Epstein-Barr virus (EBV) establishes a stable latent infection that can persist for the life of the host. EBNA1 is required for the replication, maintenance, and segregation of the latent episome, but the structural features of EBNA1 that confer each of these functions are not completely understood. Here, we have solved the X-ray crystal structure of an EBNA1 DNA-binding domain (DBD) and discovered a novel hexameric ring oligomeric form. The oligomeric interface pivoted around residue T585 as a joint that links and stabilizes higher-order EBNA1 complexes. Substitution mutations around the interface destabilized higher-order complex formation and altered the cooperative DNA-binding properties of EBNA1. Mutations had both positive and negative effects on EBNA1-dependent DNA replication and episome maintenance with OriP. We found that one naturally occurring polymorphism in the oligomer interface (T585P) had greater cooperative DNA binding in vitro, minor defects in DNA replication, and pronounced defects in episome maintenance. The T585P mutant was compromised for binding to OriP in vivo as well as for assembling the origin recognition complex subunit 2 (ORC2) and trimethylated histone 3 lysine 4 (H3K4me3) at OriP. The T585P mutant was also compromised for forming stable subnuclear foci in living cells. These findings reveal a novel oligomeric structure of EBNA1 with an interface subject to naturally occurring polymorphisms that modulate EBNA1 functional properties. We propose that EBNA1 dimers can assemble into higher-order oligomeric structures important for diverse functions of EBNA1.

IMPORTANCE Epstein-Barr virus is a human gammaherpesvirus that is causally associated with various cancers. Carcinogenic properties are linked to the ability of the virus to persist in the latent form for the lifetime of the host. EBNA1 is a sequence-specific DNA-binding protein that is consistently expressed in EBV tumors and is the only viral protein required to maintain the viral episome during latency. The structural and biochemical mechanisms by which EBNA1 allows the long-term persistence of the EBV genome are currently unclear. Here, we have solved the crystal structure of an EBNA1 hexameric ring and characterized key residues in the interface required for higher-order complex formation and long-term plasmid maintenance.

爱泼斯坦-巴尔病毒（EBV）建立了稳定的潜伏感染，可以在宿主的生命中持续存在。EBNA1是潜在附加体的复制，维持和分离所必需的，但是赋予这些功能中的每一个的EBNA1的结构特征尚不完全清楚。在这里，我们解决了EBNA1 DNA结合域（DBD）的X射线晶体结构，并发现了一种新型的六聚环寡聚形式。寡聚界面围绕残基T585枢转，作为连接并稳定高阶EBNA1复合物的接头。界面周围的取代突变使高阶复合物的形成不稳定，并改变了EBNA1的协同DNA结合特性。突变对EBNA1依赖的DNA复制和OriP维持附加体具有正向和负向影响。在体外，DNA复制中的细微缺陷以及附加体维护中的明显缺陷。T585P突变体在体内与OriP的结合以及在OriP处组装起源识别复合物亚基2（ORC2）和三甲基化组蛋白3赖氨酸4（H3K4me3）时受到损害。T585P突变体还因在活细胞中形成稳定的亚核灶而受到损害。这些发现揭示了EBNA1的新型寡聚结构，其界面具有调节EBNA1功能特性的自然多态性。我们建议EBNA1二聚体可以组装成对EBNA1的各种功能很重要的高级寡聚结构。

重要说明爱泼斯坦-巴尔病毒是一种人类γ疱疹病毒，与多种癌症有因果关系。致癌特性与病毒在宿主生命周期中以潜伏形式持续存在的能力有关。EBNA1是在EBV肿瘤中始终表达的序列特异性DNA结合蛋白，并且是在潜伏期维持病毒附加体所需的唯一病毒蛋白。EBNA1允许EBV基因组长期保留的结构和生化机制目前尚不清楚。在这里，我们已经解决了EBNA1六聚环的晶体结构，并表征了高阶复合物形成和长期质粒维持所需的界面中的关键残基。