EBNA1 SUMOylation by PIAS1 Suppresses EBV Lytic Replication and Enhances Episome Maintenance

Epstein-Barr virus nuclear antigen 1 (EBNA1) is essential for the replication and stable maintenance of the viral episome in infected cells. Here, we identify the SUMO E3 ligase PIAS1 as a key regulator of EBNA1 through site-specific SUMOylation. Our Chromatin-Immunoprecipitation Sequencing (ChIP-seq) analysis revealed that PIAS1 is enriched at the viral origin of plasmid replication (oriP), where it physically associates with EBNA1 and catalyzes its SUMOylation. Using mutational analysis, we identified three lysine residues on EBNA1 (K17, K75, and K241) as major SUMOylation sites. Disruption of these sites compromises EBNA1's ability to restrict EBV lytic replication. In addition, both PIAS1 depletion and the disruption of EBNA1 SUMOylation lead to reduced retention of EBNA1-OriP-based EBV mini-replicon, indicating the importance of EBNA1 SUMOylation in viral episome maintenance. Together, these results uncover a conserved post-translational mechanism by which PIAS1-mediated SUMOylation modulates EBNA1 function and EBV episome maintenance and suggests a broader role for SUMOylation in viral latency, lytic replication and persistence. Overall design: ChIP seq of PIAS1 in Akata (EBV+) Burkitt lymphoma

EB病毒核抗原1（Epstein-Barr virus nuclear antigen 1, EBNA1）对于感染细胞中病毒游离基因的复制与稳定维持至关重要。本研究通过位点特异性SUMO化修饰，鉴定出SUMO E3连接酶PIAS1为EBNA1的关键调控因子。染色质免疫沉淀测序（Chromatin-Immunoprecipitation Sequencing, ChIP-seq）分析显示，PIAS1富集于质粒复制的病毒起源位点（oriP），并在此处与EBNA1直接结合并催化其SUMO化修饰。通过突变分析，我们鉴定出EBNA1上的三个赖氨酸残基（K17、K75与K241）为主要的SUMO化修饰位点。破坏这些位点会削弱EBNA1抑制EB病毒裂解性复制的能力。此外，PIAS1敲低与EBNA1 SUMO化修饰的破坏均会导致基于EBNA1-OriP的EB病毒微型复制子的留存率降低，表明EBNA1的SUMO化修饰在病毒游离基因维持中具有重要作用。综上，本研究揭示了PIAS1介导的SUMO化修饰调控EBNA1功能与EB病毒游离基因维持的保守翻译后修饰机制，并提示SUMO化修饰在病毒潜伏感染、裂解性复制与持续感染中具有更广泛的作用。整体实验设计：Akata（EBV阳性）伯基特淋巴瘤细胞中PIAS1的ChIP-seq分析