# Structural Basis for TRF2-RAP1 Recruitment by EBNA1 at the EBV origin of replication

## TL;DR

This study reveals how the EBV protein EBNA1 interacts with human telomere proteins TRF2 and RAP1 to enable virus replication in infected cells.

## Contribution

The study identifies a novel acidic patch on EBNA1 that is crucial for recruiting TRF2-RAP1 to the EBV origin of replication.

## Key findings

- EBNA1 forms a dynamic complex with TRF2 and RAP1 on the EBV origin of replication.
- A unique acidic patch on EBNA1 is essential for TRF2 homodimerization domain binding.
- Disrupting the acidic patch prevents TRF2-RAP1 recruitment and oriP-dependent replication.

## Abstract

Epstein-Barr Nuclear Antigen 1 (EBNA1) is essential for the episomal maintenance and DNA replication of Epstein-Barr virus (EBV) in latently infected cells and acts through binding to oriP. The minimal replicative unit of oriP (½DS) contains four EBNA1 binding sites flanked by single telomeric nonamers that recruit shelterin proteins TRF2 and Rap1, but the structural basis for host-factor engagement is not known. Here, we integrate cryo-electron microscopy, zero-length cross-linking mass spectrometry, Alphafold3 modeling, and biochemical binding assays to define the complex formed by EBNA1-TRF2-Rap1 assembly on the ½DS. We find that a highly dynamic complex is formed, with the TRF2 homodimerization domain (TRFH) flexibly interacting with EBNA1 on the surface opposite the DNA-binding region, where there is a large acidic patch in EBNA1 that is unique amongst the herpesvirus episome maintenance proteins. Mutagenesis of this acidic patch abolishes TRFH binding and oriP-dependent plasmid replication. These findings identify a previously uncharacterized acidic patch docking surface on EBNA1 essential for coordinating TRF2-RAP1 at oriP and provide new insights into both EBV and telomere DNA replication.