1 μM，且不与A3G或Vif共价结合。它们通过抑制Vif诱导的A3G泛素化和降解来恢复A3G在Vif存在下的表达。这为新的抗艾滋病毒药物发现开辟了道路。通过本项目实施，发表标注项目基金号论文14篇，授权专利2项。培养博士生5名，硕士生8名。