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.

mpox 病毒 (MPXV) DNA 聚合酶全酶的核心由三个关键成分组成：DNA 聚合酶 F8、持续合成因子 A22 和尿嘧啶 DNA 糖基化酶 E4。由于 MPXV 在宿主细胞的细胞质中复制，全酶被认为是重要的抗病毒靶点。西多福韦和阿糖胞苷 (Ara-C) 等核苷酸类似物已显示出抑制 MPXV 复制的潜力，并且还显示出对抗其他痘病毒的潜力。然而，其抑制作用背后的机制仍不清楚。在这里，我们展示了 DNA 聚合酶全酶 F8/A22/E4 与其竞争性抑制剂 Ara-C 衍生的阿糖胞苷三磷酸 (Ara-CTP) 结合的冷冻电镜结构，整体分辨率为 3.0 Å，并揭示了其抑制机制。 Ara-CTP 在脱氧胞苷三磷酸 (dCTP) 结合位点附近充当直接链终止子。 Asn665 形成的额外氢键使其比 dCTP 具有更强的结合力。 Asn665 在真核 B 家族聚合酶中是保守的。