Center for Infectious Disease Research, Westlake Laboratory of Life Sciences and Biomedicine, Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou 310024, Zhejiang Province, China.
Beijing Advanced Innovation Center for Structural Biology, Tsinghua-Peking Joint Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing 100084, China.
Structure. 2024 Jun 6;32(6):654-661.e3. doi: 10.1016/j.str.2024.03.004. Epub 2024 Apr 4.
There are three key components at the core of the mpox virus (MPXV) DNA polymerase holoenzyme: DNA polymerase F8, processivity factors A22, and the Uracil-DNA glycosylase E4. The holoenzyme is recognized as a vital antiviral target because MPXV replicates in the cytoplasm of host cells. Nucleotide analogs such as cidofovir and cytarabine (Ara-C) have shown potential in curbing MPXV replication and they also display promise against other poxviruses. However, the mechanism behind their inhibitory effects remains unclear. Here, we present the cryo-EM structure of the DNA polymerase holoenzyme F8/A22/E4 bound with its competitive inhibitor Ara-C-derived cytarabine triphosphate (Ara-CTP) at an overall resolution of 3.0 Å and reveal its inhibition mechanism. Ara-CTP functions as a direct chain terminator in proximity to the deoxycytidine triphosphate (dCTP)-binding site. The extra hydrogen bond formed with Asn665 makes it more potent in binding than dCTP. Asn665 is conserved among eukaryotic B-family polymerases.
痘病毒（MPXV）DNA 聚合酶全酶的核心有三个关键组成部分：DNA 聚合酶 F8、延伸因子 A22 和尿嘧啶-DNA 糖基化酶 E4。全酶被认为是一种重要的抗病毒靶标，因为 MPXV 在宿主细胞的细胞质中复制。核苷酸类似物，如cidofovir 和阿糖胞苷（Ara-C）已显示出抑制 MPXV 复制的潜力，并且对其他痘病毒也有一定的效果。然而，其抑制作用的机制仍不清楚。在这里，我们展示了与竞争性抑制剂 Ara-C 衍生的阿糖胞苷三磷酸（Ara-CTP）结合的 DNA 聚合酶全酶 F8/A22/E4 的 cryo-EM 结构，整体分辨率为 3.0Å，并揭示了其抑制机制。Ara-CTP 作为一个直接的链终止子，靠近脱氧胞苷三磷酸（dCTP）结合位点。与 Asn665 形成的额外氢键使其比 dCTP 具有更强的结合能力。Asn665 在真核 B 族聚合酶中是保守的。
