African swine fever virus (ASFV) is an important animal pathogen that is causing a current African swine fever pandemic and affecting pork industry globally. ASFV encodes at least 150 proteins, and the functions of many of them remain to be clarified. The ASFV protein E301R (pE301R) was predicted to be a DNA sliding clamp protein homolog working as a DNA replication processivity factor. However, structural evidence was lacking to support the existence of a ring-shaped sliding clamp in large eukaryotic DNA viruses. Here, we have solved a high-resolution crystal structure of pE301R and identified a canonical ring-shaped clamp comprising a pE301R trimer. Interestingly, this complete-toroidal structure is different from those of the monomeric clamp protein homolog, herpes simplex virus UL42, and the C-shaped dimeric human cytomegalovirus UL44, but highly homologous to that of the eukaryotic clamp homolog proliferating cell nuclear antigen. Moreover, pE301R has a unique N-terminal extension that is important in maintaining the trimeric form of the protein in solution, while specific features in length and surface electrostatic potential of its interdomain connector implies specificity in interactions with binding partners such as the viral DNA polymerase. Thus, our data pave the way for further dissection of the processivity clamp protein structural and functional diversity and ASFV DNA replication mechanisms.

非洲猪瘟病毒（ASFV）是一种重要的动物病原体，导致当前非洲猪瘟大流行并影响全球猪肉业。ASFV 编码至少 150 种蛋白质，其中许多蛋白质的功能仍有待阐明。ASFV 蛋白 E301R (pE301R) 被预测为 DNA 滑夹蛋白同源物，充当 DNA 复制持续因子。然而，缺乏结构证据来支持大型真核 DNA 病毒中存在环形滑动钳。在这里，我们解析了 pE301R 的高分辨率晶体结构，并鉴定了包含 pE301R 三聚体的规范环形夹。有趣的是，这种完整的环形结构与单体钳蛋白同源物、单纯疱疹病毒UL42和C形二聚体人巨细胞病毒UL44的结构不同，但与真核钳同源物增殖细胞核抗原高度同源。此外，pE301R 具有独特的 N 末端延伸，这对于维持溶液中蛋白质的三聚体形式非常重要，而其域间连接器的长度和表面静电势的特定特征意味着与病毒 DNA 聚合酶等结合伙伴相互作用的特异性。因此，我们的数据为进一步剖析持续钳蛋白结构和功能多样性以及 ASFV DNA 复制机制铺平了道路。