Nucleolar Targeting and ROS-dependent inhibition of rRNA synthesis by Epstein-Barr Virus Nuclear Antigen 1

Epstein-Barr virus nuclear antigen 1 (EBNA-1) is essential for viral episome maintenance and is consistently expressed in all forms of EBV latency. In a previous study, we observed EBNA-1 localizing in the nucleolus, as well as its interaction with the nucleolar protein EBP2 in both the nucleoplasm and nucleoli. Nucleoli are dynamic nuclear domains that coordinate ribosome assembly with the regulation of cell cycle progression and stress responses. Here, we studied the mechanism by which EBNA-1 achieves nucleolar localization and functional consequences for host cell physiology. We observed that EBNA-1 nucleolar accumulation occurs specifically during late G1 and S phase and depends on a bipartite nucleolar localization signal (NoLS) composed of two cooperative Weber motifs separated by 24 amino acids. Mutations disrupting NoLS abolish interaction with the nucleolar protein EBP2, whose depletion similarly prevents EBNA-1 nucleolar import. Once in the nucleolus, EBNA1 triggers a 50 % reduction in the synthesis of ribosomal RNA (rRNA) which leads to a global reduction in cellular protein production. In addition, EBNA1 nucleolar targeting induces reactive oxygen species (ROS) production and antioxidant treatment restores both rRNA transcription and protein synthesis. These findings identify EBP2 as a viral nucleolar docking partner and establish nucleolar targeting of EBNA1 as a regulated, cell cycle-dependent process that links oxidative stress to the perturbation of ribosome biogenesis. This mechanism may contribute to the ability of EBNA-1 to promote cell survival and transformation. IMPORTANCEEBNA1 is the only Epstein-Barr virus protein that is consistently expressed in all forms of latency and in every EBV-associated tumor. Although it localizes to the nucleolus and interacts with three nucleolar proteins, its influence on nucleolar functions had never been investigated. Here, we demonstrate that EBNA1s nucleolar targeting is a strictly cell-cycle-regulated process that depends on a previously unrecognized bipartite nucleolar localization signal as well as its interaction with the nucleolar protein EBP2. Once inside the nucleolus, EBNA1 induces a ROS-dependent suppression of rRNA synthesis, resulting in a global decrease in cellular protein production. In addition, these findings uncover a new function of EBNA1: the ability to induce nucleolar stress without activating canonical apoptotic pathways, thereby promoting host-cell survival and potentially contributing to EBV-driven oncogenesis