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

Area of Science:

• Molecular Biology
• Virology
• Structural Biology

Background:

• Epstein-Barr Nuclear Antigen 1 (EBNA1) is critical for Epstein-Barr virus (EBV) DNA replication and episomal maintenance.
• The minimal replicative origin of EBV (oriP), specifically ½DS, recruits telomeric proteins TRF2 and Rap1 via nonamer elements, but the interaction mechanism is unclear.

Purpose of the Study:

• To elucidate the structural basis of host-factor engagement involving EBNA1, TRF2, and Rap1 at the EBV oriP minimal replicative unit.
• To understand the role of a unique acidic patch on EBNA1 in this complex formation and its impact on viral replication.

Main Methods:

• Cryo-electron microscopy
• Zero-length cross-linking mass spectrometry
• AlphaFold3 modeling
• Biochemical binding assays
• Mutagenesis studies

Main Results:

• A dynamic complex of EBNA1, TRF2, and Rap1 was defined at the ½DS.
• The TRF2 homodimerization domain (TRFH) interacts with a unique acidic patch on EBNA1, distinct from its DNA-binding site.
• Mutating this acidic patch disrupted TRFH binding and abolished oriP-dependent plasmid replication.

Conclusions:

• An uncharacterized acidic patch on EBNA1 serves as a docking surface for TRF2-RAP1, essential for coordinating these proteins at oriP.
• These findings offer novel insights into EBV replication mechanisms and potential connections to telomere DNA replication processes.