Identification of a Functionally Relevant Adeno-Associated Virus Rep68 Oligomeric Interface.

Area of Science:

• Virology
• Molecular Biology
• Biochemistry

Background:

• Adeno-associated virus (AAV) relies on multifunctional Rep proteins for its life cycle.
• Large Rep proteins (Rep78 and Rep68) possess DNA binding, nicking, unwinding, transcriptional regulation, DNA replication, and integration activities.
• Rep proteins feature an AAA(+) helicase domain and an N-terminal origin-binding domain (OBD).

Purpose of the Study:

• To investigate the oligomeric interface of Rep68.
• To determine the role of this interface in Rep protein oligomerization and function.
• To elucidate the molecular mechanisms regulating Rep protein activity.

Main Methods:

• Identification and characterization of an oligomeric interface in Rep68.
• Site-directed mutagenesis to disrupt the identified interface.
• Assessment of the impact of mutations on Rep68 oligomerization, DNA binding, nicking activity, and viral DNA replication.

Main Results:

• A specific oligomeric interface in Rep68 was identified and characterized.
• Disruption of this interface significantly affected Rep68 oligomerization and its enzymatic activities (DNA binding, nicking).
• Mutations compromising the interface impaired viral DNA replication and transcriptional regulation.

Conclusions:

• The four-helix bundle in Rep68's helicase domain acts as an oligomerization domain (OD).
• Key residues at the OD interface are critical for Rep68 stability, oligomerization, and function.
• Understanding Rep oligomerization dynamics is crucial for regulating AAV life cycle transitions and optimizing gene therapy vector production.