Origin binding protein-containing protein-DNA complex formation at herpes simplex virus type 1 oriS: role in...

Area of Science:

• Virology
• Molecular Biology
• Genetics

Background:

• Herpes simplex virus type 1 (HSV-1) DNA replication initiation requires origin binding protein (OBP) binding to viral origins (oriL and oriS) during productive infection.
• The role of OBP during HSV-1 reactivation from neuronal latency is unknown.
• A truncated OBP, OBPC, binds origin sequences but its function in viral DNA replication is unclear.

Purpose of the Study:

• To investigate the role of OBP and OBPC in HSV-1 DNA replication initiation.
• To characterize protein-DNA complex formation at the oriS origin in neural and non-neural cells.

Main Methods:

• Gel shift assays were performed using nuclear extracts from HSV-infected PC12 (neural) and Vero (non-neural) cells.
• Specific nucleotide mutations were introduced into the oriS binding site.
• Transient DNA replication assays were conducted.

Main Results:

• Three specific protein-DNA complexes (A, B, and C) involving OBP or OBPC were identified at the oriS highest-affinity binding site (site 1).
• Complex A contained OBPC exclusively, while complexes B and C contained OBP and other cellular proteins.
• Mutations affecting complexes B and C significantly impaired oriS-dependent DNA replication, whereas mutations affecting only complex A did not.

Conclusions:

• OBP-containing complexes B and C are required for efficient initiation of HSV-1 oriS-dependent DNA replication.
• OBPC-containing complex A alone is insufficient for efficient viral DNA replication initiation.
• These findings clarify the distinct roles of OBP and OBPC in HSV-1 DNA replication.