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<p>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 <i>oriP.</i> The minimal replicative unit of <i>oriP</i> (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 <i>oriP-</i>dependent plasmid replication. These findings identify a previously uncharacterized acidic patch docking surface on EBNA1 essential for coordinating TRF2-Rap1 at <i>oriP</i> and provide new insights into both EBV and telomere DNA replication.</p>

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Structural basis for TRF2-RAP1 recruitment by EBNA1 at the EBV origin of replication

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年4月9日

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  • Samantha Sustek,
  • Troy E. Messick,
  • Jayaraju Dheekollu,
  • Coltin Albitz,
  • Christopher Chen,
  • Anneliese Faustino,
  • Hsin-Yao Tang,
  • Hee Jong Kim,
  • Kenji Murakami,
  • Paul M. Lieberman

摘要

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.