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Interaction between the herpes simplex virus type 1 origin-binding and DNA polymerase accessory proteins
S J Monahan1, L A Grinstead, W Olivieri
1Department of Medical Microbiology and Immunology, Ohio State University, 333 West Tenth Avenue, Columbus, Ohio, 43210, USA.
Virology
|February 28, 1998
Summary
Herpes simplex virus type 1 origin-binding protein (UL9) physically interacts with the DNA polymerase accessory protein (UL42) in a DNA-independent manner, facilitating viral DNA replication complex assembly.
Area of Science:
- Virology
- Molecular Biology
- Biochemistry
Background:
- Herpes simplex virus type 1 (HSV-1) DNA replication requires a complex assembly of viral proteins at the origin of replication.
- Previous studies identified interactions between the origin-binding protein UL9 and other replication complex components, but its interaction with the DNA polymerase holoenzyme remained uncharacterized.
Purpose of the Study:
- To investigate and demonstrate the interaction between HSV-1 UL9 and the DNA polymerase accessory protein UL42.
- To characterize the nature and binding domain of the UL9-UL42 interaction.
Main Methods:
- Co-immunoprecipitation assays using specific antibodies against UL9 and UL42.
- Affinity chromatography employing glutathione S-transferase (GST) fusion proteins of UL42.
- In vitro transcription/translation of UL9 and its fragments.
- Analysis of binding affinities using GST affinity matrices.
Main Results:
- UL9 and UL42 form a stable complex in vitro, independent of DNA presence.
- The N-terminal 533 amino acids of UL9 are sufficient for binding to UL42, though with reduced affinity.
- UL9 demonstrates physical association with components of key HSV-1 DNA replication complexes.
Conclusions:
- The direct interaction between UL9 and UL42 is a crucial step in assembling the HSV-1 DNA replication machinery.
- This interaction contributes to the ordered recruitment of viral proteins to replication origins, initiating viral DNA synthesis.