Structural and Functional Basis for an EBNA1 Hexameric Ring in Epstein-Barr Virus Episome Maintenance

Area of Science:

• Virology
• Structural Biology
• Molecular Biology

Background:

• Epstein-Barr virus (EBV) establishes lifelong latent infections.
• EBNA1 protein is essential for EBV episome replication, maintenance, and segregation during latency.
• The structural basis for EBNA1's functions remains incompletely understood.

Purpose of the Study:

• To elucidate the structural features of EBNA1's DNA-binding domain (DBD).
• To investigate the oligomeric state and functional implications of EBNA1 structure.
• To characterize the impact of polymorphisms on EBNA1 function and EBV episome maintenance.

Main Methods:

• X-ray crystallography to determine the structure of the EBNA1 DBD.
• Site-directed mutagenesis to analyze the oligomeric interface and functional domains.
• In vitro DNA-binding assays and in vivo episome maintenance assays.
• Analysis of EBNA1 interactions with OriP, ORC2, and H3K4me3.

Main Results:

• A novel hexameric ring oligomeric form of the EBNA1 DBD was discovered.
• The T585 residue is crucial for stabilizing higher-order EBNA1 complexes.
• Mutations at the oligomeric interface altered cooperative DNA binding and EBNA1-dependent functions.
• A naturally occurring T585P polymorphism exhibited altered DNA binding, replication, and significant defects in episome maintenance.
• The T585P mutant showed impaired binding to OriP, reduced recruitment of ORC2 and H3K4me3, and compromised subnuclear foci formation.

Conclusions:

• EBNA1 can assemble into higher-order oligomeric structures through a novel hexameric ring interface.
• Polymorphisms within this interface modulate EBNA1's DNA-binding and episome maintenance capabilities.
• Understanding EBNA1's structural dynamics is key to comprehending EBV's persistent latency and associated pathologies.